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UNICORN 5.0 User Reference Manual

1. Evaluation 12 12 Evaluation Introduction This chapter describes e How to evaluate results with the focus on how to integrate peaks e How to automate evaluation operations e How to export data and curves In this chapter This chapter contains these sections Topic See Peak integration 12 1 Other evaluations 12 2 Automated evaluation procedures 12 3 e p 327 12 Evaluation 12 1 Peak integration 12 1 Introduction In this section 03 0014 90 ep 328 Peak integration Peak integration is used to identify and measure a number of curve characteristics including peak areas retention time and peak widths This section describes e How to perform peak integrations e How to optimize peak integrations This section contains these sub sections Topic See How to integrate part of a curve and how to exclude or skim peaks 12 1 7 Measurements 12 1 8 12 1 1 Introduction Baseline options The Calculate baseline function Baselines based on a blank curve Zero baseline Reuse an existing baseline Evaluation 12 Baseline calculation The first step when you integrate peaks is to calculate a baseline A correct baseline is crucial for accurate calculation of the peak areas This section describes the options for how to calculate baselines in the Integrate dialog box UNICORN offers several options for how to create an accurate baseline e
2. Print Chromatograms x Printer Acrobat Distiller Print Format Li Ey GL tes Chromatograms in each column P REEI EE Chromatograms in each row V Use thick lines Preview I Landscape OK Cancel Help Ceres Hep The table below describes how to print active chromatograms Step Action Open all chromatograms that you want to print in the Evaluation module e Select File Print or e Click the Print toolbar icon Result The Print Chromatograms dialog box opens Select print format and layout options e Click OK to print or e Proceed with step 5 to preview and edit the layout ep 247 10 How to view results 10 5 How to print active chromatograms 03 0014 90 ep 248 Step Action or Click the Preview button Result The Customise Report window opens Click the Edit Mode button to make changes e g change the order of the chromatograms see 10 6 1 How to create and print a customized report on page 250 for more information about how to edit Click the Preview button to return to preview mode Select File Print Click the Print toolbar icon Result The Print dialog box opens Select the print range and number of copies Click OK How to view results 10 10 6 How to create and print reports Introduction The Evaluation module provides extensive tools to create detailed reports This section describes how
3. When an error message appears in System Control follow the instructions in this table to activate the Generate Report Wizard and create a report Step Action e Click the Report button in the error message dialog box or e Choose System Report The first step is a Welcome screen Click the Next button Result The Description dialog box is displayed and shows a list of the problems errors that have occurred All the problems errors that have occurred together with help texts are automatically recorded and included in the report e If you select a specific error in the Description dialog box the appropriate help text is shown in the error message box Add the following information in the Description dialog box e A short description of the problem e The circumstances under which the problem occurs e The consequences of the problem Click the Next button Result The Reproducibility dialog box opens Specify whether the problem is reproducible or not Select one of these alternatives e Yes Provide a short description in the text box of how the problem can be reproduced e No e Unknown Click the Next button to proceed to attach example files see table below ep 473 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 2 How to generate a report from the System Control ve a toat You can attach method files and or log files to the problem report t
4. ____ Parameter___ Value _ Range Pe Opt 14 0 0 0 14 0 0 0 14 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 Number of acidic protons 0 0 3 Charge of deprotonated ion O 3 0 pKa values given at 25 C OK Cancel Help 3 Select the buffer component from the Name droplist and note the displayed pKa value Click Cancel to return to the New Recipe dialog box Use the pKa value to determine the pH range Typically a range of 0 5 units around the pKa value is useful Note Check buffer tables for the exact ranges How to definea Note Before you can define a new buffer substance you must ensure that all pKa FA sub values are available for the substance The pKa values should be true i e the pKa value at indefinite dilution and not apparent pKa values i e measured at a non zero concentration The pKa values should be given at 25 C The table below describes how to define a new buffer substance Click the Buffer substance button in the New Recipe dialog box Result The Define buffer substance dialog box opens ep 539 E How to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe How to define a new salt 03 0014 90 e p 540 Step ik Action Click the New button Result The New component dialog box opens Type a name for the new component and click OK to return to the Define buffer substance dialog box Type appropr
5. When the result file is saved it includes the molecular size table that was used for the molecular size determination You can view the table that was used by selecting Mol Size Edit Mol Size Table View Current 03 0014 90 e p456 The Analysis module 13 Ifthe molecular If the molecular size cannot be calculated one of the following signs is shown in size cannot be cal the peak table Mol size column culated Function This means that the molecular size is larger than the largest size in the molecular size curve i e outside the valid range of the curve This means that the molecular size is smaller than the smallest size in the molecular size curve i e outside the valid range of the curve This means that the retention value is negative Molecular size The table below describes the new procedure instruction for molecular size eae instruc measurement that becomes available when the Analysis module is installed Instruction Description MOLSIZE The instruction calculates the molecu lar sizes from a molecular size curve A Mol size column will be added to the peak table ep 457 14 System settings 14 Introduction In this chapter 03 0014 90 e p 458 System settings This chapter describes some of the general system settings This chapter contains these sections Topic See General information about system settings 14 1 Alarms 14 2 Curves 1
6. Recovery applied Current peak filter settings of peaks Note The checkbox Do not show global peak table data must be de selected in the Peak Table tab of the Chromatogram Layout dialog box If the recovery cannot be calculated one of the following signs is shown in the peak table Amount column Function This means that one of the amounts concentrations is higher than the highest value in the calibration curve i e outside the valid range of the calibration curve lt This means that one of the amounts concentrations is lower than the lowest value in the calibration curve i e outside the valid range of the calibration curve The Analysis module 13 Sign Function This means that the recovery factor cannot be calculated For ex ample this sign might indicate that the peak could not be identified in both runs e p 437 13 The Analysis module 13 5 Automated quantitation 13 5 Introduction In this section 03 0014 90 e p 438 Automated quantitation Some method wizards designed for quantitation are available for KTAdesign systems supplied with Autosampler A 900 or A 905 These can be used to quantitate a sample automatically or to update a quantitation table The procedures described in this chapter are designed for use with the systems mentioned above This section contains these sub sections Topic See How to set up for automated qu
7. so Function The Hold icon suspends execution of the method while liquid is still pumped at the current flow rate and eluent concentration The function of the Pause icon depends on the strategy The Pause icon suspends execution of the method and stops all pumps so that the system comes to a standstill The Continue icon resumes the execution of a paused or held method The End icon terminates the method execution and puts the system into an End state The Customise Panes icon opens the Customise Panes dialog box which is used to select the display panes that are open The View Documentation icon opens the documenta tion pages Run notes can be entered in the Notes page and settings can be changed The View Properties icon opens the Properties dialog box which is used to control the data display in the System Control panes The Connect System icon is used to connect a system The Disconnect System icon is used to disconnect the system The Take Control of the System icon is used to leave the view mode for the system and change into a con trol mode The Leave Control of the System icon is used to leave the control mode for the system and change into a view mode UNICORN concepts 2 2 2 4 The Evaluation module Introduction The Evaluation module provides extensive facilities to present and to evaluate curve data The module win Opened result files are displayed in the Evaluation modul
8. M Curve options Overlay Stack Mirror M Eise innegchiomaiogian Compare ewj e e Select the desired search criteria in the Folder Result Chromatogram and Curve name droplists of the Chromatogram selection section e Click Search and a list of found curves will be displayed based on the selected search criteria Note A new search can be performed with new search criteria without erasing curves located in the previous search e Select the check boxes for the curves that you want to import Click the Select All button if you want to import all the curves e Ifyou select the Store in new chromatogram option the curves will be imported into a new chromatogram This is recommended to keep the source chromatogram free of too many additional curves 03 0014 90 p 306 How to edit results 11 Step Action Select how to display the imported curves in the Curve options field and click OK See the options below iki 4 AWH tw Sa 4 mr ares ane J a od V V y ka Noea _ ___ The curves are presented overlaid on one another Stack The curves are presented with a given offset Y axis value so that the curves are stacked and distinct from one another Mirror For comparison of two imported curves One curve is inverted in the Y axis and thus appears to mirror the other curve e p 307 11 How to edit results 11 8 How to impor
9. Result The Normalise window is displayed where a box surrounds the curve selected to be normalised ee Lae ime i Lie owe e ep 313 11 How to edit results 11 8 How to import and compare different runs 11 8 4 How to stack and stretch curves 03 0014 90 ep 314 Step ii Action In the Normalise window you can use the following command but tons Size Allows the arrow keys to be used to stretch the selected curve along its Y axis or X axis This is useful for comparison of curves with for example different gradient lengths e Click the Size button and use the arrow keys to stretch the the curve either along its Y axis or X axis Move Allows the arrow keys to be used to move the selected curve to any position on the chromatogram Axes are automatically re scaled to accommodate the new positioning This function is useful for stacking curves e Click the Move button and use the arrow keys to move the curve into position The curve can also be moved with the mouse pointer Click the mouse button when the curve is in the correct position Note The curve can also be moved and sized with the mouse pointer Normalise The curve to be normalised will be adjusted to the help curve Thus the height of the highest peak on both curves will be the same and will occur at the same retention point e Click the Normalise button The curve to be normalised is auto matically moved along the X
10. determines which items of the Run Setup that are dis played at the start of the run displays the questions used in the method Questions provide a means for entering run specific information at the start of a run Use this tab when you want to define questions specifies how the result files will be named for the results of a run and where the result file will be saved 6 5 2 Introduction Check boxes How to change the default values Blue values Variables can also be changed in the Text Instructions Editor How to edit methods 6 The Variables tab The Variables tab lists all variables used in the method with their default values organized by method block You can change the default values to create a variant of the method Note The variables of a block are only displayed once on the Variables tab even if the block is called several times in a method Variables are displayed only if the method contains variables There are three check boxes on the Variables tab The table below describes these boxes Check box Select this box if you want Show details detail variables to be shown Detail variables are in dicated by a D in the column immediately to the left of the Variable column Show unused variables unused variables to be shown Unused variables are indicated by a U in the column immediately to the left of the Variable column Display tooltip for exten to display useful
11. How to view results 10 How to cut a The table below describes how to cut the curve between two values on the X axis curve and store as a new curve and store this part of the curve as a new curve Step Action Open a result file Choose Operations Cut curve Result The Select Curve s to Operate On dialog box opens e Select the curves to be operated on e Click OK Result The selected curves are shown in the Cut dialog box which contains two vertical cursor lines To select the region to be cut either e drag the two cursor lines to define the left and right limits of the cut area or e type the desired left and right limit values in the Left limit and Right limit boxes Note The areas outside of the Left limit and Right limit will not be saved in the newly created cut curve Thus the X axis of the new curve will not begin at zero unless this is designated as one of the limits The original curve is not changed Click OK Result The Save Cut Curves dialog box opens e Select whether to save the new cut curve in the Source chromatogram that is the current active chromato gram or aNewchromatogram if you select this option you can change the name of the chromatogram Note that it is a recommend ation not to use only numbers as names for chromatograms e Click OK Result If the destination of the cut curve was the source chromato gram the cut curve is automatically displayed
12. Integrity check When UNICORN is installed a checksum calculation is performed on the stationary files dll and exe for the system An integrity check means that a new checksum calculation is performed for the same files in their folders This new calculated value is compared with the checksum value obtained during installation The results of the comparison are presented in the report and any deviations are included e Click the Next button Result The Generate report dialog box is displayed Proceed to Step 3 How to generate and save the report below The table below describes how to generate and save the report Action By default the report is saved in the folder Unicorn Reports If you want to save the report at another location select a folder in the tree structure You also have these options e Click the Preview button to open the report in Notepad e Click the Print button to print the report without any preview Click the Finish button to generate and save the report 15 2 2 Introduction Step 1 How to create the report System maintenance and error reporting How to generate a report from the System Control 15 The Generate Report Wizard is used to generate problem reports When an error message appears in System Control you can activate the report wizard from the error message dialog box The Generate Report Wizard can also be activated anytime if you choose System Report
13. Result The sample concentration and amount for each pool is calculated in the corresponding table cell The Target conc and Target vol cells are used to calculate the pool volume at a specific concentration level The result can then be used to determine if the pool needs to be concentrated further or diluted e Type the desired concentration level mg ml in the Target conc table cell Result The corresponding target volume is calculated in the Target vol table cell using the following formula Target vol Conc Vol Target conc A protocol of the pooled fractions can be printed for use when handling the samples The table below describes how to add pools to the Pooling Protocol and send the list to a printer or export the list to a file Step Action 1 e Open a result file in the Evaluation module e Pool fractions as described in How to pool fractions above e Click the Add to Pooling Protocol button Result The pooled fractions from the active result file is added to the Pooling Protocol How to edit results 11 Step Action e Repeat step 1 to add pooled fractions from other result files Note You will be asked to save the current file when you open the next The pool table will not be saved e Click the View Pooling Protocol button Result The Pooling Protocol dialog box opens Pooling Protocol a E x Fractions to be pooled System Result SampleId Pool Conc Text T
14. Step Action Use the Browse button to find and select the folder to search for result files and chromatograms Note The search will only be performed in the selected folder You can use standard wildcard characters and define restricting search criteria for the Result and Chromatogram fields Up to 10 user defined search filters can be saved in the drop menus Click the Search button Result A list of found chromatograms is displayed Select the chromatograms you want to perform the run on e The Select All button selects all chromatograms e The Clear button removes all chromatograms from the list Click the Run button Result The batch run is performed and any created curve or peak table will automatically be saved in each result file The Batch Run The illustration below is an example of search results in the Batch Run dialog box dialog box Ta Batch Run Report_Chromatogram Of x r Chromatogram selection Folder c Defaults E Browse Result e aseline ba Browse All Chromatogram F Ma Browse All Clear r Found chromatograms d Select all VBaseline example 1 VBaseline example 2 1 Baseline example 3 1 Run Cancel Help e p 385 12 Evaluation 12 3 Automated evaluation procedures 12 3 3 How to run a procedure How to batch run Evaluation procedures combined with batch runs can be a useful tool to produce reports
15. file Evaluation Troubleshooting A This section describes how to solve the following evaluation problems e Incorrect date and time in the result file e Evaluation procedure aborts The table below describes a problem and its solution Problem description The result file shows incorrect date and time Solution Check the system clock setting The date and time recorded in the result file are taken from the PC sys tem clock setting Evaluation proced The table below describes a problem and its solution ure aborts Problem description The evaluation procedure aborts Solution Instructions in an evaluation proced ure refer to curves by identification number irrespective of the curve names Make sure that the curves processed when the procedure is ex ecuted are compatible with those pro cessed when it was recorded An eval uation procedure aborts if you try to store resulting curves at the position of an original raw data curve ep 487 A Troubleshooting A 5 AKTAdesign system specific problems A 5 AKTAdesign system specific problems In this section This section describes how to solve the following problems e Connected to a system but no system contact e Flow scheme not displayed properly Connected to a The table below describes a problem and its solution system but no sys tem contact ET Problem description Solution You are connected to a s
16. for information only This window is not updated according to system status and changes in the meth od to specify break points instructions parameters and vari ables to insert change and delete instructions to select or display blocks Gradient to display block dura tion and eluent gradient throughout the method See section 6 2 1 How to view method blocks on page 98 6 3 Method instructions on page 111 9 2 4 The Flow Scheme pane on page 204 6 3 2 How to add meth od instructions on page 113 6 2 Method blocks on page 97 6 5 5 The Gradient tab on page 133 How to edit methods 6 6 2 Method blocks Introduction This section contains a description of how to organize a method in blocks of instructions in order to make it more structured and of how to work with method blocks In this section This section contains these topics Topic See How to import method blocks 6 2 7 e p 97 6 How to edit methods 6 2 Method blocks 6 2 1 How to view method blocks 6 2 1 Instructions can be grouped into blocks The Text pane 03 0014 90 ep 98 How to view method blocks To view a method as a long list of individual text instructions can be confusing and inconvenient Text instructions can therefore be grouped into blocks of instructions that define a specific functional use For example one block might contain the instructions necessary to equ
17. quantitation table Components f Colbeation curve Ales Enter amount or concentration for every level Level maim mAU m 1 14 8827 1 500 2 14 3303 3 14 5568 The table below describes how to enter data for the standards and create a quantitation table and a calibration curve Step Action e Click the IS and Table settings button if you want to use an intern al standard or base the calibration curve on peak height see How to select an Internal Standard below this table 2 e Verify that the selected components in the Components list are correct If an internal standard is used the related component is la belled IS If relative retention has been used the reference component is labelled Ref e Click the Define components button to change the components 03 0014 90 ep 418 The Analysis module 13 Step Action e Select the first component at the top of the Components list Note Do not select an internal standard component if available as the amount for this has already been entered and does not change between the levels e Highlight the Amount Concentration for Level 1 e Type the amount or concentration of the component in the standard at this level Note This is the amount corresponding to the injected volume not the total amount used when the standard level was prepared e Repeat this for the other levels for this component Clic
18. Changes made in the Text pane are automatically updated on the Variables tab and vice versa The figure below illustrates the relationship between variables in the Text pane and on the Variables tab of Run Setup 0 00 Block Eluent_A_Inlet tEhent A_Inlet ri Base SameAsMain en O a E 0 00 Block Eluent B_inlet Event B_Intet x00 Base SameAsMain 0 00 it sll B1 Pump_B_ Inlet eeren Mash eA If a breakpoint or gradient length is defined as a variable changing the variable value in the Variables tab when the method run is started will shift other instruction breakpoints accordingly This functionality is equivalent to using the Change button to alter a breakpoint or gradient length see 6 3 4 How to change or move method instructions on page 116 for how the Change button affects instructions within gradients Only one variable that affects block length breakpoint or gradient length may be defined within each block However any number of parameters may be defined as variables within a block The table below describes how to define a new variable Step Action Text pane of Text instructions box box e Click the Var button Result The Variable Name Definition dialog box opens Select the instruction where you want to define the variable in the Result The parameters for the instruction are shown in the Instruction Locate the breakpoint or the required parameter in
19. How sae the You can optimize the exported curves to only the parts that you want to focus on exported curves in the Export Curves dialog box The table below describes how to use these editing options Dialog box option Instruction Enter retention values in the text boxes to limit the curve to only a portion of the original curve Cut graphically This button opens the Export Cut dialog box Move the vertical markers to the correct cutoff points Reduce number of samples Adjust the factor value or the maxim um number of samples To reduce the number of samples by a factor of five means that only every fifth point will be sampled for export Normalise retention Select the Normalise retention checkbox to have all exported curves normalized to a common X axis 03 0014 90 ep 322 How to export curves in AIA format How to export peak tables How to edit results 11 The table below describes how to export curves in AIA format Action Select File Export Export curve to AIA Result The Export curve in AIA format dialog box opens e Select the source chromatogram and the curve you want to export e Click the Export button Result The Export Curves to File dialog box opens e Select a destination folder e Type a file name e Click OK Action Choose File Export Peak Table Result The Export Peak Table dialog box opens e Select the source chromatogram and the peak table
20. In System Control for manual runs the Frac 950 tab cannot be used Instead use the manual fractionation instructions starting with Man_ e Choose Manual Frac to open the Frac Instructions dialog box The total number of tubes sampled may differ if a last tube has been chosen The Number of tubes equation in the bottom left corner of the Frac 950 dialog box of the Start Protocol shows the current number of available tubes chosen for fractionation followed by the total possible number in parentheses ep 149 6 How to edit methods 6 5 Run Setup 6 5 13 The Frac 950 tab How to select the last tube How to set the last tube to default setting 03 0014 90 ep 150 You can select a position for the last tube to be used in the fractionation process If the process attempts to go further than the selected last tube during a method run an alarm will be executed The last tube position can only be selected on the Frac 950 dialog box in the Start Protocol when you start a method run or when you do an instant run The illustration below shows an example of this dialog box Frac 950 ial Rach ach ig esomm Tubes z Enen AXIY a EES 00000000000 cRwy gt PA ATT TTT D 000000000000 see IPU 000000000000 000000000000 ago INN 900000000000 000000000000 Ree Reset to defauk OOQOOOOCOO0O000O Pode este 0101010010000000 78 120 amp 8 Help The lower right box within the Last tube field
21. Select the unspiked sample peak table from the Peak table s list to the left Repeat step 2 in the Addition chromatogram section to the right to select the addition peak table for the spiked sample Edit the default unit mg in the Unit label field if necessary Type the amount of the component that was added as the spike in the Added amount field Click OK Result The Identify Peak dialog box opens To locate and select the peak of the unspiked sample do the follow ing Click its triangle marker black or select its reference in the Source table Repeat step 5 to select the spiked sample The triangle color is blue Use the Addition table Click the OK button ep 433 13 The Analysis module 13 4 How to quantitate the sample 13 4 2 Standard addition quantitation The Identify Peak The illustration shows the Identify Peak dialog box described in the table above dialog box Identify Peak 8 6 4 2 0 2 4 8t 10 12 14 16 138 0 Source table Addition table Retention Retention min min 584 585 6 53 6 53 7 78 7 76 How to view the The amount of the component of interest is displayed in the peak table Amount oe res columns of the Evaluation module area mAU min Height mAU Amount mg 52 9263 282 754 6 53 4 8881 30 365 7 06 6 4036 38 687 7 78 11 3656 42 744 Total number of detected peaks 220 Total area mAU min 92 9166 Area in evaluated peaks mAU min 75 5839 Ratio
22. You can choose to place a method in a MethodQueue if the system is already busy with another method run See 8 1 How to create a new MethodQueue on page 183 In a similar manner you can also start a new MethodQueue while another MethodQueue is in progress It will be placed in queue and executed when the first queue is completed e p217 9 How to perform method runs 9 5 How to perform a MethodQueue run How to display Definition A pending MethodQueue is one for which Run has been requested but and edit pending hich has not yet started either because the system is not available or because the and running MethodQueues setup time has not been reached The table describes how to display running and pending MethodQueues Step Action Click the Running MethodQueue icon Result The Running MethodQueue dialog box is displayed Active MethodQueues LE MOTest4 AE Tuesday 1 39 PM Example2 Example3 ExampleS ExamplePeakFrac Example2 Select a MethodQueue in the Active MethodQueues list box Result Information on the selected MethodQueue is displayed in the Details for field of the dialog box Choose from the following e The Restart button restarts the currently running MethodQueue if a Start Protocol has been terminated by Cancel e The End button terminates a running MethodQueue after the cur rent step Any methods currently in operation will continue to run and must be terminated with the End but
23. e p 133 6 How to edit methods 6 5 Run Setup 6 5 5 The Gradient tab How to use the A vertical marker line can be dragged from the Y axis with the mouse As you drag vertical marker the marker line the current position is identified at the top of the tab in terms of line ar the block name X position in the currently displayed base and eluent concentration in per cent of eluent B How to change You can change the base shown on the Gradient X axis The alternatives are time the base shown on volume and column volumes Changing the base for the display does not affect the the X axi A i RRT base in the method instructions which means that you can check how long a method will take simply by setting the axis scale to time even if the method blocks are written in volume or column volume base The list below describes two ways to change the base shown on the X axis e Click the X axis to toggle between the base types or e Right click anywhere on the Gradient tab Result A sub menu is displayed e Select Base and make the appropriate choice Time Volume or CV How to view You can display a hatched background on the Gradient tab The table below hatch marks describes how to do this Step Action Right click anywhere on the Gradient tab Result A sub menu is displayed Select Hatch Result The Gradient background is hatched 3 To hide the hatch marks repeat steps 1 and 2 03 0014 90 ep 134
24. total area 0 514893 10 Totar peak width min 2 49 11 Column height icm 5 00 32 calculated from ID 265002 10 UV2_21Snm 33 Baseline ID 265002 10_UV2_21Snrij0z BASEN 14 Peak rejection on 15 Naximum number of peaks 20 16 Ine standard applied 17 The quantitation table used for the quantitation When the result file is saved it includes the quantitation table that was used for the quantitation You can view the table that was used by selecting Quantitate Edit Quantitation Table View Current If the amount can If the amount cannot be calculated one of the following signs is shown in the peak not be calculated table Amount column Function This means that the value is higher than the highest value in the calibration curve i e outside the valid range of the calibration curve This means that the value is lower than the lowest value in the calib ration curve i e outside the valid range of the calibration curve This means that the value cannot be calculated For example this sign might indicate that the peak could not be identified ep 431 13 The Analysis module 13 4 How to quantitate the sample 13 4 2 Standard addition quantitation 13 4 2 Standard addition quantitation es in standard Standard addition is performed in five stages addition Description Perform two runs Copy the curves into one result file Integrate the curves to produce the peak tables Sele
25. 109 id1 48Quantitate001 1_Flow 10 id148Quantitate001 1_ Temp 17 id1 48Quantitate001 1_UV1_280nm 01 gt I 3 Select one or more Help curves Select e minimum and maximum values for the Y axis e appropriate units from the Unit list Note The help curve determines the minimum and maximum values for the X axis Click OK Result The Create Curve chromatogram window opens e p 373 12 Evaluation 12 2 Other evaluations 12 2 4 How to create curves How to create In the Create Curve dialog box you can also create new units for the curve The NEW units table below describes how this is done Step Action Click the New unit button Result The Create New Unit dialog box opens Type a new unit name and a number of decimal places Click OK Result The Create New Unit dialog box is closed The new unit is now available in the Create Curve dialog box How to create The new curve is created in the Create Curve window The table below describes curves step 2 how to work in this window Step Action Click the Set Curve Points icon poo e Click to insert curve points in the chromatogram e Add more points to draw the curve Result A green square marks the new curve point The curve is drawn from the previous point Hold the cursor over the inserted point to see the coordinates displayed Curve mode e The regular s
26. 2 2 The UNICORN user interface 2 2 4 The Evaluation module Function The Report icon opens the Generate Report dialog box which is used to select a report format The View Documentation icon opens the Documentation dialog box which is used to view and edit the result documentation The Peak Integrate icon opens the Integrate dialog box which is used to select peaks to integrate in a modified peak table The Chromatogram Layout icon opens the Chromatogram Layout dialog box which is used to select and format curves and display items in the chromatogram The Multifile Peak Compare icon opens the Multifile Peak Compare Wizard which is used to compare peak data from different result files 03 0014 90 ep 36 2 2 5 Introduction Search the Folder list Search the Result list Search the Chro matogram list Search the Curve name list Searches for Sample ID UNICORN concepts 2 Search functions This section describes the general search functions that can be used to locate for example chromatograms curves and text strings in UNICORN These functions can be used in several program modules dialog boxes and wizards The search will take place in the displayed folder only To select another folder click the Browse button and open the desired folder e The search will take place in all result files within the selected folder as denoted by the asterisk To select specific result file s
27. 214 Measurements How to make direct 363 Messages Usage 160 How to issue 160 How to add a message instruction to a method 555 Method blocks Description 98 Blocks in the Text pane 98 How to show or hide instructions 98 Blocks in the Block pane 99 Blocks in the Gradient pane 99 Calls 100 Unconditional calls 100 Conditional calls 100 Use the Instruction box to add 101 Use the New Block dialog box to add 102 Fields of the New Block dialog box description 102 Use the Delete Block dialog box 104 How to use the Delete Block command 105 How to delete unused blocks 105 How to rename blocks 106 How to find text strings 107 How to copy a block 107 How to move a block 108 How to import general information 109 How to import 109 Base instructions descriptions 155 Block length 158 Method Editor Modes 28 Text instructions display panes 29 The Block pane 29 The Flow Scheme pane 30 The Gradient pane 30 Index e pix Index The Text pane 30 The Instruction box 31 Icon descriptions 94 Log Format 159 Method files How to open in the UNICORN Manager 69 How to connect a method to a system 77 Method instructions Instruction markings 112 How to add an instruction 113 Pause Hold and Hold_until instructions 113 How to delete instructions 114 Undo delete 114 How to change 115 Difference in function between Change and Replace command 115 Move an instruction within the same breakpoint 117 Move an i
28. 523 Quadratic through origin 523 Point to point 524 Curves How to copy into the Temporary chromatogram 227 Run curves default appearance 231 How to choose the Y axis scale 231 Default curve names 236 Peak labels 238 Fraction text alignment options 238 Logbook text alignment options 238 How to change the color and style 238 How to filter logbook information 238 How to set a hatched background 239 How to change and fix the Y axis 240 How to add a second Y axis 240 How to change and fix the X axis 241 How to save a layout 242 How to apply a layout 242 How to use the zoom function 244 How to cut a curve and store as new 245 How to reduce noise 274 How to remove ghost peaks 274 How to import a blank run curve 276 How to subtract a blank curve 276 How to add 277 How to rename 286 How to compare peaks in different curves 288 Multifile Peak Compare Wizard 288 Manual peak identification 294 Commands to import curves into a chromatogram 305 How to use the Open to compare command 306 How to import using File Open 309 How to copy curves into one chromatogram 310 How to align with Normalise 312 How to move using the Shift function 315 How to stretch or shrink using Multiply 315 03 0014 90 epiv How to produce a mirror image 316 How to shift a mirror image 316 How to import 319 Export options 321 How to export 321 How to export in AIA format 323 How to delete unwanted curves 326 How to divide 366
29. 6 5 6 Introduction Sub tabs Recommended us age How to write method notes How to search for text strings How to edit methods 6 The Notes tab Notes are descriptive comments that form part of the method documentation Method templates are supplied with notes describing the system requirements for running the method Read through these notes carefully before using a method There are four sub tabs e Method Notes e Start Notes e Run Notes e Evaluation Notes Only the Method Notes can be edited from the Method Editor the other notes are accessible at the respective stages in a run We recommend that you use Method Notes to describe the system setup required by the method for example eluent and sample inlets outlets and column connections Use the Start Notes or Run Notes for run specific information Note Method Notes are saved with the method and apply to all runs made with the method To write method notes in your own methods place the cursor in the white area of the Notes tab and type the relevant text Use standard Windows editing functions to edit the notes You can search for text strings in the method notes The table below describes how to perform a search Action Click the Find button Result The Find dialog box opens e Type the text string in the Find what text box e Select search criteria and click OK Result The located text string is highlighted in the text
30. Click the Close button to close the Import Reference Curve dialog box How to delete ref The table describes how to delete reference curves erence curves Step Action Select the curves you want to delete 2 Click the Delete button and confirm the action when prompted Note Deleting curves from the method does not affect the curves in the result file from which they were imported 03 0014 90 ep 140 How to edit methods How to rename The table below describes how to rename a reference curve in a method reference curves e Select a curve from the list e Change the name in the Rename item to field e Click the Rename button Repeat steps 2 and 3 if you want to rename more reference curves Click the Close button Result The reference curve name is changed e p141 6 How to edit methods 6 5 Run Setup 6 5 9 The Columns tab 6 5 9 The Columns tab Display of the The Columns tab shows the parameters of the column selected for your method oo paramet The column parameters are displayed in the Column Data field If you perform scouting runs with different columns all of these will be listed Select the appropriate column to display the parameters Illustration The illustration shows an example of the Columns tab Evaluation Procedures Method Information Stait Protocol Questions ResukName Columns Frac 950 l Variables Scouting Noles Gradient Bulte
31. Delete System Insert Row Before Inset Row After Double cick on a cell to change ts content E Here Bun Gore Hep The default selection for Start MethodQueue is As soon as possible e Click the At time radio button and select a time and weekday for the start of the MethodQueue if desired e Double click the cell in the first row of the System column Result The Method for row number 1 System dialog box opens Note See How to set up MethodQueues on several systems below if you have more than one system available e Select a method and click OK Result The method is displayed in the System column ep 183 8 MethodQueues 8 1 How to create a new MethodQueue Step Action 5 e Click the Insert Row After button and repeat steps 3 and 4 to add more methods to the MethodQueue Note The timing of MethodQueue steps performed on different sys tems can also be controlled by the Ready instruction in the method see Relative timing of steps below By default each method step will start as soon as possible ASAP after the completion of the previous method step Use the Condition cell of the chosen method to set another time interval for starting a selected step e In the Conditions column double click the cell for the method to be delayed Result The Condition for row number X dialog box opens Condition for row number 4 x As soon as possible Wait Hours fe xh Minutes p 4 Prev
32. Description Exports an evaluation log in ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question Exports an evaluation log in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question Exports an evaluation log in XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question Exports a method to the file defined in Export to file in ASCII format If is entered as File name the current Result file will be used If all paramet ers are OFF then no method is expor ted If Main is ON then the main meth od is included and if Blocks is ON then all blocks are included in the exported file Evaluation functions and instructions B Instruction Description EXPORT_METHOD_WKS Exports a method to the file defined in Export to file in WKS format If is entered as File name the current Result file will be used If is entered follow
33. How to define different columns for scouting You can define different columns for use in the various scouting runs However in selecting a different column other variables may also be changed between runs The table describes how to scout columns Step Action Choose a method with a column not Any Alternatively you can have a method with CV as the main base and a column not Any selected as a variable called column Click the Define button on the Scouting tab Result The Scouting Variables dialog box is displayed Select Column and click OK Click the Column drop down menu item within the desired run Result A menu is displayed Select a column Result The Column Value Update dialog box is displayed The dialog box asks you whether you want to update the instructions with column default values Select one of the following Yes The method for the scouting run is updated with variable parameter values for the selected column consisting of UV average time pres sure limit flow rate etc These parameter values are added to the scouting variables on the Scouting tab Note that the updated para meter values may differ from the values for the same variables in other scouting runs No No changes are made The method retains the parameter values corresponding to the column that were either originally selected during creation of the method or included in an earlier version of the method on the Scouting
34. I z Peak table s Peak toble s idi 4SQuanitateQ01 1 UVI 230ren 01 PEAR N THE ep 435 13 The Analysis module 13 4 How to quantitate the sample 13 4 3 How to calculate the recovery factor How to view the recovery factor calculation results If the recovery cannot be calcu lated 03 0014 90 p 436 Step Action Select Global or Personal quantitation tables Select a quantitation table on the Quantitation table droplist Note Only external standard quantitation tables will be shown Select the chromatogram that contains the unspiked sample peak table on the Source chromatogram droplist Select the unspiked sample peak table from the Peak table s list to the left Repeat step 2 to select the peak table for the spiked sample on the Addition chromatogram fields Select the component that was added prior to the sample prepar ation on the Addition component droplist Type the injected amount of this component in the Added amount field Click the OK button The recovery factor calculated by the software is placed at the bottom of the peak table in the Evaluation module You need to scroll to the end of the table to see it Recovery Factor Component name Calculated from Baseline Maximum number Maximum number Area Peak area time Height m u 0 Chymotrypsinogen A id 186501 1_UV1_280nn id 18501 1_UV1_280nm 01 BASEC Peak rejection on of peaks
35. Introducing UNICORN 1 About the UNICORN user documentation The user documentation for UNICORN is divided into three separate manuals This section is an overview of the contents and the relationship between the manuals The three manuals are e Getting Started with UNICORN e UNICORN User Reference Manual See 1 2 About this manual on page 14 e UNICORN Administration and Technical Manual The questions and answers in the table below describe the features of the Getting Started manual Question Answer Who should read Getting Started Users that are new to the UNICORN system and with limited experience from other chromatography systems What do I need before I start A basic knowledge of PC and Windows functions and an understand ing of the concepts and terminology of liquid chromatography What are the contents of Getting Basic descriptions of UNICORN and Started its use based on a model system How should I use Getting Started Read in front of your computer and test the instructions at the same time The questions and answers in the table below describes the features of the User Reference Manual Question Answer Who should read the User Reference Users that are experienced with Manual previous UNICORN system ver sions e Users with vast experience from other chromatography systems e p17 03 0014 90 1 Introducing UNICORN 1 3 About the UNICORN user documentatio
36. Model Fan to point Model function values Number of peint s 4 03 0014 90 ep 524 C 2 Introduction Correlation Too few data points Correlation calcu lation Explained vari ance Curve fit models and statistics C Statistics This section explains the correlation and explained variance calculations that are used by the Analysis module The Analysis module calculates the correlation coefficient for linear models This shows how well the data are linearly related The correlation is displayed in the Statistics table If you are producing a calibration curve that relates peak area or height to amount or concentration you aim to achieve a high positive correlation coefficient A value of 1 indicates a perfect fit of all the data to the straight line A molecular size curve has a negative slope so the aim is towards a correlation coefficient of 1 If you only have two data points for a Linear model or only one point for a Linear through origin model the fitted straight line will inevitably pass exactly through the points By definition this leads to a correlation of exactly 1 but this does not indicate a good fit but instead indicates too few data points In these cases the Statistics table will display a symbol instead of the correlation value The correlation is derived as follows gt eao Correlation ze pe Where eX is the average of the x value ey is the
37. Preview Cancel Help Click the appropriate tabs and select the check boxes for each item that you want to include in the report 03 0014 90 ep 264 How to view results 10 Step Action Click the Chromatogram tab and select the chromatogram s you want to include e Select the Current option in the Layout field to apply the current layout in the Evaluation module or e Click the Define button in the Layout field to open the Curve tab in the Report Chromatogram Layout Select the curves that you want to include in the report and click OK Click the Contents tab to see a list of all the selected items Click the Preview button to see the entire report layout Click the Close button to return Click the Print button to print a test report e Click the Save As button Result The Save Report Format dialog box opens e Type a name in the Report format name text box Select the Save as global format check box to make the format available to other users Select the Save as default report format check box if desired The format is saved as DEFAULT e Click OK Result The Generate Report dialog box opens again The new report is saved and available in the Format list e e e e e Click the Close button or e Click the New button to create another Standard report How to print a The table below describes how to print a Standard report format in the Evaluation standard report nodule
38. Result The instruction parameters are displayed in the Instruction and Parameter fields A short definition of the selected instruction is displayed at the bottom left corner Type new values in the Parameter text boxes and click the Replace button Result The old parameters are replaced by the new parameters Add a new instruction e Select the instruction in the procedure immediately before where you want the new instruction e Select a type and an instruction in the Instruction field e Type parameter values in the Parameter field e Click the Insert button Result The new instruction is inserted after the selected instruction Remove an instruction Select an instruction in the procedure and click the Delete button to remove the instruction from the procedure Choose File Save and click the Close button to close the dialog box Appendix B 4 Procedure instructions on page 504 contains a list of procedure instructions with descriptions How to add in structions toa procedure when recording Invalid instruc tions Address the right curves Default values for classic baseline in structions Global procedures Evaluation 12 If you start recording again you can add more instructions to a procedure that is already open in the Procedure Editor e The new instructions will be added to the end of the present procedure or e The new instructions will be inserted after the selected instruction
39. Step Action Open a result file e p 265 10 How to view results 10 6 How to create and print reports 10 6 2 How to create and print a standard report Step Action e Select File Report or e Click the Report icon a Result The Generate Report dialog box opens Generate Report x Eomat Contents From resit Cheomatofocusing p Global BP_Chromatogr_Report R Global 8P_Ful_Report an vie i Global Chiomatofocusing un date amp time Global Che q Report tile Result file name Method file name Page number S Chromatogram Active chromatogiam e Select a Standard report format Note The contents of a Standard report format is displayed in the Contents field e Verify in the Contents field that the report format contains all the elements that you want to include Click the Edit button to modify the report format if needed e Click the Print button Result The Print dialog box opens e Choose what pages and how many copies to print e Click OK Note Printers are set up in the File menu of the UNICORN Manager 03 0014 90 ep 266 How to view results 10 10 6 3 How to edit an existing report format Introduction This section describes how to edit an existing report format How to edit a The table below describes how to edit a standard report format in the Evaluation standard report nodule Step Action Open a result file e Select File Report or e Click t
40. The New Block dialog box How to edit methods How to add method blocks 6 You can add method blocks to a method in two ways using either e the Instruction box of the Text Instructions editor or e the New Block dialog box reached via the New Block icon Both these alternatives are described below The table below describes how to add blocks with the Instruction box Action In the Text pane of the Text Instructions editor select the instruction or block that you want to precede the new block Select Other Block in the Instruction box e Enter a name for the block in the Block field e Click the Insert button Result The block is inserted after the block that was selected in step 1 The illustration below shows the New Block dialog box that can be used when adding new method blocks Nm Base Same as main C Time Volume C Column volume F 10 ml m Length Length 10 00 Cy Call Erom Main X At 0 00 cy Cancel Help e p 101 6 How to edit methods 6 2 Method blocks 6 2 3 How to add method blocks ae to The table below describes how to add blocks with the menu options of the New ocks with the i New Block dialog Block dialog box box Step Action Choose Block New in the Method Editor or click the New Block icon 8 Result The New Block dialog box is displayed Enter the relevant information in the
41. The block from which the newly created block should be called e At The breakpoint at which the call is to be made If you do not want to call the block for example when the block being created is to be activated by a Watch instruction choose the lt Unused gt line from the From drop down list Blocks using this line are placed last in the method in the Unused category Note You should not call a block from within itself If you do you will generate a potentially infinite loop that exceeds the maximum number of calls allowed in a method A loop symbol is displayed at the beginning of the line if this occurs e p 103 6 How to edit methods 6 2 Method blocks 6 2 4 How to delete method blocks 6 2 4 Four ways lete blocks to de Delete options 03 0014 90 ep 104 How to delete method blocks There are four ways to delete blocks e To right click a block and choose Delete from the shortcut menu e To select a block and click Delete in the Instruction box e To select a block and press the lt Delete gt key on the keyboard e To select a block and use the Block Delete Block command Note When you use any of the first three ways the Method Editor dialog box will give you the option to transfer the block to the Unused section The Delete Block dialog box is displayed when you delete a block with one of the first three options mentioned above Delete Block x How would you like to proceed Delete
42. The parameter values will be updated continually during the run if the Auto update checkbox is selected Your user attributes may include a requirement to always set pressure alarms Action When you try to execute a pump instruction the Column protect mode dialog box opens e Click the Yes button in the dialog box to select a column and re trieve the correct maximum pressure value e Click OK to close the column list e Click the Insert button to add the Alarm_Pressure instruction e If necessary repeat step 2 to add an Alarm_SamplePressure instruc tion How to use manu al instructions The buttons of the manual instruc tions dialog box How to perform method runs 9 Manual instructions are entered in the same way as method instructions from the dialog box in the Method Editor The table below describes how to add a manual instruction Select an instruction group and a component in the Instructions field Select instruction parameters in the Parameters field Click the Insert or Execute buttons as needed See the descriptions of the different functions below The table below describes the functions of the manual instructions buttons Close Function This button places the current instruction in the list at the bottom left of the dialog box This button deletes the selected instruction from the current list only One instruction can be deleted at a time This b
43. To execute the instructions contained within a block in a method the block must be called by the program When a block is called the instructions in the block are executed in the order that they are written until the block is finished or the End_Block instruction is executed Any settings made in a block are valid throughout the method until the settings are changed There are two types of calls e Unconditional calls which are made with a Block instruction e Conditional calls which are made with a Watch instruction This makes it possible to call a specified block or an instruction when a particular monitor signal meets a given condition As long as the condition is not met the block is not activated Watch instructions are indicated by a green line that show the start and duration of the watch These instructions can use various conditions to respond to absolute signal values or to rate of signal changes The breakpoint when the Watch instruction is issued determines when the watch begins not when the block is activated Once set a watch remains active until the condition is met or a new Watch instruction is issued for the same monitor The watch is cancelled automatically when the condition is met A watch can also be turned off with the Watch_off instruction See F Method examples on page 545 for more details on Watch instructions 6 2 3 Two ways to add method blocks How to add blocks with the Instruction box
44. To reduce the scale of the zoom right click in the Curves pane and select one of the following options e Undo Zoom reverses each zoom in action a step at a time e Reset Zoom reverses all zoom in actions to the default scale How to select If the Pressure curve is displayed in the Curves pane you can set the displayed units Ae PRESS MLE The table below describes how to do this Action Right click in the Curves pane and select Properties in the displayed menu Result The Properties dialog box is displayed Select the Y Axis tab Select the Pressure curve and select the appropriate Pressure unit button Click OK How to edit text You can select the way that text is aligned for the Logbook and Fraction curves in the Curves pane You can also select to show only part of the Logbook information The table below describes how to do this Step Action Right click in the Curves pane and select Properties in the displayed menu Result The Properties dialog box is displayed Select the Curve Style and Color tab 03 0014 90 ep 202 How to perform method runs 9 Step Action Select the following e Logbook or Fraction curve in the Curve list as appropriate e Select the appropriate Logbook text alignment or Fraction text alignment option Horizontal Vertical Fly over displays the text if you place the mouse pointer over the generated mark To filter the type of Logbook i
45. above The illustration below is an example of the statistical information for an applied Linear curve model Statistics x Model pane Model function values Number of point s 4 The table below describes the features of the Linear through origin curve fit model Feature Description Mathematical model The constant A is determined by linear least squares regression Minimum number of required points 1 at least 2 points recommended Measuring range for the calibration From the point with the highest value curve down to the origin The illustration below is an example of the statistical information for an applied Linear through origin curve model Model F SAR Corel ion 0 9949 Number of point s 4 The Quadratic model The Quadratic through origin mode Curve fit models and statistics C The table below describes the features of the Quadratic curve fit model Feature Description Equation y Ax Bx C Mathematical model The constants A B and C are determ ined by linear least squares regression Minimum number of required points 3 at least 6 points recommended Measuring range for the calibration Within the highest and lowest values curve for the points Note A variant of this model is available for the production of a molecular size curve This uses the logarithm of the molecular size as the x value in the expression above The illustratio
46. click the Browse button and select the result file s e You can use wildcard characters to search for chromatograms within result files with a specific name profile represents any number of characters represents any single character y Wildcard character examples gt iex will search files named iex gt iex will search all files with names that begin with iex iex will search all files with names that end with iex iex will search only 4 character names that end with iex The asterisk indicates that all chromatograms within a result file will be selected Click Browse to select one or several specific chromatograms The UV curves are identified by number and sometimes wavelength For example UV1_280 UV2_280 and UV1_254 are all different curves To search for all UV curves select UV in the Curve name text field A Sample ID can be used as a search criteria if it has been defined as a variable The Sample ID can be entered in searches for result files both in the UNICORN Manager and in the Evaluation module e p37 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 5 Search functions Find a text string The Find command is used to search for text strings General informa tion about searches 03 0014 90 ep 38 Find what UF I Match whole word only pe Cancel I Match case Up Down I Search from top of document Field Descripti
47. e 1 2 etc inserts the corresponding chromatogram e Select the desired Settings e If desired change the Fonts Note Separate fonts can be selected for the Chromatogram the Peak table and the Header text 03 0014 90 ep 256 How to view results 10 Step Action e Click the Define button in the Layout field if you want to re define the layout of the chromatogram Result The Report Chromatogram Layout dialog box opens e Make the appropriate changes and click OK to return to the Setup Chromatogram dialog box Note The changes that you make will only affect the report and not the view of the chromatograms in the Evaluation module Click OK Result The chromatogram is inserted onto the page Note All curves can be de selected in the Report Chromatogram Layout dialog box leaving only the selected peak table s in the report aaa a includea The table below describes how to include a method in the report metho Step Action e Click the Method icon iz e Press and hold the left mouse button on the report page and drag out a box to the size of the item Release the button Result The Setup Method dialog box opens Select the items to be included in the report e Main Method is the method on which the run was based e Blocks are the blocks that were used in the method e Select the appropriate Settings Note Expand main displays the expanded method view e If desired c
48. e Ifa level has not been set the Select Level dialog box opens Select 1 on the Level menu and click OK e Click another result file in the Results field and select the new source chromatogram Result The peak tables associated with this chromatogram are dis played on the Peak table s list 03 0014 90 ep 412 Standard concen tration levels The Define Com ponent s dialog box The Analysis module 13 Step Action 5 e Repeat steps 3 and 4 until all the standard peak tables have been selected Note Increase the level number for each new standard concentration in consecutive order of decreasing or increasing concentration e Click the Current button at any time to return to the chromato gram that was active before you activated Quantitate e Highlight unwanted tables on the list and click Remove e Click OK to finish the selection Result The Define Component s dialog box opens Continue to Step 2 How to select and define components below this table It is useful to think of each level as an alias for a specific concentration of the standard You can incorporate up to 10 peak tables at each level prepared from runs repeated at the same concentration Quantitate will later allocate each with an incrementing suffix e g 1 1 1 2 etc The components that will be used to produce the calibration curves are selected in the Define Component s dialog box Quantitate must be able to identify
49. e Right click in the Curves pane and select Marker in the menu Result A vertical line is displayed Click the marker line and drag it to the desired point where you want to take a Snapshot Right click in the Curves pane and select Snapshot in the menu Result The Snapshot is displayed in the Snap Shot dialog box Retention Arpitude 01 125200101 1_UV1_280nm 11 69 MAU 02 125200101 1_UV2_250nm 237 85 MAU 03 125200101 1_UV3_Onm 0 00 mAU 04 125200101 1_Cond 0 47 mSicm 05 125200101 1_Cond 0 50 06 125200101 1_Conc 100 00 8 5 89 1 00 mimin 26 60 sC 0 00 mimin 07 125200101 1_pH 09 125200101 1_Flow 10 125200101 1_Temp 14 125200101 1_SampleFlow e Click the Save to File button if you want to save the information as an Excel file xIS or a tabbed text file txt e You can also copy the information to the clipboard Click and drag the mouse in the table to select the information you want to copy Press CTRL C The information can now be pasted in a text editor e Click the Print button if you want to print the information e Click the Close button Repeat steps 2 to 4 if you want to view more Snapshots ep 43 2 UNICORN concepts 2 3 Quick Start Guide 2 3 Introduction Quick Start in structions 03 0014 90 ep 44 Quick Start Guide This guide is intended for users who are fully familiar with the safety precautions and operating instructions that are described in al
50. epg 03 0014 90 1 Introducing UNICORN 1 1 About UNICORN 1 1 Introduction What is UNICORN Operating environ ment Windows func tions Bar code reader Compatible chro matography sys tems ep10d About UNICORN This section is a general overview of the UNICORN system UNICORN is a complete package for control and supervision of chromatography systems It consists of control software and a controller card for interfacing the controlling PC to the chromatography liquid handling module Liquid chromatography is used in separation processes for analytical purposes or in the biochemical process industry UNICORN is a trademark of Amersham Biosciences Limited UNICORN runs on a PC under Microsoft Windows 2000 or Microsoft Windows XP It is designed to run under English keyboard settings Note Microsoft and Windows are registered trademarks of the Microsoft Corporation in the United States and or in other countries Most Windows functions are also available in UNICORN including e cut and paste e right click short cut menus Note Drag and drop is not available File and folder handling in UNICORN also differs from the general Windows file manager standard You can connect a bar code reader to the PC and use the reader to enter information instead of using the keyboard This can be useful for example when entering information like batch IDs UNICORN can be used with a number of system
51. method e Instructions that apply to a different system strategy can occur if a method is written for one system and saved for another e Instructions for components that have not been selected in the System Setup Instructions that will not be executed because e they are positioned after the end of a block or method or e they constitute a block to which there is no call When a block is called from within itself this will generate a potentially infinite loop which might ex ceed the maximum number of calls allowed in a method 6 3 2 Instruction Pause Hold and Hold_until instruc tions How to edit methods How to add method instructions 6 The table below describes how to add a method instruction in the Text Instructions Editor Step 1 Action Select a block in the Text pane and display the instructions within the block Select an instruction line in the block Make sure that the selected instruction line is in the block not the call to the block Open the Instruction box if it is not already displayed View Panes Do the following e Set the desired breakpoint in the Breakpoint field e Choose the instruction type and the instruction in the Instructions field For basic help on each instruction click the instruction and press lt F1 gt e Type values for instruction parameters in the Parameters fields If a scroll bar appears at the right side of the Parameters field addi
52. monitored values exceed or fall below specified limits The system will be paused You can perform a Batch run of a number of result files in the Evaluation module The files do not have to be open and the run operates in the background The procedure is useful if you want to print a number of results with the same settings or if you want to perform integration with the same parameter settings on many results BufferPrep is a function to prepare a buffer of different pH and salt concentrations online from four stock solutions This eliminates the need to manually prepare new buffers every time the pH needs to be changed Note BufferPrep is only available for some AKTAdesign systems A chromatogram is a collection of data represented by a number of curves that have been created during a separation run including UV conductivity pH fraction marks etc The original raw data curves cannot be deleted or modified They can be used as a basis for evaluation procedures and subsequent creation of new curves A chromatogram can also contain curves that have been created and saved during an evaluation session The monitor signals from the chromatography run are displayed graphically as curves The program instructions for a chromatography run are defined in a Method A Method can be divided into blocks that represent steps in the separation process Each block consists of a series of instructions that request specific operations i
53. name as password Click OK or press the Enter key CE xi ae UNICORN logon User name default M Password Cancel Help Note We recommend that the default user is deleted when regular user profiles are created General system operations 3 How to open User All user administration is performed in the User Setup dialog box in the Main Menu Setup module It is accessible only to authorized users and the default user User Setup is found on the Administration menu e Choose Administration User Setup Administration Tools Window He EH Audit Trail i System Setup Create System Report Generate File List Expansion Card Settings Rescue Diskette Change Password Change User Attributes The User Setup The illustration below shows the User Setup dialog box dialog box n x Go Define users and their security attributes m Access groups Each user is assigned to an access group This group defines access to different functions in UNICORN When changing a group all users assigned to that group are affected Users Full name Group default default Administrator Neue Q OPC user OPC user Administrator Eas Edit Delete Print How to createa The table below describes how to create a new user new user Click the New button in the User Setup dialog box Result The Create New User dialog bo
54. or choose File Method Wizard 03 0014 90 ep 94 How to edit methods 6 6 1 2 Text Instructions editor How to select You have a choice of four panes that can be open together with the Instruction box plays r be dis in the Text Instructions editor all at once or one at a time Follow the steps in this table to select the panes to be displayed Step Action e In the Method Editor choose View Text Instructions or e click the Text Instructions icon e Choose View Panes Customize or select additional panes here or e click the Customize Panes icon Select panes e Select panes in the dialog box and click the OK button Customize Panes Text V Flow scheme Instruction box Cancel Help Deselect panes e Deselect panes in the Customize Panes dialog box and click the OK button or e right click a window and select Hide e p95 6 How to edit methods 6 1 The Method Editor interface 6 1 2 Text Instructions editor Method editing operations per formed in the dif ferent panes 03 0014 90 ep 96 This table shows the method editing operations that can be performed in the different panes The pane Instruction box Is used e to display instruc tions to display and hide block instructions to select current in struction to edit instructions to cut copy and paste instructions to move instructions within a breakpoint
55. required peak tables Result The Molecular size table dialog box opens Use one of the following ways to select a peak e Click the peak in the curve e Click the peak entry in the Retention Mol size table 17 Double click in the Mol size column cell and type the known mo lecular size from the standard 03 0014 90 p 452 The Analysis module 13 Step Action 10 Repeat step 8 and 9 for all components of known molecular size 11 To remove unwanted entries click the peak entry in the table and click the Exclude button 12 Select the appropriate curve model in the Curve model field see The molecular size curve below 13 Click the Save as button Result The Save molecular size table dialog box opens 14 e Choose if the table is to be globally accessible to any user or re stricted to your personal user ID The default is global e Type a name in the Molecular size table name field e Click OK The molecular size The molecular size curve shows the relationship between molecular size and the CULV corresponding retention The curve is plotted from the Retention Mol size data that you have typed in the table as described above Before this can be done a curve model is needed which describes the relationship between molecular size and retention Each of the peaks selected is represented by a point in this curve which is drawn according to the best fit that can be achieved using the selected model S
56. shortterm Average particle diam Code no Typical loading range Mol weight range Scan rate mand mand mand mand mand mand Anion_Exchange Cation_Exchange RPC HIC Size_Exclusion kDa Spectra sec Cancel Help Howtoaddanew The table below describes how to add a new column to the Column List column Step Action e Choose Edit Column List in the Method Editor Result The Column List dialog box opens Note Select a column from the list to display the parameters in the field to the right Most column parameters are displayed in the Nor mal Parameters tab Additional parameters for special columns may be displayed in the Advanced Parameters tab e Click the New button Result The New Column dialog box opens e Select the appropriate parameter tab e Type the desired parameter values e Click the Save as button Note Mandatory parameters are labelled mand The column cannot be saved unless all mandatory parameters are filled in Result The Save as dialog box opens ep 529 D The Column list D 1 How to edit the Column List Step Action 4 e Type the name of the new column e Click the Save as global checkbox if the column should be avail able to other users Note You must have Edit global lists authorization to save a column for global use A global column cannot have the same name as a personal column e Click OK Result The new
57. size Exclude Pim Save Save as Help More ep 451 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size How tocreateand The table below describes how to create and save a molecular size table in the save a molecular Eyajyation module size table Step Action Open a result file and select Mol Size Edit Mol Size Table New Result The New Molecular Size Table dialog box opens New Molecula Size Table Ura Molecular size unit label ros Squice cheomsalogiam fr 141480 uanktaheOOh 1_UV1_ZoOremS01 PEAK Peak tablets Ae fe id1 480 ustadi UVI Zare PEAK 1i 125200101 i AT2001Aprt Sno00 AJ AT 200M ay 1610001 e Double click the result file in the Select peak table s list e Select the source chromatogram on the Source chromatogram droplist Highlight a peak table that was prepared from the standard in the source Peak table s list and click the Select button Repeat step 3 to select more peak tables Note The runs must all have been made under the same conditions To deselect a table highlight the table in the Peak table s list to the right and click the Remove button Repeat steps 2 to 4 to select peak tables from other result files e Type the appropriate size measurement unit in the Molecular size unit label field default kDa e Click OK when the Peak table s list to the right contains all the
58. templates or wizards To do this either e click the Instant Run icon in the UNICORN Manager toolbar Ee or e select File Instant Run in System Control If the method is defined with a Start Protocol this will be displayed before the method actually starts The table below describes how to use the Start Protocol Step Action e Start the method run e Work through the start protocol answering questions as required The start protocol items that can be displayed are described in 6 5 14 The start protocol tab on page 151 e As each screen is completed click the Next button to move to the next screen or the Back button to return to the previous screen Click the Start button in the last window to start the run Confirm Sign authorization for the Start Protocol If there are any questions in the Start Protocol that require authorized confirmation you will be asked for a user name and password when you attempt to leave the screen containing the questions Only users with Confirm Sign authorization may authorize answers to such questions Each question that requires an authorization must have a separate authorization If the system is busy with a method run in progress you can still start a new method You will have the option to place the method in a MethodQueue which can be executed as soon as the system becomes available again The table below describes how to do this Step Action 1 e While
59. will remove the block completely from the method Move will place the block in the lt unused gt section of the method Options Choose from the following options e Delete The block is totally removed from the method If the block is called several times in the method all the blocks will be deleted Blocks deleted in this fashion cannot be called again in the method Note If the block contains sub blocks another dialog box is displayed asking you if you want to delete the sub blocks as well e Move The block is deleted from the method and transferred to the Unused section If the block is called several times in the method however only the row with the block currently marked in the Text pane will be deleted In this case the block will not be placed in the Unused section since the block is still used in the method Blocks deleted in this fashion can be called again in the method How to use the Block Delete Block command How to delete un used blocks How to edit methods 6 The table below describes how to delete a block using the Block Delete Block command Step Action Select the menu command Block Delete Block in the Method Editor Result The Delete Block dialog box is displayed with all blocks listed in alphabetical order Delete Block x Select the block s to delete jAutoZero_UY a QBufferalve_A1_Inlet OClean_after_Elution C Column_E quilibration C Column_Pressure_Limi
60. 1 e Choose Operations Pool Result UNICORN will automatically pool suitable fractions The pooled fractions are listed in a table below the chromatogram and the pooled peaks are numbered sequentially in the chromatogram Cv aaation tracmpbRirwdk OII V LMICERY Local EE Nacho aru Deni be aai GOAN Ecama CLT ined jE GK Yow pegas yesim Domare Quirciate Mi Sew aion Help alfj DEPA SBOkwS PTET E RN E Recort Pros tie Fna Skah wt PVA A oe fester fet Se ee ee a A A a Tit tid Deets Osean Odearoa tee Vol Aawa acot Cont Amount Tert Target conc Como 10e 10u05 100000 01343 COO VE 3655 10 0000 Om 05880 O97 160D wwo oa OO OMS 1138 0 1139 Tage val 100 G78 150 13 1 7800 587 2635 120 aana TAN 3 aes 46 Foon ume NEY Ladia Pastry pve Mirea Ol Von Poio pusod Nm Note Only adjacent fractions will be pooled The fraction numbers for each pool are listed in the table as a range in retention order e g A6 A7 etc e p 279 11 How to edit results 11 5 How to pool fractions Step Action The pooled fractions can be adjusted manually To include or exclude adjacent fractions in a pool e Click the numbered marker under the pool and drag the sideline To add more pools e Click between the droplines under a fraction to create a new pool and drag the sidelines to include more adjacent fractions To delete
61. 163 6 6 7 Gradients and eluent concentrationS ss sssssserssrrerrrrrrrsrrerrsrrrrerrrrrerrerne 164 6 7 Standard Watch conditionS s ssssssrereennnsnnrrsrsrrirrrrrnrnnrsrsrrrrererernrrsrsrrrrrrerne 166 6 8 How to save or delete a method template cccceccsecssceseeeseseeeeeeeeeeeeeeneeeneers 171 6 9 How to printa method siers ap a a a a 172 6 10 How to export a INCLIOs wanstern re acheter ene eae erie ena 174 Fe SCOUTING occa site erg E E os cued aes acini dual vse te Mc ema hen ds cea T 175 7 1 How to set up a Scouting SCHEME accaidisaccvrssgupiecenpiue rs ysei sand avdeamcadat eis tenteaneteacses 176 7 2 How to define different columns for SCOUTING ccccccceeceeeeeeeeeeeeeerseeneeeeeenaees 181 By TEE OU GU NGS 5 Fes Sos So Soa eae vaca vac asinaa tans an taacutancuauecsnetos A 182 8 1 How to create a new MethodQueue ccecccecseccsereserseeeseteeesseeeeeeeeeaeeeneeegeees 183 8 2 How to edit a Method QUCUGs x se crc ciccvees dcnedsehcebenilscuneabastste rence iaiaesosseiciecs 187 9 HOW to perform method NUNS ccce ccc cecectteessaedssvnasvtecetescnivceawssendesteetoendsduttinvr dere irene 189 9 1 How to start a method TUMauaicse ciated inte enero 190 9 2 How to monitor a method run 222 cccsssssssnsceeesessssnesseeeessssnnnecseeseesssnnnseeeessesens 193 9 2 1 How to customize System Control Panes ccccsccseesseeeseseeeseeeeeeeeeeneeeneees 194 9 22 25 RuN Data DANG wasvenceccs
62. 50 Variables Scouting Notes Questions Gradient ButfePrep Columns Block Variable Value Range a a e fon Rae Pow Rae Gand T Jaoo 0000 Gotan Presawe_ta Cohen Present Pa 4oo 000 1000 Stet Inowacons id Cavett dCi a Cf Tavera 3m meat SSCS a ufferValve_A1_Iniet luent_A_ Inlet ta wih PuneWoah Ergo wonen O N a e e a Pesiant Bi Fonos Facionaion Powhcugh FiacSae oat foo 000 10000000 D G G E E iuert_B_Inlet Pump Binet E E E Samole_iniection _ Empty_loop_wih mi 2 500 0 000 999999 000 w I Show detaie I Show unused variables F Display tooltip for extended variable cells Edt Variable The Variables tab The method is represented by a number of blocks on the Variables tab The blocks are typical steps in a chromatographic run Each block contains a number of Method Variables with suitable default values that can be changed to suit your application Only the most commonly used variables are initially shown on the page Click the Show details check box to display all variables in the method Default values for When you select a column default values will be set for several parameters including columns the following e the correct column volume e the recommended flow rate e the correct pressure limit Note If you exceed the recommended values for the selected column you will receive a warning when you save your method 03 0014 90 ep 82 How to create a
63. Alarms and warnings Alarms and warnings are displayed regardless of the activity currently in progress in UNICORN You will be notified of an exceeded limit in a running system even if you are developing a method evaluating data or monitoring a method run ona different system Warnings and alarms are also recorded in the logbook for the run The system settings determine the acceptable limits of monitor signals during a run The limits can also be set for the current run by an instruction in the method Limits set with a method instruction override the limits set in system settings If these limits are exceeded in a run a warning or alarm dialog box is displayed on the screen Alarms and warnings have different effects on the system e Warning The run continues e Alarm The system is paused In a network installation alarms and warnings are displayed on the controlling station and all stations viewing the system An alarm can be acknowledged only from the computer connected in control mode Alarms are displayed but cannot be acknowledged on computers connected in view mode e p215 9 How to perform method runs 9 4 How to perform a scouting run 9 4 How to perform a scouting run More information See 7 1 How to set up a scouting scheme on page 176 for information on how to on Scouting runs set up scouting runs Instruction The table below describes how to perform a scouting run Step Action Start th
64. Classic algorithm parameters How to set a Clas sic baseline 03 0014 90 ep 340 How to optimize the baseline with a classic algorithm The first choice when you want to optimize the peak integration is to change the baseline parameters This section describes how to optimize the baseline with a classical algorithm The Classic algorithm searches for all parts of the source curve that are longer than a defined minimum baseline segment and fall within limiting parameters Together the parameter values define the limits for a rectangular box A part of the source curve must fit entirely inside this rectangular box to be identified as a baseline segment The Classic algorithm is particularly useful when you need to integrate curves with negative peaks and when quantitative data from negative peaks are important The parameters for the Classic algorithm are e Shortest baseline segment e Noise window e Max baseline level e Slope limit See more information about the parameters below The table below describes how to set a Classic algorithm and define a baseline Step Action Click the Baseline settings button in the Integrate dialog box Result The Settings dialog box opens e Select the Classic algorithm e Change the Baseline parameters See more information about the parameters below this table e Click OK Note The same settings can be edited in the Calculate Baseline dialog box when a new
65. Close button 03 0014 90 ep110 How to edit methods 6 6 3 Method instructions Introduction This section describes how to work with the individual method instructions in order to edit method blocks and methods In this section This section contains these topics Topic See How to read method instructions 6 3 1 How to add method instructions 6 3 2 How to delete method instructions 6 3 3 How to change or move method instructions 6 3 4 e p111 6 How to edit methods 6 3 Method instructions 6 3 1 How to read method instructions 6 3 1 Description of in struction markings 03 0014 90 e p112 How to read method instructions Method instructions are displayed in the Text pane of the Text Instructions Editor The table below explains the meaning of the markings Marking Blue square beside text Blue square with a red cross Bold text Red dot L Normal text Text with a loop sym bol Q Explanation Valid call instructions that is Block and Watch in structions to other blocks in the method Call instruction that contains one or more invalid instructions Valid instructions Instructions with invalid syntax All such instructions must be deleted or changed before a method can be run See 6 3 4 How to change or move method instruc tions on page 115 The instructions may be of the following types e Calls to blocks which are not defined in the
66. Disconnect Connect system a The Disconnect button is used to disconnect the system and leave it in a locked or unlocked state va The Connect button connects the system Leave Take control of the system F The Leave control button leaves the system in a locked or unlocked state Ed The Take control button takes control of the system The status bar displays a message indicating the connection status of the window The table below describes the different messages Message Connection status Controlled by lt user gt The indicated user has a control mode connection to the system Other users can establish a view mode connection How to perform method runs 9 Message Connection status Locked by lt user gt The indicated user has left the system in a locked state Users who can supply the required password can unlock the system and establish a connection The password is case sensitive Note It is possible to unlock with the lock pass word or with the UNICORN logon password Any one who uses the UNICORN logon password must have Unlock systems access rights The lock pass word is the password entered by the user who locked the system System is available Any user can establish a connection Status bar Watch The status bar displays a message indicating if a Watch is active in the method status e Click the Active watch status message to open the Watch dialog box wit
67. End Block 20 00 End Block Effects of the The Length parameter in the Gradient instruction affects the length of a gradient oe a ted Depending on which button you use the change will have different results The button on adi table below describes this ent length Command Function If this button is used to change the length of a gradi ent the breakpoints for any instructions issued during the progress of the gradient will be adjusted propor tionately so that they are always placed at the same relative position within the gradient Instructions is sued after the end of the gradient will be shifted by the amount of the change Since the gradient works over time any instruction that you want to insert after a gradient should be placed after the combined breakpoint and gradient length Note Moving the End_block instruction in a gradient block with the Change button does not affect the length of the gradient Replace If this button is used to change the length of a gradi ent other instructions are not affected 03 0014 90 e p116 How to edit methods 6 Illustration of the The illustration shows the different effects of the Change button and the Replace effects of the button on instructions within and after gradients Change button vs the Replace but ton on gradients Message at 10 End pr at 30 _ End ge at 30 Message oi 4 at 10 o0 10 20 30 Gradient_
68. Evaluation Log should start on a new page e Click OK Result The Evaluation Log is inserted into the report How to view results 10 How to include The table below describes how to include Quantitate and Molecular Size data in the Quantitate and report Molecular Size pork data Note This option is only available if the Analysis module has been installed e Click the Quantitate and Mol Size icon e Press and hold the left mouse button on the report page and drag out a box to the size of the item Release the mouse button Result The Setup Quantitate dialog box opens e If desired change the Fonts e The default option is that the Quantitate and Molecular Size data will start on a new page e Click OK Result The Quantitate and Molecular Size data is inserted into the report How to include The table below describes how to include Frac 950 data in the report Frac 950 data os l Note This option is available only if a Frac 950 has been installed and if the result file contains data from the Frac 950 Step Action e Click the Frac 950 icon g e Press and hold the left mouse button on the report page and drag out a box to the size of the item Release the mouse button Result The Setup Frac 950 dialog box opens e If desired change the Fonts e Select if the Frac 950 data should start on a new page e The Include rack layout option is selected by default This w
69. Export Dialog to export the view Note You can also click the Export button from the Customization dialog box How to use the 3D Data View dialog box How to edit results 11 The 3D Data View dialog box presents a three dimensional plot of a selected peak See also How to use the 3D Data View shortcut menu below The illustration below shows the dialog box 3D Data View c 2 p 2 o a Select peak Select psoas Select pans Select z axis Fie name aj Reterton Run Date Area cues Peak name Heig Retention Resohsion Resolution Heaght Resohition Tj The list boxes Use the list boxes to select which peak to plot and the units of the x y and z axes The command buttons The table below describes how to use the command buttons of the dialog box Command button Function Returns to the Data View dialog box Print Prints the spreadsheet Copy to Clipboard Stores a figure for transfer to an external program Save Wizard Settings See How to save the Wizard Settings below Cancel Ends the Multifile Peak Compare wizard Displays the data in 2 dimensional plot See How to use the 2D Data View dialog box above e p 299 11 How to edit results 11 8 How to import and compare different runs 11 8 1 How to use the Multifile Peak Compare wizard How to use the Click the right mouse button in the plot area of the 3D Data View dialog box to 3D Data View open the shortcut menu See
70. Found chromatograms field and a list of chromatograms will be displayed based on the designated search criteria e A new search can be performed with new search criteria without erasing the first found chromatograms from the list e Select the chromatograms that you want to import If you click the Select All button all the displayed chromatograms will be imported e If you want to clear the list of displayed chromatograms click the Clear button e Click OK Result All the selected chromatograms are shown in the Evaluation workspace Note If the names of the imported chromatograms already are used they will be sequentially numbered for identification purposes Up to 10 chromatograms can be made available at the same time in the Evaluation workspace How to import The table below describes how to import chromatograms one by one using the chromatograms command File Open with the command j i File Open Step Action Choose File Open Chromatogram in the Evaluation module Result The Open Chromatograms dialog box is displayed Double click a result file to select it Result All the chromatograms contained in the result file will be displayed in the Available field e Select the chromatogram s of interest and click the Select button Result Selected chromatograms are added to the Selected chromato grams list Note Chromatograms can be deselected with the Remove button e p 303 11 Ho
71. Function A running system can only be con trolled from one connection Systems may be locked with a password to prevent other un authorized users from changing parameters Result files from an ongoing separa tion run can be saved automatically at preset intervals to minimize data loss if the system fails The results are saved locally if the network communic ation fails Method and result files can be signed electronically for enhanced security and accountability 1 ep 13 1 Introducing UNICORN 1 2 About this manual 1 2 About this manual Introduction This section is a general description of the manual the contents and the pre requisites for the examples and instructions that are presented in the User Reference Manual The purpose of The purpose of the User Reference Manual is to present a comprehensive guide to Saher Reference the UNICORN system for a user either with previous experience of this system or from other similar chromatography systems The system is presented in detail along with practical instructions of how to operate a model system Systems covered This manual and the corresponding version of Getting Started with UNICORN by this manual covers the following systems e AKTAexplorer e AKTApurifier e AKTAFPLC e AKTAbasic e AKTApilot e Ettan LC ted h TE C ae a Note Adapted versions of this manual are available for AKTAxpress AKTA oligopilot
72. If it has been closed select View File Navigator in the Evaluation module Quick View is a preview function for result files to make it easier to select the correct result file You can preview the first curve in the first chromatogram You can also select to view another curve as default by selecting another curve number in your User Attributes settings see 3 4 How to change user attributes on page 57 Several files can be opened for comparison How to use Quick The table below describes how to preview result files in Quick View View 03 0014 90 e p70 Step Action 1 Select one or more result files in the Result window of the UNICORN Manager Files and folders in UNICORN Step Action e Choose File Quick View or e Right click and choose Quick View from the short cut menu Result The Quick View dialog box opens Quick iew c Default Example files Example Result Example Res 102 x Example Result002 1_U 1_215nm Cancel Help e Click the Next and Previous buttons to move between the result files if more than one is selected e Click the Open button when the right file is displayed Result The result file that is displayed in the dialog box opens in the Evaluation module ep71 4 Files and folders in UNICORN 4 3 How to arrange and locate your files 4 3 Introduction Different view modes How to change the view mode Sort order in de tailed vi
73. New Block dialog box and click OK Result The new block is added to the method and placed last of all blocks Note The block can be placed in other positions by selecting some thing other than Main in the From droplist The fields of the The table below describes the fields of the New Block dialog box New Block dialog box l Field Description Block names can be up to 30 characters long and can contain letters A Z digits 0 9 and the underscore character Block names must be unique within the method The case of letters is retained but not significant the names Start_Frac and START_FRAC are treated as identical One of the following options can be selected e SameAsMain the new block will inherit the base from the Main block in the method The corresponding Base instruction will be inserted in the block at breakpoint 0 e Time The block will be based on time e Volume The block will be based on volume e Column volume The block will be based on column volume Length A block continues until the breakpoint for the End_Block instruction has been reached An End_Block instruction will automatically be inserted in the block at the defined breakpoint This field must not be left blank 03 0014 90 ep 102 How to edit methods 6 Field Call Description You can call the new block from an existing block for example the Main block Select values in the two fields e From
74. On Result The Procedure Editor dialog box opens in record mode Minimize the Procedure Editor dialog box Perform the evaluation steps that the procedure is to contain Result The steps are recorded in the order that they are performed e p 379 12 Evaluation 12 3 Automated evaluation procedures 12 3 1 How to create a new procedure How to create a Global procedure How to build a procedure with in structions 03 0014 90 e p 380 Step Action Stop the recording e Choose Procedures Record Off or e Restore the minimized Procedure Editor dialog box and click the Stop button or e Restore the minimized Procedure Editor dialog box and select Control End Record Choose File Save or File Save As in the dialog box Result The Save As dialog box opens e Type a name for the new procedure in the Procedure name text box e Select the Global procedure checkbox if desired see further inform ation below e Click OK Result The procedure is saved and available for future use Click the Close button to close the dialog box You can choose to save the new procedure as a Global procedure This makes the procedure available to all users The procedure will have Global before the name to designate that it is available to all users You must have Edit global list s authorization to be able to save Global procedures You can select instructions in the Procedure Editor dialog box to
75. Panes icon opens the Customise Panes dialog box which is used to select the panes that are open in Text Instructions mode The Text Instructions icon opens the Method Editor in Text Instructions mode The Run Setup icon opens the Method Editor in Run Setup mode The Log Format icon opens the Log Format dialog box which is used to display the accumulated time or volume for a method The Method Wizard icon opens the Method Wizard which is used to create new methods ep3l 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 3 The System Control module 2 2 3 Introduction The System Con trol panes The Run Data pane The Curves pane 03 0014 90 ep 32 The System Control module The System Control module is used to perform and monitor separation runs The System Control module contains four different display panes that can be opened all at once or in any combination e The Run Data pane e The Curves pane The Flow Scheme pane The Logbook pane The Run Data pane displays the current values for the selected run parameters The values are updated at regular intervals which are defined in the system strategy See the illustration below The Curves pane displays monitor signal values graphically See the illustration below UNICORN concepts 2 The Flow Scheme The Flow Scheme is a graphical representation of the chromatography system During a run the Flow Scheme displa
76. Result The Edit Texts tab of the Chromatogram Layout dialog box is displayed e Select the text that you want to edit and make the appropriate changes in the Selected text field e Click the Change text button or the Delete text button e Use the Font and Set Orientation buttons if needed and make the desired changes in the resulting dialog boxes e Click OK to apply the changes Shortcut option You can also right click outside the text box and select Edit Text Mode from the shortcut menu This activates all the text boxes in the chromatogram The list below describes how to edit the text e Click the text and type the new text e Click outside the text box to set the text 03 0014 90 ep 278 11 5 Introduction How to view the contents of a frac tion How to pool frac tions How to edit results 11 How to pool fractions Fractions are collected sequentially during a separation Each fraction contains a set volume of sample This section describes how to pool the information on several fractions into a new curve Each fraction is numbered according to its order in the sequence The information is saved as a curve under the name Fractions e Select this curve on the Curve tab in the Chromatogram Layout dialog box to display the contents of each fraction in relation to the information displayed on the UV detection curve The table below describes how to pool fractions Step Action
77. Runs tab Result The Recent Runs list opens in the File Navigator ix Recent Runs Fies Find Recent Results g E 1057001 o latta Wizardver Op6 Hia MU iF Samplel HisGFP F7 IDS5 Fz Samplez F71D58 fz Sample3 F7ID58 1D32001 o latta Instruction verification P2 0p80p 1D32002 o lotta Instruction verification P2 0ps0p 1D33001 o latta Instruction verification P2 0p80p 1033002 o lotta Instruction verification P2 0p80pi li ID34001 o latta Instruction verification P2 0p80p df ID3S001 o latta Instruction verification P2 0p80p ID35001 o lotta Instruction verification P2 0ps0p ID37001 o latta Instruction verification P2 0p80p di 1037002 o latta Instruction verification P2 0p80p bef H Se wap Note Until the files and chromatograms in the list have been opened and saved they are noted in bold text When they are opened and saved the text is changed to plain text e If needed click the Refresh button in the bottom of the File Nav igator Result The Recent Runs list is updated with all runs that were per formed since the File Navigator was opened the last time e Locate the desired run e Double click the file Result The result file opens in the Evaluation module Note Click the signs to view or select individual chromatograms from the result files Individual result files can be selected and removed fr
78. Scouting TextMethod Notes Questions Columns Reference Cuves Evaluation Procedures Method information ResultInfcimation Stait Protocol Settings Cealiretion Logbook EvalustionLog Result Neme Information Signatures Run Summary Snapshots eR vam BE Colurnn_Equiibeation System_Volume_Compensation Flowthvough_Fiactionation Autosampler Injection Wwash_Oul_Untound Sample Fractionation Step Chromatefecusing_Elulion Reequiibeation CF Initiol_Ehaent_Condilions_CF Save the method You can save the method and the variables that were used for the run as a new used for the run as method a new method Step Action e Select the Text Method tab in the Documentation dialog box e Click the Save as button Result The Save As dialog box opens e Select the appropriate destination folder e Type a name in the Method name text box e Select a system in the For System field e Select a technique in the Technique field e Click OK Result The method is saved 03 0014 90 ep 272 How to edit results 11 11 How to edit results Introduction This chapter describes e how to edit the results that are presented in the Evaluation module e how to import and compare runs e how to import and export results For more information about how to view results see chapter 10 How to view results on page 220 In this chapter This chapter contains these sections Topic See H
79. See How to use selected method instructions Standard Watch conditions How to save or delete a method template How to print a method How to export a method 6 10 03 0014 90 ep 92 How to edit methods 6 6 1 The Method Editor interface Introduction This section contains a general description of the Method Editor user interface and the editing operations that can be performed in the different parts of the module In this section This section contains these topics Topic See The Method Editor module 6 1 1 Text Instructions editor 6 1 2 ep 93 6 How to edit methods 6 1 The Method Editor interface 6 1 1 Method Editor module 6 1 1 Method Editor module Two modes The Method Editor interface operates in two modes e Text Instructions editor for entering and editing method instructions see 6 1 2 Text Instructions editor on page 95 e Run Setup for defining method properties see 6 5 Run Setup on page 122 How to open the The table below describes how to open the dialog boxes in the Method Editor Method Editor dialog boxes If you want to open then the Text Instructions ed click the Text Instructions icon itor 1 or choose View Text Instructions the Run Setup click the Run Setup icon or choose View Run Setup the Log Format click the Log Format icon or choose View Log Format the Method Wizard click the Method Wizard icon a8
80. Strategy file error Unable to log on The table below describes some log on problems and their solutions to UNICORN Problem description Solution You have forgotten your password Ask the system administrator to supply a new password Username and password not accepted Restore the file USERS30 MPM You cannot log on although you use from the latest back up copy your correct username and password oy Reason The file USERS3 0 MPM in the folder UNICORN SERVER FIL could be corrupt e reinstall the default user No user names Remote station Make sure that the computer is logged on to the network before you start UNICORN Note A remote station accesses the user list directly from the network server Both these conditions must apply e The User name drop down box in the Logon dialog box is empty You are trying to log on from a re mote station in a network installa tion No user names Local station Make sure that the computer is logged The user list on a local station in a on to the network before starting UNICORN network installation is not up to date Note The user list is stored locally on a local station and is updated automat ically from the network server if the computer is logged on to the network e p477 A Troubleshooting A 1 Logon Error message The table below describes some problems and their solutions Strategy file er ror Problem des
81. The Flow Scheme pane The flow scheme is a graphical representation of the chromatography system that shows the current status of the run During a run the flow scheme displays open flow path s in color and monitor signals with numerical displays The illustration below shows an example of a flow scheme for a run Injection Valve The flow scheme can be stretched to fit the screen To do this right click in the pane and select Stretch in the shortcut menu Some strategies link specific manual instructions directly to the components in the flow scheme pane The components in the flow scheme that are associated with instructions are indicated with double arrows gt gt A particular component can have one or more instructions attached to it In cases where there is more than one instruction one of the instructions is the main instruction To display and select instructions e double click a component or e right click a component select Instructions and an instruction in the shortcut menu Result The manual instructions dialog box for the selected instruction type opens 9 2 5 Introduction Illustration Autoscroll How to filter the logbook contents How to perform method runs 9 The Logbook pane All actions including method start and end base instruction method instructions and manual interventions such as Pause or Hold and unexpected conditions such as warnings and alarms a
82. User Fokler Method Queue Folder S7SKB Method File 12 7 2001 11 0 12 7 2008 11 0 12 7 2008 11 0 12 7 2008 11 0 12 13 2001 9 4 11 21 2001 4 2 Note The icons for MethodQueue folders are different from the regular folder icon ep25 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 1 UNICORN Manager ae Results win The Results window contains all the saved results and all the result folders ow 5 Results 12 7 2001 11 06 AM 12 7 2091 11 0 demo Results 2 12 7 2001 11 05 AM 12 77 2001 11 Example results Scouting Folder 11 26 2001 10 17 11 26 2001 10 ed Test Results 1 User Fokler 12 7 2001 11 05AM 12 7 2001 11 0 a Test Resutts 2 User Folder 12 7 2001 11 05 AM 12 7 2001 11 0 d 125200101 1146KB Resu File 1 25 2001 2 04PM 1 25 2001 2 04 4 AT2001 Apr 190001 GSKB Resuk File 5 16 2001 3 11AM 5 16 2001 3 11 Note The icons for Scouting folders are different from the regular folder icon Toolbar icons in The table below describes the toolbar icons in the module the UNICORN Manager Icon Function The Logon Logoff icon is used to log on or log off the system Note The arrow in the Logoff icon points away from the door The Instant Run icon immediately starts a run from a selected template or from a wizard The New Method icon opens the Method Editor module and displays the New Method dialog box The System Control icon activates the first connected System Control modul
83. a method fe Double cick on a systema Cancel Hep Result The method is connected and the system name is added after the method name in the Method files list Repeat step 1 until all methods are connected to a system Click OK The table below describes how to rename files and folders in the Methods or Results windows in the UNICORN Manager module Step Action Select the item that you want to rename e Select File Rename or e Right click and select Rename from the shortcut menu Result The Rename dialog box opens Type a new name Click OK The table below describes how to delete files and folders in the Methods or Results windows in the UNICORN Manager module Note Home folders cannot be deleted this way Step Action 1 Select the item that you want to delete e p77 4 Files and folders in UNICORN 4 4 How to copy delete rename and backup files and folders Step Action e Select File Delete or e Right click and select Delete from the shortcut menu or e Press the Delete key Confirm the delete action in the confirmation dialog box Backup security Backup copies should be taken regularly to avoid data loss in the event of hard disk failure or accidental deletion You can use the function Copy to External to save your files on the network server Note Amersham Biosciences cannot accept responsibility for the replacement of method programs that were l
84. a method run is in progress right click on the next method you want to run and select Run System Result The System Busy dialog box opens ep 191 9 How to perform method runs 9 1 How to start a method run Step Action e Select the Add the method to a MethodQueue that will execute as soon as the system is free option e Click OK Result A MethodQueue will automatically be created in the default queue folder The name of the MethodQueue will be the same as the method name followed by a five digit sequence number The method will be executed as soon as the system is free Note A warning note is displayed in the System Busy dialog box if the method includes a Start Protocol The Start Protocol must be completed at the start of the method run before it can be executed Note See 8 2 How to edit a MethodQueue on page 187 for more information 03 0014 90 ep 192 9 2 Introduction In this section How to perform method runs 9 How to monitor a method run This section describes how to monitor a method run by using the System Control module and how to customize the different panes The table shows the topics that can be found in this section Topic See How to customize System Control panes 92 1 The Run Data pane 9 2 2 The Curves pane 9 2 3 The Flow Scheme pane 9 2 4 The Logbook pane 9 2 5 e p 193 9 How to perform method runs 9 2 How to monitor a method run 9 2 1 How
85. and other results see 11 8 How to import and compare different runs on page 287 poe subtracta You can subtract the blank run curve or the baseline from the sample curve The blank run curve table below describes how to do this Step Action Select Operations Subtract Result The Subtract dialog box is displayed Select the sample chromatogram and curve in the left field and the baseline or blank run curve to be subtracted in the middle field Click OK Note All resulting curves from the subtract operation receive the SUB suffix by default The default curve name can be changed as needed 03 0014 90 ep 276 11 3 Introduction Instruction How to edit results 11 How to add curves In some method runs several sequential chromatograms might have been created This can occur for example when the instruction New chromatogram has been used in the method thus creating different chromatograms during the run In order to view and evaluate the resultant curve of all the chromatogram parts the curves must be added together Usually you have a number of chromatograms within the same result file and you want to add the curves In some circumstances curves might need to be imported from other result files The table below describes how to add curves Step Action 1 Select and view the first chromatogram in the sequence Choose Operations Add Result The Add dialog box is displayed e Select the fi
86. area ep 135 03 0014 90 6 How to edit methods 6 5 Run Setup 6 5 7 The Evaluation Procedures tab 6 5 7 Introduction Changes in the Evaluation mod ule References to curves How to print eval uation results How to define and view evaluation procedures How to select pro cedures to run ep 136 The Evaluation Procedures tab The Evaluation Procedures tab lists all evaluation procedures associated with the method Evaluation procedures can be called automatically at the end of a method to evaluate and or print the results Many UNICORN strategies are supplied with method templates or wizards that include a number of evaluation procedures User defined procedures are created in the evaluation module and can be saved in method files see 12 3 Automated evaluations procedures on page 378 A procedure in a method will not be updated when a procedure with the same name is changed in the Evaluation module The same applies to report formats saved in a procedure Evaluation procedures that process chromatogram data rely on consistent identification of curves in the result file for correct operation If you include evaluation procedures with a method make sure that references to curves in the procedure will be valid when the procedure is executed at the end of the run see 12 3 Automated evaluation procedures on page 378 for more details If you use an evaluation procedure to print results aut
87. available e Curves e Export curve to AIA e Peak table e Method e Documentation e Evaluation log The table below describes how to export curves in the Evaluation module Step Action 1 Choose File Export Curves Result The Export Curves dialog box opens Sasco cheematog acm Chaves to egot 01 125200101 1_UV1_280nm 02 125200101 1_U 2_250nm 403 125200101 1_U 3_Oom Gh 1252001 01 1 UVI 220m JOZ 1252001 01 1_UV2_2500m TOR 1252001 01 1 UVI Ona 05 1252001 01 1_Cone 07 1252001 01 1_sH 08 1252001 01 1_Pressure 08 1252001 01 1 Few 10 125200101 1_Tomo z Tesoro Reduce number of samples T Nome gt Reduce by factor 19 All Maxmo sample 905 EEN Carcel Hep ep 321 11 How to edit results 11 9 How to import and export results 11 9 2 How to export results Step Action e Select the curve s you want to export e Enter parameters to limit the curve s if necessary e Click the Select button e Repeat Step 2 to select more curves Click the Export button Result The Export Curves to File dialog box opens Select the export file format from the Save as type droplist e ASCII files asc e Lotus 1 2 3 files wks e Excel files xIs e AIA files cdf e Select a destination folder e Type a file name and click OK Note Curves are exported as series of numerical coordinates that refers to the time volume and signal respectively
88. axis and stretched along the Y axis e Click OK to save the new normalised curve Result The Save Curve dialog box opens Choose a curve position to save the curve in and click OK e Choose Edit Chromatogram Layout to open the Chromatogram Layout dialog box e Select the normalised curve for viewing on the Curve tab e Click OK Repeat steps 1 5 for all curves you want to stack or stretch How to move a curve with the Shift function How to stretch and shrink a curve with the Multiply function How to edit results 11 If you want to position a curve more precisely the Shift function should be used The function is similar to Normalise Move but each curve is repositioned by a precise value instead of by eye and the instruction is logged in the evaluation log The table below describes how to use the Shift function Step Action e Make sure that a chromatogram with the relevant curves is open in the Evaluation module e Choose Operations Shift Result The Shift dialog box is displayed e Select the curve to be shifted in the Source chromatogram list e Select a curve position in the Target chromatogram list e Type a new Curve name or accept the default e Select the axis axes along which the shift is to be made along the X axis Shift retention along the Y axis Shift amplitude e Type the shift value s e Click OK Curves can be stretched or shrunk on the x or y plane with the Mu
89. column is added to the Column List Note See column instruction to determine the back pressure over the system and the column The normal The table below is a list of all the available normal column parameters column paramet ers Parameter Unit Comment e Mandatory e Calculation of N m Diameter cm e Mandatory nl pl ml or liter e Mandatory e Automatically calcu lated from Height and Diameter e User cannot set this parameter directly Column volume unit nl pl ml or liter e Not mandatory e The column volume is calculated in the set unit Technique e Mandatory e Decides which tech nique the column should be available for 03 0014 90 e p 530 The Column list Parameter Default flowrate Max flowrate Typ peak width at base pH high value longterm Unit nl pl ml or liter nl pl ml or liter nl min pl min ml min or liter min nl min pl min ml min or liter min nl pl ml or liter Comment Not mandatory Total liquid volume Used to calculate the capacity factor after an integration Not mandatory Void volume Used to calculate K after integration Mandatory Used for setting pres sure limit in a meth od automatically Mandatory Used to set the flowrate in a method automatically Not mandatory Used to give a warn ing if a higher flowrate is chosen when saving or start ing a method Not
90. curve fits completely within the box The found baseline segments are joined by connecting adjacent seg ments provided that the slope of the joining lines does not exceed the Slope limit When the baseline segments have been defined and joined they are replaced by baseline points at the start and end of each segment The line between these is also filled with points Note The baseline points are shown as green squares in the Integrate Edit baseline function of the Evaluation module The baseline points are used to create the baseline curve using a spline interpolation The spline function ensures that the baseline curve is guided by the baseline points However the curve does not necessarily pass through the baseline points The baseline will be a smoothly curved function passing close to or through the points To reduce the effect of noise at the peak integration the created baseline is forced equal to the source curve in every position where the difference between the baseline and the source curve is small enough Choose Integrate Calculate Baseline If the Accept negative peaks option is off the baseline will be forced down to the level of the source curve whenever the created baseline goes above the source curve You can try to measure the Shortest baseline segment length directly on your chromatogram The table below describes how to do this Step Action Locate the shortest segment of the curve that you cons
91. defines available method and manual instructions system settings run data curves and Method Wizards UNICORN gt a System Note The examples in this guide are generally based on the E100F400 strategy Templates are basic methods that can be used as a starting point for developing customized methods The method variables in a suitable Template is adjusted to create a method for another application Instruction parameters and values at breakpoints in the Methodmay be defined as Variables Variables makes it easy to adapt a method to a particular chromatography run e A framework Method with default parameters can be changed to create variants e A Method can be used in automatic Method Scouting where one or more parameter Variables are changed systematically UNICORN concepts 2 Warnings Systems settings or method instructions specify acceptable limits for monitor signals during a separation run A Warning dialog box may be displayed on the screen if a specified limit is exceeded The system will still continue to run after a Warning ep 23 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 The UNICORN user interface Introduction This section is an overview of the four UNICORN modules with descriptions of some of the elements of the user interface The section also contains a description of the search functions in UNICORN Note A user profile can be set up so that the user only has limited a
92. delete on the Quantitation table s list e Click the Delete button e Click the Yes button to confirm e Click the Close button Note You must have Edit global list s rights to be able to delete a global quantitation table ep 427 13 The Analysis module 13 4 How to quantitate the sample 13 4 Introduction In this section 03 0014 90 ep 428 How to quantitate the sample This section describes how to use calibration curves to quantitate samples Calibration curves are applicable to external and internal standard quantitation and to recovery factor measurement Standard addition measurements are also described This section contains these sub sections Topic See External and internal standard quantitation 13 4 1 Standard addition quantitation 13 4 2 How to calculate the recovery factor 13 4 3 13 4 1 Introduction Method for the sample runs How to prepare for the quantita tion The Analysis module 13 External and internal standard quantitation This section describes how to perform quantitation in the Evaluation module using either an external standard or an internal standard The processes involved in both external standard and internal standard quantitation of a sample are very similar The procedural differences mainly concern the creation of the quantitation tables A quantitation table is specific to either external standard or internal standard q
93. do not use a template or wizard define ap propriate variables in the method In the Run Setup click the Scouting tab Result If no scouting variables have been previously defined the Scouting Variables dialog box is displayed If not click the Define button e p177 7 Scouting 7 1 How to set up a Scouting Scheme 03 0014 90 e p 178 Step Action Select the variables you want to scout If you cannot find the variable you want use the following options e Show details to display variables created with the Visible in details only option e Show unused variables to display all variables including those that are not used in the method Click OK Result The selected scouting variables will appear in a column with default valued inserted Make any required changes in the scouting variable values To add a new Run column click the Add button to copy the values from the last run column and then change variable values as re quired Repeat steps 3 to 5 as required until you have defined all the scouting runs you need To exclude scouting runs from the default scouting scheme right click the heading of the run To include the scouting run again right click it again Click the Start Protocol tab in Run Setup Select from the following options e The Scouting box Select this to display the Scouting page at the start of a run This allows the operator to adjust the values for scouting variab
94. edit results 11 9 How to import and export results 11 9 1 How to import results Copy from a floppy disk When you import SMART or FPLCdirector files from a floppy disk it is best to first copy the files to the hard disk and then import the files How to import The table below describes how to import AKTAprime data in the Evaluation module data from AK TAprime Step Action Choose File Import Result Result A menu box with the available data sources opens This box opens immediately after Import if no result file is open in the Evalu ation module Choose AKTAprime Result The AKTAprime Data Collection wizard opens e Click the Next button and follow the instructions to connect AK TAprime to the correct serial port on your computer e Click the Finish button to complete the setup Result All open result files are closed and UNICORN is set up to receive sample data from AKTAprime 03 0014 90 p 320 11 9 2 Introduction Data formats Export options How to export curves How to edit results 11 How to export results This section describes how to export curves in different formats and how to copy data and curves to the clipboard You can export data in the following formats e AIA cdf e ASCII asc e Lotus 1 2 3 wks e Excel xls e XML xml Select File Export in the Evaluation module to export data from an open result file The following export options are
95. ep 231 10 How to view results 10 3 Basic presentation of chromatograms 10 3 2 The chromatogram window Use the command if you want Window Tile to view several chromatogram windows side by side Window Cascade to stack the open windows like a deck of cards How to displaya The table below describes how to display a vertical marker line vertical marker line Step Action Right click the Curves pane and select Marker Drag the marker line with the mouse Result Where the line bisects the curve the X axis and Y axis values are displayed at the top right corner of the pane Note Right click and select Snapshot to record the marker position values See 2 2 7 Snapshots on page 41 for more information about the Snapshot function How to set a refer The table describes how to set a reference point ence point Step Action e Display a Marker in the Curves pane e Right click and select Set Marker Ref Point to define a reference point for the marker position 2 When the marker is moved from the reference point the X axis and Y axis values for the new position are displayed together with e the new position in relation to the reference point e the minimum maximum and average values for the curve interval between the reference point and the new position How to display The table below describes how to display the logbook entries as an overlay in the Ee logbook over chromatogra
96. filter Smooths the source curve with a moving average filter and stores the result in the Resulting Curve The Filter width parameter decides how many samples wide the filter is Smooths the source curve with a medi an filter and stores the result in target curve position The Filter width para meter decides how many samples wide the filter is Smooths the curve with the Savitzky Golay algorithm Subtracts two curves to produce a third curve which is the difference of the two curves The two source curves must have the same Y axis unit and not be fraction or injection curves Divides two curves to produce a third curve which is the quotient of the two curves The two source curves can have any Y axis unit The threshold values are used to avoid division of numbers close to zero At those points where source curve 1 has an amplitude less than Threshold1 or the source curve 2 has an amplitude less than Threshold2 the result of the division is defined to be 1 0 Integration Evaluation functions and instructions B The table below contains a list of instructions for integration Instruction CALCULATE_BASELINE CALCULATE_BASELINE_ MORPH CLEAR_PEAKTABLE COPY_PEAKTABLE NEGATIVE_PEAKS PEAK_INTEGRATE PEAK_WINDOW Description Calculates a baseline from the source curve The baseline is stored in the target curve position DEFAULT can be selected in the Baseline parameters which w
97. from the Right Axis droplist Click the OK button 03 0014 90 ep 240 How to view results 10 How to change The table below describes how to change and fix the X axis and fix the X axis Step Action Open a result file Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed Click the X Axis tab Select the appropriate option in the Base field e Time of retention e Volume e Column Volume Note Some calculated curves for example baselines exist in only one base and might seem to disappear when the base is changed Curves are collected in time and recalculated for display in volume Thus switching the base between Time and Volume can slightly alter the resolution e Click the Fixed option in the Axis scale field to set the axis limits manually e Type the desired minimum and maximum values e If desired de select the Adjust retention zero to injection number checkbox This checkbox is selected by default The function sets the time volume to zero at the injection mark that is when the sample was injected The time and volume before injection will become negative values e Click OK ep 241 10 How to view results 10 4 How to optimize the presentation of a chromatogram 10 4 5 How to save and apply a layout 10 4 5 How to save and apply a layout Introduction All configurations that you make in the Chromatogram Layout dialog box c
98. his or her passwords and some user attributes even if user administration is handled exclusively by the system administrator The changes are made in the UNICORN Manager How to change The table below describes how to change your logon and signature passwords passwords Step Action 1 Select Administration Change Password Result The Change Password dialog box opens Type your old logon password in the Old text box Note Your passwords will only be shown as asterisks Type a new password in the New text box Repeat the new password exactly in the Confirm text box Repeat steps 2 to 4 in the Signature password section if necessary Click OK About passwords The list below is a summary of facts and advice about UNICORN passwords The system can be set up to operate without required passwords The minimum number of password characters is set up at installation Passwords can be any combination of letters and numbers Passwords are case sensitive Avoid using obvious passwords e g your username your telephone number etc The settings in the User properties determine the expiration for a password Change passwords regularly even if your user profile is set up without password expiration 03 0014 90 ep 56 General system operations 3 How to change The table below describes how to change your user attributes user attributes Step 1 Action Select Administration Change User Attribu
99. i I Show details I Show unused variables IY Display toollip for extended variable cells The tabs The table below contains brief descriptions of the tabs of Run Setup If you want more detailed descriptions see sections on the respective tabs Tab This tab allows the user to choose rack type and the fraction ation order for the Frac 950 fraction collector Variables lists all variables used in the method with their default values organized by method block shows the scouting scheme used for the method The scouting scheme can also be set up from this tab Notes shows the descriptive comments that form a part of the method documentation ep 123 6 How to edit methods 6 5 Run Setup 6 5 1 Overview of Run Setup 03 0014 90 ep 124 Tab BufferPrep Reference curves Evaluation Procedures Method Information Start Protocol Result name This tab provides a graphical overview of the block structure and eluent gradient tab in the current method displays information about the selected buffer prepar ation recipe for the current method displays the columns used in the current method displays the reference curves that will appear in the System Control curve dialog box during the run of the current method shows the evaluation procedures that will run at the end of the current method displays information about the method such as method name target system and last date of change
100. if an instruction has been selected The procedure will stop and display an error message if an instruction calls for an invalid operation when the procedure is run Any subsequent instructions in the procedure will not be executed Curves are identified only by their storage position An instruction can become invalid if it addresses the wrong curve Example e The instruction ADD 01 02 03 will try to add curve 01 to curve 02 and store the result in position 03 e A curve in position 03 that is not a raw data curve will be overwritten e A raw data curve in position 03 cannot be overwritten and the procedure will be stopped at that point When a classic or morphological algorithm is used to calculate a baseline UNICORN will suggest default values for the four control parameters based on the appearance of the curve To instruct UNICORN to use default values appropriate for the curve every time the procedure is run choose the default setting in the appropriate fields for the parameters Example e CALCULATE BASELINE 01 06 XXX XXX XXX XXX Can be changed to e CALCULATE BASELINE 01 06 DEFAULT DEFAULT DEFAULT DEFAULT It is not advisable to edit existing global procedures Open the global procedure instead and save a copy under a new name Use this copy for editing purposes e p 383 12 Evaluation 12 3 Automated evaluation procedures 12 3 3 How to run a procedure 12 3 3 Introduction How
101. in the source chromatogram If the destination of the cut curve was a new chromatogram this will be represented as a new open chromatogram window ep 245 10 How to view results 10 4 How to optimize the presentation of a chromatogram 10 4 7 How to change the size of Fraction Injection and Logbook marks 10 4 7 How to change the size of Fraction Injection and Logbook marks Introduction The sizes of Fraction Injection and Logbook marks are all determined by your user settings The settings are applied for all your chromatograms Instruction The table below describes how to change the size of the Fraction Injection and Logbook marks Step Action e Choose Administration Change User Attributes in the UNICORN Manager module Result The Change user attributes dialog box opens Select the unit for the Fraction mark height e Percent of window height e Character Heights e Pixels Type a new size value in the Fraction mark heightbox e Repeat step 2 for the Injection and Logbook marks if necessary e Click OK 03 0014 90 ep 246 10 5 Introduction How to view results 10 How to print active chromatograms This section describes how to print the chromatograms that are open in the Evaluation module The Print Chroma This is an illustration of the Print Chromatograms dialog box tograms dialog box Instruction Note The selected print format is outlined in red
102. individually all with the same scale click the All with this unit button e Click OK to display the curves e p 309 11 How to edit results 11 8 How to import and compare different runs 11 8 3 How to import and compare curves How to copy A practical way to compare curves is to create a chromatogram and copy curves Cur veS mto one from different chromatograms into the new chromatogram The comparisons are chromatogram then performed in the new chromatogram The table below describes how to copy curves into a chromatogram Step Action Perform either A or B below A Create a new chromatogram e Choose File New Chromatogram to create a new chromatogram B Use the Temporary chromatogram e Choose Window Temporary Open the source chromatogram s Choose File 0pen Chromatogram to open the chromatogram s that contains the curves you want to copy Result The Open Chromatogram dialog box opens e Select the result file e Click the check box for the source chromatogram in the Available list e Click the Select button e Click OK Result The source chromatogram opens Copy the curves e Choose Edit Copy Curves Result The Copy Curve dialog box is displayed e Select the source chromatogram and a curve of interest in the Source Chromatogram field e Select the target chromatogram the one you created or Tempor ary in the Target Chromatogram field e Click the Copy button e Repeat
103. information In the Evaluation module select Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed e p 229 10 How to view results 10 3 Basic presentation of chromatograms 10 3 2 The chromatogram window Step Action e Click the Header tab e Select the options you want in the header e Click OK 4 e Inthe chromatogram window place the cursor at the top of the curve window just below the toolbar until the window sizing tool appears e Drag the cursor down to display the header window How to view peak The table below describes how to display peak table information if the result has table information been integrated Step Action Open a result file e Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box opens e Click the Peak Table tab e Select a peak table in the Select peak table to display list e Select what peak table columns to display e Check if global peak table data should be displayed or not e Click OK How to view the If fractions are pooled the Pool Table is displayed in the same pane as the Peak Pool table Table e Click the Pool Table tab to display the Pool Table information See 11 5 How to pool fractions on page 279 for more information on how to create the Pool Table 03 0014 90 p 230 Run curves de fault appearance and information How to view results 10 The first time a result
104. instruction description 162 Hold_until instruction description 162 Linear flow rates 163 Gradient instruction parameters 164 How to print 172 How to export 174 How to export 324 Monitor signal limits 460 Molecular size calculation Overview 450 Preparations before curve creation 451 How to create a size table 452 How to open an existing table 454 How to rename a molecular size table 454 How to delete a molecular size table 455 How to calculate the size 455 Error signs 457 Procedure instruction 457 Molecular size curve Overview 449 Description 453 e pxi Index Morphological algorithm Description 336 How to set 336 Structure width 337 Incorrect structure width 338 Noise window 338 Minimum distance between points 339 Definition 493 Multifile Peak Compare Wizard How to select the operation 288 How to select data to compare 290 How to select the peaks 292 Manual peak identification 294 How to select the Peak Data 295 The Data View dialog box 296 How to use the 2D data view 297 How to use the 3D data view 299 How to save the settings 301 How to open saved settings 301 P Peak integration How to perform 330 Differences between to filter peaks and to reject peaks 333 How to display peak labels 334 How to select part of a curve for peak integration 358 Peak skim Compared to drop lines 360 How to select a ratio 360 Peak table How to display information 230 How to rename 286 Ho
105. instructions The table below contains a list of instructions for other operations Instruction Description BASE Sets the X axis base that the following calculations will be made in If the value of the X axis base is DEFAULT then the default base is used usually the base the method was run in This instruction should be the first in the evaluation procedure otherwise it will have no effect at all 03 0014 90 ep 516 Evaluation functions and instructions B Instruction ENDLOOP LOOP MOLSIZE QUANTITATE REPORT RUN_PROGRAM UPDATE Description Inserts a comment below the marked instruction Marks the end of a LOOP statement The instructions between this state ment and the ENDLOOP statement are repeated n times It is possible to have loops within loops as long as the number of LOOP statements matches the number of ENDLOOP statements Calculates the molecular sizes from a molecular size curve A Mol size column will be added to the Peak table Calculates the concentration and amounts in the sample from a quantit ation table Amount and Concentration columns will be added to the Peak table Prints a report with the specified named report layout and title If Title is then the title in the report lay out is used If Report Layout is then a default layout is used Starts a program as a separate process The Program name string contains the program name and paramet
106. is saved and available in the Format list e Click the Close button or e Click the Edit button to edit another report format How to edit a cus The table below describes how to edit a customized report format in the Evaluation tomized report mo uale 1 Open a result file 03 0014 90 ep 268 How to view results 10 Step Action 2 e Select File Report or e Click the Report icon m Result The Generate Report dialog box opens 3 e Select a Customized Report Format to edit e Click the Edit button Result The report format opens in the Report Editor 4 e Double click the report item you want to edit e Make the desired changes in the dialog box e Continue to edit all items until the format is complete Note See 10 6 1 How to create and print a customized report on page 250 for more information 5 e Select File Save As Result The Save Report Format dialog box opens e Type a name in the Report format name text box Select the Save as global format check box to make the format available to other users Select the Save as default report format check box if desired The format is saved as DEFAULT e Click OK Result The new report format is saved and available in the Format list e p 269 10 How to view results 10 7 Run documentation 10 7 Run documentation Introduction The full documentation for a method run is stored in the result file This section des
107. is used to compare the results from scouting runs The scout ing variables can be displayed This option features different default values than the Compare peak data option specially selected to measure changes in the column media This option is used to run several quantitations This is an alternative to Quantitate Calculate Amount and Conc which is used to quantitate single res ults A quantitation table must be cre ated before this option can be used This option is available only if the Analysis module has been installed e p 289 11 How to edit results 11 8 How to import and compare different runs 11 8 1 How to use the Multifile Peak Compare wizard Operation Description Batch Mw determination This option is used to batch run mo lecular size calculations This is an al ternative to Mol Size Calculate Mol Size which is used for single cal culations A molecular size table must be created before this option can be used This option is available only if the Analysis module has been installed The Data Selec The illustration below shows the Data Selection dialog box tion dialog box Data Selection x 220010 12200101 1 UV 28am 26200101 125200101 1_UV2 250m 125200101 126200101 1U 3_ Orm JAT 2001Ap11 S001 AT2001 Ape In0001 1 UY AT2001May16ec001 AT 2001Mayl n001 1_UV 1412001 May Bre001 AT2001May16n0001 1_UV O1BASEM Barelire example 1 ATIJMF006 1_LV1_21 See
108. it easier for you to quickly scan a page to find the exact topic you are looking for Menu commands field names and other text items from the software are quoted exactly as they appear on the screen in a bold typeface Example Run Setup Search paths are shown in a bold typeface with a separating colon between each level Example View Panes Customize i e the menu command Customize in the sub menu Panes from the View menu Text entries that UNICORN generates or that the user must type is represented by a monotype typeface Example Connection change ep15 1 Introducing UNICORN 1 2 About this manual Pre requisites The following pre requisites must be fulfilled before you can use this manual the way it was intended e You need to have a general understanding of how your PC and Windows works In most cases universal computer functions will not be explained e UNICORN must be installed and configured correctly on your computer e You need to understand the concepts of liquid chromatography Terminology and functionalities will be explained only when they differ from normal practise e Before you try to operate a chromatography system based on the instructions in this manual you need to study and understand the safety information that is part of the system documentation 03 0014 90 ep 16 1 3 Introduction The manuals User info about Getting Started User info about the User Reference Manual
109. it is possible to equilibrate the column without the risk of running out of buffers F 3 Introduction Example instruc tion This is what hap pens Method examples F Equilibration with extra safeguard This section contains an example of how a Watch instruction for extra safeguard can be inserted into a method This is an example instruction as it would be presented in the Text pane 0 00 Block EQUILIBRATE Equilibrate 0 00 Base SameAsMain 0 00 Block COND LESS THAN Cond_less_ than 0 00 Base SameAsMain 0 00 Watch Cond Less than 5 mS cm END BLOCK 6 00 Message Low conductivity not reached Screen No sound 6 00 Pause INFINITE Minutes 6 00 End Block 0 00 Block COND STABLE Cond_stable 0 00 Base SameAsMain 0 00 WatchPar Cond 0 500 mS cm 2 mS cm 0 00 Watch Cond Stable Baseline 5 Minutes END BLOCK 10 00 Message Conductivity not stable Screen No sound 10 00 Pause INFINITE Minutes 10 00 End Block 0 00 End Block Note If you are not using AKTA instruments a delay should be added after the Hold Pause instruction so that the following instruction will not be executed simultaneously with the Hold Pause instruction The table below describes what happens in the above example Description The column is equilibrated until the conductivity is below 5 mS cm If this value is not reached within 6 column volumes the method is paused and a message is displaye
110. list The table below contains a list of instructions for curve operations Instruction AMP_MUL AMP_SHIFT CLEAR COPY CUT DERIVATE Description Adds two curves to produce a third curve which is the sum of the two curves The two source curves must have the same Y axis unit and not be fraction or injection curves or else a run time error will occur Multiplies the amplitude of the source curve by the multiplication factor and stores the result in the target curve position Shifts the amplitude of the source curve by the shift factor and stores the result in the target curve position Clears the specified curve from the working memory of the computer Copies the source curve to the target curve position Cuts out the part of the source curve between the Left and Right limits and stores the result in the target curve position Differentiates the source curve first or second order and stores the result in the target curve position The Y axis of the target curve position will be a normalized scale without unit Evaluation functions and instructions B Instruction HISTOGRAM INTEGRATE POOL_FRACTIONS RET_MUL RET_SHIFT SIMULATE_PEAK_FRAC Description Divides two curves to produce a third curve which is the quotient of the two curves The two source curves can have any Y axis unit The Y axis of the target curve position will be a normalized scale without unit Cre
111. maximum and peak end A F and G in the diagram above The capacity factor will only be calcu lated when the chromatogram is in volume base The total liquid volume Vt must be entered in the Integrate dialog box for this parameter to be calculated See definition below this table Values calculated by the Analysis module Only available if the Quantit ation module is installed Fraction number at peak start peak maximum and peak end Maximum amplitude above the baseline C F in the diagram above Gel phase distribution constant in gel filtration Kav will only be calculated when a gel filtration column was used and when the chromatogram is in volume base The void volume VO must be entered in the Integrate dialog box for this parameter to be calcu lated See definition below this table Values calculated by the Analysis module Only available if the Quantit ation module is installed Height equivalent to theoretical plate and plates meter The column height must be entered in the Integrate dialog box for this parameter to be calcu lated See definition below this table Amplitude above the baseline at left A in the diagram above and right peak limits E G in the diagram above 03 0014 90 ep 498 Evaluation functions and instructions B Parameter Peak endpoint retention Peak name Percent of total area Percent of total peak area Type of peak limits Width at half h
112. method 5 The Gradient tab The Gradient tab shows the method graphically How to save the new method Evaluation Procedures Method Infomation Stet Protocl Questions Resuk Name Frac 950 Variables Scouting Notes Gradient BufleiPiep Columns Reference Curves Gradient Block name 0 00 Main The length of each block is marked at the bottom of the graph e Click the X axis to view the method in time volume or column volumes e Drag the Y axis to display a marker in the gradient The Base value Gradient value and Block name at the current marker position will be displayed in the upper part of the graph A new method created from a Wizard is untitled and must be saved under a method name before it can be used The table below describes how to save a new method e If required save the method in a folder other than the default home folder e Enter a Method name for the method The total path can be up to 256 characters long The method name must be unique for the chosen system within the folder e If you have more than one system connected to the computer choose the System for which the method is intended The method can be run on any system that uses the same strategy Remember that different systems may have different configurations and control capabilities e Choose the Technique for which the method was written ep 83 5 How to create a method 5 1 How to use the Method Wizard Step
113. on the same system the timing set in Condition applies from the completion of one method to the start of the next If successive methods are run on different systems you can use the Ready instruction in one method to trigger the start of the next method In this way you will be able to start the next method before the current method has ended The Condition setting then applies from the Ready instruction to the start of the triggered method This is useful in situations where a method on one system prepares the starting material for the next and then continues to wash the system See the example below Instruction to System 1 Instruction to System 2 Apply sample Apply sample Wash Eluate The Start Protocol for each method step in the MethodQueue is displayed when the corresponding method is run If you want the MethodQueue to operate unattended you must ensure that the methods do not include a Start Protocol See 5 How to create a method on page 79 for more information ep 185 8 MethodQueues 8 1 How to create a new MethodQueue How to hold a The table below describes how you can create a MethodQueue if you try to start a method in queue pew method run while the system is still busy with another method run while the system is busy Step Action Right click on the method in the UNICORN Manager module and select Run system name on the shortcut menu Result The System Busy dialog box opens Cannot star
114. operation The table below describes how select peaks in the Peak Selection dialog box Step Action 1 Choose a curve in the curve window e Double click the peak or click the peak once and then click the Select peak button Result The peak is assigned a letter A B C and the peak para meters are displayed in the Peak identification settings table How to edit results 11 Step Action Set the desired peak identification criterion e Click the desired parameter value in the Peak identification settings table Example If you have selected the highest peak in the curve and want to compare the highest peak among all curves select the Height check box In the illustration below the initial A peak and the Height check box have been selected Curve quantitation 1_UV1_280rm Peak Table qarita UVI ZOE PEAR 5 7 Pest wane Retention min Area aadvain Height C aav 41 49 1 9113 8 863 13 04 3 4960 10 242 28 8611 If desired you can assign a name to a chosen peak e Click the name of the row for example A e Click the Set peak name button e Type anew name and click OK Note This can be useful when you compare multiple peak parameters and you wish to have peak names other than Peak A Peak B etc to simplify peak identification and clarity f ex when comparing peak data between batch quantitated results Repeat step
115. peak table Sets the column height for the peak integration calculation of the HETP value The Column height parameter is the height of the column in centi metres If Column height is OFF then the HETP value is not calculated for the following integrations Sets void volume for Kav peak integra tion calculation Sets the total liquid volume for peak integration calculation of the capacity factor Sets the Skim size ratio to be used in the following peak integration s Integrates the curve within the peak window All curve parts outside the peak window remain unchanged File operation The table below contains a list of instructions for file operations Instruction CURVE_OPEN IMPORT_CURVE Description Opens the curve specified in the Result file defined in File name and stores it in target curve position If is entered as File name the current result file will be used The File name para meter may include a path from the users root folder Imports a curve to the current chroma togram from another chromatogram in the current file and stores it in the target curve position 03 0014 90 ep 508 Evaluation functions and instructions Instruction IMPORT_PEAKTABLE PEAKTABLE_OPEN Description Imports a peak table to the current chromatogram from another chroma togram in the current file and stores it in the target curve position Opens the specified Peak table in th
116. point e Determine the highest slope value of the baseline non peak part of the curve e Add 10 e Select Integrate Calculate Baseline and use this value as the Slope limit Note If the differentiated curve is very noisy it can be filtered with a light Moving average filter in the Operations Smooth function 03 0014 90 ep 496 Evaluation functions and instructions B B 3 Peak table column components Introduction This section contains a list of peak parameters with explanations and calculation formulae when applicable Peak parameters The diagram below illustrates the peak parameters See the parameter list below illustration for explanations Peak parameter The list below contains descriptions of the peak parameters descriptions Parameter Description Amount Values calculated by the Analysis module Only available if the Quantit ation module is installed Calculated as the area between the curve and baseline between the peak start and peak end time or volume base Gray area in the diagram above Asymmetry Peak asymmetry indicator of column packing See definition below this table e p 497 B Evaluation functions and instructions B 3 Peak table column components Parameter Baseline height Capacity factor Concentration Kav Molecular size Plate height HETP Peak endpoint heights Description Baseline amplitude at peak start peak
117. s list Note By default the list will show the quantitation tables that are globally available Click the Personal radio button to display the tables that are restricted to your own user ID Click OK Result The Quantitation table dialog box opens Note Quantitate includes an update function that can be used to add new peak size data to an existing quantitation table in a simplified way This function does not allow you to redefine components in the Define Component s dialog box The update function can be used to add new peak size data to an existing quantitation table This enables precision to be improved through the use of data from a number of standard runs It also simplifies the process of renewing the calibration curves before each analysis Note The injection volume must always be the same for the new run as it was for the previous standard runs The Analysis module 13 The Update Quantitation Table dialog box The illustration below shows the Update Quantitation Table dialog box Update Quantitation T able X i mols03 m Quantitation table l Ext_Std_Chym z C Global Personal Select peak table Besult Source chromatogram Level o Default Name o 3 B Example files a 125200101 i AT2001Apr1 9n0001 L AT2001May 60001 i Baseline example 1 L Baseline example 2 ai Baseline example 3 ti id 48QuantitateOOt a Peak skim example Li quantitatio
118. set a reference point 232 How to make chromatogram layout changes general 235 How to exit the module 326 Evaluation logs How to export 324 Evaluation procedures How to delete 137 How to rename 138 How to edit 138 Explained variance Definition 525 External standard quantitation How to perform 397 F File Navigator How to open result files 69 How to open 222 How to locate files from the Files list 222 How to use Find to locate a file 223 How to open a recent run 224 How to change preference settings for Recent Runs 225 How to close 225 03 0014 90 epvi Index Files and folders How to copy 75 How to move 75 Copy to external 75 How to copy from external 76 Flow Scheme pane Description 204 Stretch to fit screen 204 How to view manual instructions 204 Folders How to create a user specific 68 FPLCdirector How to import data 319 Frac 950 Defining the number of available tubes 149 How to select the last tube 150 Fraction Histogram How to create a curve 377 Fractions How to set up the Frac unit 149 How to view the contents of a fraction 279 How to pool fractions 279 How to create a pooled fraction curve 280 Show only the pooled fraction curve 281 How to calculate protein amounts and concentrations in pooled fractions 282 How to determine the volume of a pooled fraction with a specific concentration 282 How to use the Pool Fraction print list 282 G Generate Report Wizard How to generat
119. settings authorization to assign a new value to an actual system setting e p 459 14 System settings 14 1 General information about system settings How to assigna The table below describes how to assign a new value to a system setting in the new value to a System Control module system setting Step Action Select System Settings Result The Instructions dialog box for the connected system opens The illustration below shows the dialog box opened with the Alarms group of settings selected Instructions Alam Piesswe Paraenetets o G Aams Mode Mode Enabled F 3 HighAloem 10 00 MPa C Disabled Enabled C Specists LowaAlarm 0 00 MPa a Wath Alam Pressure High lam 0 00 10 00 Mode Enabled po o C Monitors HighAlsen 3 00 MPa jal hPo Aen ce paeaaad Alam 10 00 5 00 Mlm tt foco MPa C Curves sium anann Set Selected Parametes To Strategy Defauit Value toed Hee Click the radio button to select one of the following instruction groups e Alarms Specials Monitors Curves Result The instructions for the group are displayed The parameters are listed below each instruction The title bar of the dialog box shows the selected instruction group e Select a parameter from the list e Change the setting value in the Parameters field Result The parameter is updated with the new value in the list Click the Set Selected Parameter To Strategy Default Value button to re
120. shows one way to create a BufferPrep method Step Action In the Text Instruction editor e Insert a BufferPrep pH block at breakpoint zero at the beginning of the method e Define BufferPrep pH as a variable Change to the Run Setup e Select the BufferPrep tab e Click the ON radio button in the Status field e Select a Recipe from the drop down list box There are two main alternatives AIJEX or CIEX which are recipes covering a broad pH range single buffer recipes for more narrow pH ranges Result All information relevant to the selected recipe will be dis played on the tab Prepare the required stock solutions Do one of the following e Select the Variables tab Set the required pH for the method run in the variable BufferPrep_pH or e If you want to perform pH scouting click the Scouting tab and select BufferPrep_pH as a scouting variable Enter the pH values for the different runs BufferPrep recipes The recipe saved in the method the one selected on the BufferPrep tab cannot be edited although fine tuning is possible However the recipes on the list of all BufferPrep recipes can be edited New recipes can also be created see E How to create and edit BufferPrep recipes on page 536 03 0014 90 ep144 How to fine tune the BufferPrep re cipe with correc tion factors How to edit methods 6 In order to obtain high pH accuracy the recipe can be fine tuned around a spe
121. small cross in the top right hand corner of the File Navigator Result The File Navigator closes e p 225 10 How to view results 10 3 Basic presentation of chromatograms 10 3 Introduction In this section 03 0014 90 e p 226 Basic presentation of chromatograms This section describes how to access result files and optimize the presentation of a chromatogram and its curves via the Chromatogram Layout dialog box This section contains these topics Topic See Introduction and temporary chromatograms 10 3 1 The Chromatogram window 10 3 2 10 3 1 Contents of a chromatogram Temporary chro matograms How to copy curves into Tem porary How to clear a temporary chroma togram How to view results 10 Introduction and temporary chromatograms Chromatograms can be viewed in the Evaluation module A chromatogram includes a number of curves that have been created during a method run such as UV conductivity pH fraction marks etc A chromatogram also contains the curves created and saved during an evaluation session The original raw data curves cannot be deleted or modified but they can be used as the basis for evaluation procedures and subsequent creation of new curves A Temporary chromatogram is essentially an empty chromatogram that is specific to the Evaluation module It is also user specific so that all users have their own Information contained within a Temporary c
122. tab for which the default values for that column were accepted Note If the method contains a linear flow rate instruction the user can keep the linear flow rate by selecting a check box in the dialog box ep 181 8 MethodQueues Introduction In this chapter 03 0014 90 ep 182 MethodQueues MethodQueues provide a means for linking several methods together on the same or different systems For example if a system wash procedure is programmed in a separate method it can be linked in a MethodQueue to a series of different process methods ensuring that the same wash procedure is used before every process Alternatively the product of a separation on one system might form the starting material for a separation on the next allowing fully automated multi step processing This chapter contains these topics Topic See How to create a new MethodQueue How to edit a MethodQueue 82 8 1 Instruction MethodQueues 8 How to create a new MethodQueue The table below describes how to create a MethodQueue in the UNICORN Manager module Step 1 Action e Select File New MethodQueue or e Right click in the Methods window and select New MethodQueue on the shortcut menu or e Click the MethodQueue icon Result The MethodQueue Editor dialog box is displayed Mcthod ucue Editor Untitled x Start MethodQueue AsSoonAsPoratld C Atime 0203PM iy Dx f z R
123. tables that are globally available Click the Personal radio button to display the tables that are restricted to your own user ID e Click OK Result The Molecular size table dialog box opens How to renamea_ The table below describes how to rename an existing molecular size table molecular size table 03 0014 90 ep 454 Step Action 1 Select Mol Size Edit Mol Size Table Rename Result The Rename molecular size table dialog box opens How to delete a molecular size table How to calculate the molecular size The Analysis module 13 e Select Personal to display the tables that are restricted to your own user ID if needed e Select the molecular size table you wish to rename in the Molecular size table s list e Click in the Molecular size table name text box and type a new name e Click the Rename button Note You must have Edit global list s rights to be able to rename global tables The table below describes how to delete an existing molecular size table Action Select Mol Size Edit Mol Size Table Delete Result The Delete molecular size table dialog box opens e Select Personal to display the tables that are restricted to your own user ID if needed e Select the molecular size table you wish to delete in the Molecular size table s list e Click the Delete button e Click the Yes button to confirm Note You must have Edit global list
124. the chosen system within the folder e If you have more than one system connected to the computer choose the System for which the method is intended The method can be run on any system that uses the same strategy Remember that different systems may have different configurations and control capabilities e Choose the Technique for which the method was written e Click OK Result The method is saved but remains open in the Method Editor so that you can continue editing if you wish Note You might want to sign your method If you do so you can choose to lock the method so that nobody will be able to change the method See 5 4 How to sign the method on page 91 for further instructions e p89 5 How to create a method 5 3 How to use Text instructions How to display descriptions of in structions How to print de scriptions of in structions How to add a Snapshot 03 0014 90 ep90 A dedicated strategy is available for each system in the AKTAdesign platform Although the majority of the instructions are general some of them differ slightly between the individual strategies The list below describes two ways to display descriptions of the instructions in your particular strategy e Select the instruction in the Instruction Box of the Method Editor and press lt F1 gt or e Right click the instruction in the Text pane and choose the menu option What s This The table below describes h
125. the Delete System button to remove a system and all associ ated methods from the MethodQueue Click the Insert Row Before or Insert Row After buttons to add new rows before or after the selected row Click the Delete Row button to remove the selected row Click the Move Row Up or Move Row Down to move the selected row one step up or down in the queue Click the Save button Click the Run button to execute the MethodQueue immediately or the Close button to close the dialog box How to perform method runs 9 9 How to perform method runs Introduction This chapter describes how to perform and monitor different kinds of method runs from the System Control module It also describes how to control the system with manual commands and instructions In this chapter The chapter contains these sections Topic See How to start a method run How to monitor a method run Manual system control How to perform a scouting run How to perform a MethodQueue run If the network connection fails 96 e p 189 9 How to perform method runs 9 1 How to start a method run 9 1 Before you start How to start from the UNICORN Manager How to start from System Control How to start a method run Before you start a method make sure that e the correct system is connected in control mode Note If the system is connected via a CU 950 Advanced unit the Ethernet connection must not be broken during the s
126. the Instruction e p119 6 How to edit methods 6 4 How to use method variables Variable names Step Action e Enter a name for the variable e Select the Visible in details only check box if you want to set the variable as a details variable Detail variables only become visible on the Variables tab if the Show details check box is selec ted This option is useful for hiding less important variables e Click OK Result The Var button changes to VAR to confirm the new variable The variable is displayed in the Text pane Variables are defined with names that can be explicit descriptions of the variable function for example Sample_volume and Gradient_length Suitable choices of variable names can make the method easier to read and understand and also help the operator in setting variable values at the start of a method run The names can be up to 32 characters long and the following characters can be used e Letters A Z e Digits 0 9 e The underscore character _ The case of letters is retained but not significant The names Flow_Rate and FLOW_RATE are treated as identical How to rename a The table below describes how to rename a variable variable 03 0014 90 ep 120 Step Action Select the instruction that includes the variable you wish to rename in the Text pane of Text instructions Result The parameters for the instruction are shown in the Instruction box e Locate the r
127. the Moving Average smoothing algorithm is used Stage Description For each data point in the source curve the processed curve is calcu lated as the average of the data points within a window centered on the source data point e The width of the window is determined by the parameter value expressed as number of data points When the source point is less than half the window size from the beginning of the end of the curve the average is calculated symmet rically round the source point over as many data points as possible e If you increase the window width the smoothing effect is also increased Note The filter algorithm only accepts odd integer parameter values between 1 and 151 If an even number has been given it is incremented by one 1 Autoregressive The table below describes the process when the Autoregressive smoothing algorithm is used Description The first data point in the source curve is copied to the processed curve For each subsequent data point the previous processed point is multiplied with the parameter value and added to the current source data point 03 0014 90 ep 490 Evaluation functions and instructions B Stage Description 3 The result is then divided by the parameter value plus 1 according to the following formulae t 5 t p t nit Sn pt 1 Where t current processed point t 1 previous processed point S Current source point p smo
128. the System Control module where you can take Snapshots during a run using the Marker How to view recor The table below describes how to view Snapshots which have been recorded during ded Snapshots a method run using the Snapshot text instruction Note How to insert the Snapshot text instruction in a method is described in 5 3 How to use Text instructions on page 90 Step Action In the Evaluation module e choose View Documentation or e click the View Documentation icon Result the Documentation dialog box is displayed e Select the Result Information tab e Select the Snapshots sub tab Result The recorded Snapshot information for a chromatogram is displayed in a list You can e select other chromatograms in the Select chromatogram drop down box e select the Rows or Columns radio button to display each Snapshot as a row or a column e select the Time or Volume radio button depending on which quantity you want as a base e p41 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 7 Snapshots Action To print the Snapshot information e click the Print button e select the Snapshot check box in the Print dialog box e click OK Click OK or the Cancel button to exit the Documentation dialog box How to take Snapshots in the The table below describes how to take Snapshots in the Evaluation module Evaluation mod ule Step 4 Action e Open a result file in
129. the blank run curve Example ofa UV The illustration below shows the UV curve with baseline prior to subtraction of curve with the baseline baseline Example ofa UV The illustration below shows the UV curve after subtraction of the baseline curve after subtrac tion of the baseline e p 275 11 How to edit results 11 2 How to subtract a blank run curve How to importa If a blank run curve was made this might have been stored in another result file blank run curve f there is no blank run curve you can create one with Integrate Calculate baseline The table below describes how to import the blank run curve Step Action Ensure that the destination chromatogram has been opened and is the active window on the screen Choose File Open Curves Result The Open Curves dialog box is displayed Double click the result file that contains the blank run curve Result The curves in the first chromatogram are displayed e Select the appropriate chromatogram in the Chromatogram list Result The curves for that chromatogram are displayed on the Available list e Select the curves that correspond to the blank run curve and click the Select button Result The selected curve is displayed on the Selected curves list e If you want to remove a curve from the list select it and click the Remove button e Click OK to import the curve Note For more detailed information on how to import curves chromatograms
130. the exported file Chromatogram The table below contains a list of instructions for chromatogram functions functions Instruction COPY_CHROM Description Creates a copy of the specified chroma togram If is used as source then the current default chromatogram is used If is used as destination then a default name will be created for the copy ep515 B Evaluation functions and instructions B 4 Procedure instructions Instruction Description CREATE_NEW_CHROM Creates a new chromatogram with the given name If is used for the chromatogram name a default name will be generated and used Note It is a recommendation not to use only numbers as names for new chromatograms DELETE_CHROM Deletes the named chromatogram If trying to delete the current default chromatogram a run time error will be caused OPEN_CHROM Opens the specified chromatogram from the specified file RENAME_CHROM Renames the specified chromatogram If is used as From then the current default chromatogram is used RESTORE_DESTINATION_ CHROM Resets the destination for the sub sequent curve and peak table opera tions to the default chromatogram Used in pair with the SET_DESTINA TION_CHROM instruction SET_DESTINATION_CHROM Opens the named chromatogram as destination for the subsequent curve and peak operations Used in pair with the RESTORE_ DESTINATION_CHROM instruction Other
131. the folders that you have access to on the network e Method files or folders cannot be copied to the Results window e Result files and folders cannot be copied to the Methods window If you copy a folder you will also at the same time copy all files and folders that it contains The table below describes how to copy files and folders Note Follow the same steps but select Move to move files and folders Select one or more files and folders in either the Methods or Results window of the UNICORN Manager e Select File Copy or e Right click and select Copy from the short cut menu Result The Copy dialog box is opened Select a target folder or floppy disk drive Click OK Use the function Copy to External when you need to copy files and folders outside of your own user folders Copy to External should be used specifically when you need e to copy a method to another system the method can then be connected to the appropriate system e to copy toa floppy disk drive The files are automatically compressed into a zip file The file will also automatically be spanned across several disks if necessary e p75 4 Files and folders in UNICORN 4 4 How to copy delete rename and backup files and folders How to Copy to External The function Copy from Extern a How to use Copy from External 03 0014 90 e p76 The table below describes how to use the function Copy to External
132. there is only one or a few noise spikes for example caused by air bubbles or if the noise is confined to only a small part of the curve It can flatten peaks and affect peaks areas slightly but does not affect retention e Savitzky Golay Use this to calculate the smoothing and differenti ation of data by a least squares technique Select an appropriate smoothing parameter value from Light to Hard for the selected filter in the Filter Parameters field Use the slider or insert a value manually in the text field The smoothing effect in creases with increasing parameter values e Click OK Tip Start with a low parameter value for example the default value and increase it until the best result is achieved A useful strategy is to increase the parameter value by the default value for each try Note By default smoothed curves are given the suffix SMTH The default curve name can be changed as needed How to edit results 11 11 2 How to subtract a blank run curve Introduction Subtracting a blank run curve is a frequently used function in presentations especially if the curves have a drifting baseline or ghost peaks Ghost peaks If the ghost peaks come from impurities in the eluents all equilibration of the columns should be the same from method run to method run If for example the equilibration volume with buffer A is larger before a blank run curve than before a separation your ghost peaks might be higher in
133. these components on all levels This dialog box is used to set the criteria by which peaks are identified The illustration below shows the Define Component s dialog box Deline Component s aira Double click on a peak to select it 0 4 pan H Black peak markers Component name Possible to change 0 60 Internal standard 0 53 Ribonuclease A 5 0 56 Cytochrome C 11 01 1 33 ep 413 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Examine the com The Define Component s dialog box initially displays the components from level ponents 1 1 that is the peak table from the highest or lowest concentration of the standard The Show curve for level list is used to examine the curve for each standard run The size of the components are reduced or increased progressively as you select levels further down on the list which reflects the decreasing or increasing concentration of the standard If an internal standard has been incorporated its peak remains about the same size on each level Peaks detected during the peak integration Each component peak that was detected during the peak integration i e that is present in the peak table is identified by a lower triangle black in level 1 1 green in other levels There may be different peaks detected for different levels Upper triangles will later identify the peaks that are selected for quantitation Step 2 How
134. this step for as many curves as you want from the same or other chromatograms Note You can open more source chromatograms with the File Open Chromatogram command e Click the Close button when you have copied all curves 03 0014 90 ep 310 How to edit results 11 Step Action Change some comparison settings e Make sure the target chromatogram is open and that its window is active e Choose Edit Chromatogram Layout to display the Chromatogram Layout dialog box e Select the curves that you want to view on the Curve tab and click OK e The curves can be scaled individually or all with the same Y axis scale Use the All with this unit button on the Y Axis tab to scale all curves with the same scale 7 If you used the Temporary chromatogram e If you used the Temporary chromatogram you can perform evalu ations in the Temporary chromatogram and transfer the final curves to other destination chromatograms e All of the contents in the Temporary chromatogram can be re moved with Edit Clear Temporary Chromatogram Alternative way to copy curves An alternative way to copy curves into one chromatogram is to e create a new chromatogram by copying an existing chromatogram and saving it under a new name e import more curves into the new chromatogram according to the instructions described above in this section e p311 11 How to edit results 11 8 How to import and compare different runs 11 8 4 How
135. three sets of buttons e Manual Direct Commands buttons for starting and stopping the run e Windows buttons to access dialog boxes for pane selection documentation and layout properties e System Access buttons to control the system connection Show and hide The toolbars can be shown and hidden by choosing View Toolbars and selecting the relevant boxes The figure below shows the toolbar Run Hold Continue End 3 PI EA The available Manual Direct commands buttons in System Control are dependent on the control status of the connection The table below shows when each button is available Control Status Available buttons Hold Pause End Run Pause End Pause Continue End Hold Continue End Manual pause Run Continue End The table below describes the functions of the Manual direct command buttons Button Function Run Opens the Run dialog box which shows all available methods as the first step in a method run If a method is loaded Run Setup opens The run will start imme diately if a start protocol isn t part of the method How to perform method runs 9 Button End Function Suspends execution of a method but continues to pump liquid at the current flow rate and eluent concentration settings All settings remain un changed e to increase accumulated time and volume Method instructions are not executed until the Contin ue button is pressed Behavi
136. to The table below describes how to select and define the components select and define components Step Action Select level 1 1 in the Show curve for level list and click a peak Result The peak is highlighted in the table e Double click the peak or e Click the Include button Result The peak is selected for quantitation marked with an upper triangle and component no is listed as the Component name The selected peak is affected on all levels Note More than one peak can be selected to produce calibration curves for several components Highlight the component name and type a new name Double click the internal standard peak if applicable and type a new name Continue to Step 3 How to identify the peaks below this table 03 0014 90 p414 The Analysis module 13 The Define com The peak table within the Define Component s dialog box has three columns ponent s peak table columns e The absolute Retention value of the component in level 1 1 e The width of each component s window If you change the width of the window by adjusting the cursor lines this is reflected in the Window column e The Component name with the currently selected component highlighted Component name Possible to change 0 53 Ribonuclease A 0 56 Cytochrome C Step 3 Howto Description identify the peaks Cae When a component is selected vertical cursor lines show the current i
137. to create and print reports that are based either on a standard or a customized layout In this section This section contains these topics Topic See How to create and print a customized report 10 6 1 How to create and print a standard report 10 6 2 How to edit an existing report format 10 6 3 ep 249 10 How to view results 10 6 How to create and print reports 10 6 1 How to create and print a customized report 10 6 1 Introduction How to open the Report Editor in edit mode 03 0014 90 e p 250 How to create and print a customized report You can choose from a variety of objects to include in a report including chromatograms methods documentation free text and more in the customized report interface You can also place align and size the objects as you please This section describes how to create a customized report format Should you need to store store your reports in an electronic format you can save them as PDF files This section also describes how to do this The table below describes how to open the Report Editor in Edit mode to create a customized report format Step Action Open a result file in the Evaluation module e Select File Report or e Click the Report icon Result The Generate Report dialog box opens e Click the New button Result The Create New Report Format dialog box opens e Select the Customised format and click OK Result The Report
138. to edit 3 Rename e Type anew variable name in the New name text box e Click the Rename button Result The variable is renamed Delete e Click the Delete button e Confirm that you want to delete the variable Result The variable is deleted How to change a Detail variables are indicated with a D to the left of the Variable column on the scouting variable Scouting tab The table below describes how to set up a detail variable into a detail vari able Step Action Click the Edit Variable button on the Scouting tab Result The Edit Variables dialog box opens The variables are listed alphabetically Choose the variable to be changed e p179 7 Scouting 7 1 How to set up a Scouting Scheme Step Action 3 e Select the Set visible in details only checkbox e Click the Close button Result The variable is indicated by a D Note De select the checkbox to make the variable fully visible again How to copy con The contents of the Scouting tab can be copied and pasted into a third party factorial ae to factorial design program Processed values can then be pasted back into the Scouting tab esign programs i i The table below describes how to do this Step Action Select the text etc that you want to copy Press Ctrl C Place the cursor where you want to insert the copied text Press Ctrl V 03 0014 90 p 180 7 2 Instruction Scouting 7
139. to set up a Scouting Scheme This section describes how to set up a method for scouting Any parameter can be scouted provided that it can be defined as a variable in the method Scouting is a facility for automatically repeating a run with systematic variation of one or more parameters Some typical situations where scouting is useful are when you want e to screen for the best column e to find the optimal pH e to test column capacity sample volume e to find the optimal flow rate for binding and elution e to optimize gradient length and slope to optimize step gradients The variables that appear in the scouting scheme are usually a subset of those on the Variables tab of the Run Setup The values in the scouting scheme can only be set on the Scouting tab while the default values in the method can be set either on the Variables tab or in the Text instruction pane Changing variable values in the scouting scheme does not change the values on the Variables tab or in the text instructions Values for variables selected for scouting are grey on the Variables tab and cannot be changed there Any changes that you make to variable values when a scouting scheme is run are saved in the result file Results from a scouting run are saved in a scouting folder There are seven buttons on the Scouting tab of the Run Setup plus the Help button The table describes the functions of these buttons Click the button if you
140. to stack and stretch curves 11 8 4 Functions How to use the Normalise func tion 03 0014 90 ep 312 How to stack and stretch curves You can stack and stretch curves from different runs to better visualize the differences To achieve this you can use the following functions e Normalise e Shift e Multiply Note All the functions require the curves to be present in one chromatogram The Normalise function provides the simplest method to align curves with respect to the X axis or the Y axis for easier visualization The table below describes how to use the Normalise function Step Action 1 e Make sure that a chromatogram with the relevant curves is open in the Evaluation module e Choose Operations Normalise Result The Normalise dialog box is displayed a E Select curve to nomealise Select help curve to nomalise against o a yvi 280nm 50 01 125200101 1_ UV1_ 280nm 1_pH 08 125200101 1_Pressure 09 125200101 1_Flow 10 125200101 1_Temp zj 13125200101 1 SamolePres a 13 125200101 1_SamolePres Co i ca i e How to edit results 11 Step Action 2 e Select the curve you want to normalise in the left Select curve to normalise field e Select the reference curve you want to normalise against in the right Select help curve field Example If you want to stack the curves select the curve at the bottom of the stack as the reference curve e Click OK
141. unless you save the changes under a new column name since other users may not appreciate the changes Note It is recommended that only a limited number of users are given access to the right to edit global columns This is essential to avoid unintentional changes Columns Fiter an Columns E Data for Za HiTrap_Desating Noimal Parameters Advanced Pacameters Parameter Height Diameter Column volume Column volume unit Techrique vw Vo Max pressure Defauk flowrate Max flowrate Typ peak width at base HiLoad_16 10_SP_Sepharose_HP Global pH High value longterm HiLozd_ 16 60_Superdex_200 prep_crade Global pH Low value longterm pH High value shoitlermn pH Low value shoelterm Average perlicke diam 34 Code no 17 1408 01 Typical loading range 0 1 1 5 m Mol weight range kDa Scan rate Specira sec The table below describes how to print the column list data Step Action e Click the print button Result The Print Column List dialog box opens e Select to print a global or your personal column list e Click OK Result The column list is printed on the default Windows printer The New Column The illustration below shows the New Column dialog box dialog box The Column list D x Column name Normal Parameters l Advanced Parameters Parameter Max flowrate Typ peak width at base pH High value longterm pH Low value longterm PH High value shortterm pH Low value
142. up on the peak slopes The illustration below is an example of noise detected as peaks A and the result of a second peak integration with an increased Noise window B tvahsstion Example Result0O2 1 v UNICORN Loca IN Nas result IEeample Resull 002 feannple these resi ajoj uh Film Edt Wew Integrate Operations Procedures Window Hep aig sisal saosa oT No Peak name Retention min Area mAU min Height mAU 9 41 3 4405 42 176 9 14 0 2736 2 037 8 86 0 1840 1 715 8 46 0 3746 2 324 0 85 2 0 053 156 706 No Peak nane Retention min Area mAU min Height mAU 3 interna st 5 85 60 5051 336 828 2 Peak A 0 2 6 53 14 6082 95 761 3 Peax B 0 2 7 06 20 4377 125 560 Dear r in 7 7R ar aan 1a Aa Note You can also use the Reject peaks function in the Integrate dialog box to reduce the number of peaks based on the total number of accepted peaks or the minimum peak height Missing peaks Evaluation 12 Sometimes obvious peaks are not detected in the peak integration The probable cause is that the Noise window is set too high See the illustration below NANA All peaks are detected if the Noise window is decreased see example below Note Missing peaks can also be caused by improper settings for Reject peaks in the Integrate dialog box or Filter peaks in the Chromatogram layout dialog box ep 345 12 Evaluati
143. will be lost the next time you log on to the network e Result files that are directed to a network drive will be stored in the Failed folder on the local station Connection modes Several workstations can connect to a single chromatography system at the same time but only one workstation can be in control mode The other connections are in view mode and the connected workstations can only monitor the system activity but not issue any commands The system status is indicated on the status bar at the bottom of the System Control window The table below describes the different connection modes the corresponding status texts and some of the various actions you can take to change the connection mode Connection mode Not connected Not Connected Status Text Controlled By default Controlled By Eric Locked By Eric System is available Possible action to change connection mode Connect to a system Disconnect from or leave control of the sys tem The system is controlled by you No connection possible The system is controlled by another user Click the Connect to sys tem icon and supply a password The system is locked by another user Connect to the system The system has been left unlocked ep 59 3 General system operations 3 5 How to connect to the chromatography system ae leave con The table below describes how to leave control of a system so that it is avail
144. your files How to copy delete rename and back up files and folders 4 4 e p67 4 Files and folders in UNICORN 4 1 How to create folders 4 1 Introduction UNICORN folders How to create a user specific folder 03 0014 90 ep 68 How to create folders This section describes how folders are organized in UNICORN and how to create a new user specific folder for the user s methods and results The files and folders are displayed in the two UNICORN Manager module windows All method files and corresponding folders are listed in the Methods window The result files and folders are listed in the Results window You can only see folders that you have access to You can only see method files that are written for systems that you have access to The table below describes how to create a user specific folder Step Action Select the window you want to create the folder in Methods or Res ults Result The window title bar is highlighted e Select File New Folder or e Right click and select the New Folder shortcut Result The Create New Folder dialog box opens Type a name for the new folder Click OK 4 2 Introduction How to opena method file How to opena result file in UNICORN Man ager How to opena result file in the Evaluation mod ule Files and folders in UNICORN 4 How to open and preview files This section describes how to open your s
145. 0 ep62 General system operations 3 3 6 How to back up and restore system data Introduction You can create a backup file with system information and store it on a diskette or another drive The backup file will contain information about e Global Files e Personal Files e System Files Afterwards you can use the backup file to restore the system definitions in case they are corrupted How to createa The table below describes how to create a backup file and store it for example on backup file a rescue diskette Step Action Insert a diskette into the computer if you want to store the backup file on a diskette Choose Administration Create Restore Backup in the UNICORN Manager to display the Create Restore Backup dialog box Create Restore Backup x Action fe C Restore Browse Global Files Global BufferPrep Recipes Global Columns Global E valuation Procedures Global Report Formats Personal Files System Files User Setup System Setup Information Create Cancel Help cme He ep 63 3 General system operations 3 6 How to back up and restore system data Step Action 3 e In the Action field make sure that the Create option is selected e Click the Browse button to select where to store the backup file Note Select A to store the file on the diskette e In the Items field select which information to include on the backup file e Click the Cre
146. 014 90 e p 326 How to save results and exit the Evaluation module After you have finished the evaluation process you can save all the changes you have made to the chromatograms including newly created curves and chromatograms that you have imported and created All the curves that you created during your manipulations will be saved in the chromatogram If some of these curves are not be needed anymore select Edit Delete Curves in the Evaluation module to remove the curves Note The original curves that were created during the run can never be deleted You can either save your edited results in the original file or in a new result file The table below describes how to save the results in the Evaluation module If you want to save the then edited results in the original result e select File Save file or e click the Save toolbar icon in a new result file e select File Save as Note The previous version of the result file will be overwritten if you save the changes This cannot be reversed However the raw data curves remain unchanged The table below describes how to exit the Evaluation module Step Action Choose File Exit Result If there are unsaved changes a dialog box opens with an option to save the changes before exit Select Yes if you want to save the changes Result The result file is closed in the Evaluation module and the UNICORN Manager module is displayed
147. 1 9 1 Introduction Curve formats How to import curves How to import data from SMART Manager and FPLCdirector How to edit results 11 How to import results This section describes how to import curves in different formats and how to import result data from SMART Manager FPLCdirector or AKTAprime You can import curve files in the following formats e AIA cdf e ASCII text e Lotus 1 2 3 spreadsheet wks The table below describes how to import curves Action Choose File Import Curve Result A menu with the available curve formats opens Choose the correct curve format Result The Choose File to Import From dialog box opens Locate the file that contains the curve and double click the file Result The Import Curves dialog box opens e Select the curve s to import and click the OK button Result The curves are opened in the Evaluation module The table below describes how to import data from SMART Manager and FPLCdirector Action Choose File Import Result Result A menu box with the available data sources opens This box opens immediately after Import if no result file is open in the Evalu ation module Choose FPLCdirector or SMART Result The Import FPLCdirector Result dialog box or the Import SMART Result dialog box opens Locate and double click the result file Result The result file is opened in the Evaluation module ep 319 11 How to
148. 18 11 9 1 How to import TOSUNES vanausw aurrciavattnies dcr uaiwalten obiwutalscuvadiandaltaapie uated eia 319 11 9 2 How to export RESINS 1a e a a ccksin chee S cat icry tsa uenisties abaclssegunauadiss agehaas os gectebes 321 11 10 How to sign results ClECIrONICAllWissciessavepeacetacestadignslacetcaietiavieuaeieeedetanneten 325 11 11 How to save results and exit the Evaluation module ssssssssssrsrrerserrrrrerren 326 BZ ee AE a is ers EAA EE E E ATT 327 12 1 Peak Mt QtatlOMN iveissescaccotisndencscwntestisasduceevssevecandeascnauasusaredeceduasduasdutavsdurebvectuandeduas 328 12 1 I Baseline caleulatio Mnsan taster e A A E EEEE 329 12 1 2 How to perform a peak integration sssesrsrserrsrerrrrrrrrsrrtrrsrrrrsrrrrrsrrrrre 330 03 0014 90 epiv Table of Contents 12 1 3 How to optimize the baseline with a morphological algorithm 0c 000 336 12 1 4 How to optimize the baseline with a classic algorith ccceceeeeeneeeeeees 340 12 1 5 How to edit the baseline manually cscs vaveshcis cdesiah etre Beart na tater cciaen 348 12 1 6 How to edit the DOAKS cw uveitaietarus uatbvion ete verce tet ieaunaiena vend temas lienuivetaaent 351 12 1 7 How to integrate part of a curve and how to exclude or skim peaksS 358 TDL By WICASUPEMICNIES f c5 c5r E areddatas EA E E AAE 363 W252 OUG CV AINA ONS ico cedar sie ceute veteenes E EE tee taylusnaeteeket te venutadwudewneead 365 12 2 1 Peak purity and p
149. 32 How to prepare for standard addition 432 How to select the standard addition component 433 How to prepare for recovery calculation 435 How to calculate the recovery 435 Automated sample runs 441 Automatic update with Replace 442 Automatic update with Average 443 Automated in scouting runs 444 Automated update with Average for scouting runs 447 Quick View How to preview result files 70 R Recovery calculation How to perform 406 Reliability 407 Recovery factor calculation How to prepare for recovery calculation 435 How to calculate the recovery 435 Error signs 436 Reference curves In Run Setup 140 Rename files and folders 77 Reports How to create a blank customized report 250 Edit mode toolbar buttons 251 How to add or delete pages 252 How to change the page setup 252 How to add objects to a report 253 How to add free text 254 How to add picture objects 255 How to include chromatograms 256 Index epx Index How to include a peak table 256 How to include a pool table 256 How to include Method objects 257 How to add documentation 258 How to add the Evaluation log 258 How to include Quantitate and Mol Size data 259 How to include Frac 950 data 259 Toolbar icons in Report Edit Mode 260 How to print 261 How to save the report in PDF format 262 How to save the report format 263 How to create a Standard report 264 How to print a standard report 265 How to edit a standard repor
150. 4 3 14 1 System settings When to change the system settings How to change the default settings System settings 14 General information about system settings The system settings e define settings for alarms and warnings e select the data that will be stored in result files Each system has a set of default settings e Changes to the default settings should be made when the system is installed Certain system settings may need to be adjusted in the following cases e If system components are changed e g the alarm and warning limits e For specific separation runs e g the monitor and curve settings Note Only the settings for the selected components will be shown for strategies where you select the system components The table below describes the two different ways to change the default system settings Change Effect To assign a new value to a parameter The specific change is valid only until within a method End in the method After End the parameter returns to its default setting Note Only some parameters can be changed in the method To assign a new value to the system The new value is valid for all runs and setting remains until you change the value again or return the setting to its de fault value See How to assign a new value to a system setting below Like the default values the new value can be changed temporarily in a method Note You must have System
151. 535 E How to create and edit BufferPrep recipes E How to create and edit BufferPrep recipes Introduction The BufferPrep function is available for some AKT Adesign systems This appendix describes how to create and how to edit the recipes for BufferPrep In this appendix This appendix contains these sections Topic See How to create a BufferPrep recipe How to edit a BufferPrep recipe 03 0014 90 ep 536 How to create and edit BufferPrep recipes E E 1 How to create a BufferPrep recipe About BufferPrep New BufferPrep recipes are created in the Method Editor The list of recipes is not ies Ai linked to a specific method Which recipe to use in a certain method is selected on the BufferPrep tab in the Run Setup How to createa The table below describes how to create a new BufferPrep recipe in the Method TECIpE Editor Step Action Choose Edit BufferPrep Recipes Result The BufferPrep Recipes dialog box opens The illustration below shows the BufferPrep Recipes dialog box with a recipe selected i p Stock soiton Inlet A1 A11 A18 Buffer DIM Acetate ID SOLUTION 1000mt Inlet A2 Acid Base Use ampoule 0 1M HCI 01M Ha SALT SOLUTION 2000mE 233 89 Mw 58 44 Inlet B1 Water Defauk correction factees 0 0 pH at 0 B and 0 1 pH at 100 8 Inlet B2 Sek zi 2M Neti Edt p New Delete Help Click the New button Result The New Recipe dialog bo
152. 8 How to edit the baseline manually You can edit the baseline manually in the Edit Baseline dialog box in the Evaluation module e Select Integrate Edit Baseline to display the dialog box The Edit Baseline dialog box displays the baseline and the curve it was calculated from The baseline points are marked with green squares Hold the cursor above the baseline point to display its coordinates See the illustration below Q Eda Baseline lot 7 2800 Example result 003 1 UV Example tenult 003 1_UVG O1 BASEM Example terslt 003 1_Edtesiareline mA 60 B00 so 150 100 s0 o o O 0 o D 0 o 0 o g O O O C O CH o 100 20 0 20 0 0 0 60 0 00 0 70 0 20 0 min 17 233 min Deere Delete al 537 976 mAU Diaw straight to pet point cera The table below describes how to use the zoom function in the Edit Baseline dialog box Step Action e Click the Zoom icon Result The cursor is changed into a magnifying glass e Press and hold the left mouse button e Drag the cursor over the area you want to zoom in on e Release the mouse button Result The area is enlarged Right click and select Reset zoom to restore the full view How to edit and insert data points Evaluation 12 The table below describes how to edit and insert baseline data points Step Action Select Integrate Edit Baseline Result If there are more than one baseline available the Select Baseline to Edit di
153. 8 id148Quantitate001 1_Pressure A ID AI a ee hh he UIT A ee xl iH rm Peak label m Fraction text alignment r Logbook text alignment Text alignment I Number C Vertical Vertical Vertical MS Peak Name a Horizontal Horizontal Horizontal I Retention C Fly Over C Fly Over Filter Cancel Help How to display The table below describes how to display peak labels peak labels Step Action e Choose Edit Chromatogram Layout or e Click the Chromatogram Layout icon ci Result The Chromatogram Layout dialog box opens Click the Curve Style and Colour tab 03 0014 90 p 334 Evaluation 12 Step Action Select one or more of the following labelling options in the Peak label field e Number Result The peaks will be numbered sequentially e Peak Name Result Peak names will be displayed See 12 1 6 How to edit the peaks on page 351 for information about how to name the peaks e Retention Result The retention volume or time will be displayed e Click OK e p 335 12 Evaluation 12 1 Peak integration 12 1 3 How to optimize the baseline with a morphological algorithm 12 1 3 Introduction The Morphologic al algorithm How to set a Morphological baseline 03 0014 90 e p 336 How to optimize the baseline with a morphological algorithm The first choice when you want to optimize the peak integration is to chang
154. Action 4 Click OK Result The method is saved but remains open in the Method Editor so that you can continue editing if you wish Note You might want to sign your method If you do so you can choose to lock the method so that nobody will be able to change the method See 5 4 How to sign the method on page 91 for further instructions 03 0014 90 ep 34 9 2 Introduction How to create a new method How to create a method 5 How to use the Method templates This section describes how to create methods based on an existing template Note A custom system for example a process system requires that the users create their own templates by saving methods as templates Each method is written for a specific strategy The function of the method cannot be guaranteed on systems having other strategies The table below describes how to create a method from the UNICORN Manager module Note The New Method dialog box is also accessible from the Method Editor module using the same commands Step Action e right click in the Methods window and select New Method from Result The New Method dialog box opens in the Method Editor module Result The method template will be opened as an untitled method in the Run Setup in the Method Editor Note If Any is selected in the For column list you can use any column but must enter the column volume in the method on the Variables tab It is recommended tha
155. Alignment for the logo if necessary Note The logo file must be in bitmap format bmp and smaller than 64 kB Larger logo files or files in other formats must be inserted as Picture objects If you want to have a line under or over the header select the appro priate option in the Layout field e Repeat steps 3 to 6 on the Footer tab and the subsequent pages Header tab Note All Header and Footer tabs contain the same options You can have all information in either the header or footer or split informa tion between the header and footer as required e Click OK How to add ob The table below describes how to add objects to the report The various objects jects to the report 4e described below this table Step Action e Click the appropriate icon in the Report items toolbar or e Choose an object from the Insert menu sim Layout Help T Free text 5 Text Method E Chromatogram Documentation a Evaluation log 1 Quantitate and Mol Size GS Frac 950 e Press and hold the left mouse button on the report page and drag out a box to the size of the item you want to insert Note The mouse pointer shows a symbol for the type of item you have selected e Release the mouse button Result A Setup dialog box opens The dialog is specific to the type of item that you want to insert e p 253 10 How to view results 10 6 How to create and print reports 10 6 1 How to c
156. Changes will override the default settings and values for the particular run and be saved in the result file Text Method method instructions They cannot be changed from this display the Notes tab the gradient BufferPrep the recipe selected in the method The recipe cannot be changed during the start of a run the available column definitions Reference curves the reference curves associated with the method Evaluation procedures the evaluation procedures set to be executed at the end of the method Method information the method information the settings Calibration the monitor calibration settings Questions questions defined in the method You are recommen ded to always use this option since the answers to questions can form an important part of the UNICORN run documentation e p 151 6 How to edit methods 6 5 Run Setup 6 5 14 The Start Protocol tab Checkbox Displays Result name the result name which is changeable if this option has been selected Click the Browse button to change the result folder If the box is not selected the result name will still be displayed but you will not be able to change the name or folder Scouting start pro The table below describes the options in the Scouting start protocol field tocol field Option If you check this option First run only parameters for the scouting runs can be adjusted at the beginning of the first run only After t
157. Click one or both of the Reset buttons to reset the counters e Click OK e p 467 15 System maintenance and error reporting 15 1 System maintenance functions How to acknow Once a specific Periodicity parameter has been reached a warning message will be ledge a warning displayed The table below describes how to acknowledge the warning Step Action The Warning dialog box opens Lamp check e Click the Acknowledge button if you have corrected the problem e Click the Ignore button if you haven t corrected the problem Note You will be reminded later about the unsolved problem if you click the Ignore button 03 0014 90 ep 468 System maintenance and error reporting 15 15 2 How to generate problem reports Introduction UNICORN contains a Generate Report Wizard for registration of errors or problems that you have detected or that occur during a run The Generate Report Wizard takes you through the steps to generate your report There are two ways of accessing the Generate Report Wizard e From the UNICORN Manager e From the System Control In this section This section contains these sub sections Topic See How to generate a report from the UNICORN Manager 15 2 1 How to generate a report from the System Control 15 2 2 e p 469 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 1 How to generate a report from the UNICORN Manage
158. Definition 329 Zoom function How to enlarge parts of a curve 244 03 0014 90 p xxii
159. Double click the reading box Result The dialog box for manual instructions is displayed showing the instruction or main instruction if there is more than one Option 2 e Right click the reading box Select Instructions in the displayed menu Another menu shows the specific manual instruction s e Click an instruction to select it Result The dialog box for manual instructions is displayed in which you can execute the appropriate command e p 197 9 How to perform method runs 9 2 How to monitor a method run 9 2 2 The Run Data pane For more details on how to use manual instructions please see 9 3 2 Manual instructions on page 212 03 0014 90 ep 198 9 2 3 Introduction How to select curves to be dis played How to display a vertical marker line How to perform method runs 9 The Curves pane The Curves pane of the System Control module displays monitor signal values graphically The figure below shows an example of the Curves pane nay WIS Cond cond Conte Con resmme how Caves Teno Sargierres SurgieFlow Ainin ADO You can decide which curves you want to display in the Curves pane Curves will only be shown for components present in the chromatography system The table describes how to select the curves to be displayed on the screen Action In System Control select View Properties Result The Properties dialog box is displayed Select t
160. Editor opens in Edit mode The Edit mode window Toolbar button functions in the Report Editor How to view results 10 The illustration below shows the Report Editor window in Edit mode with a blank report open ae Dra Paoa Zecmin IZAC adaa Dette Pose ER PUAN Se ewe Ts Fose Text Ponidi aM o Bi Gus ve oun Gua soe Tater a we See Bes aye Ean f aro N iam The table below describes the different functions of the Edit mode toolbar buttons in the Report Editor This button toggles between a print preview of the report and the Edit mode This button displays the next page or pair of pages where there are more than one page This button displays the previous page or pair of pages where there are more than one page This button toggles between single page view and pairs of pages view when there is more than one page Exit This button closes the Customize Report window e p 251 10 How to view results 10 6 How to create and print reports 10 6 1 How to create and print a customized report How to addand The table below describes how to add or delete report pages in the Report Editor delete report pages If you want then to add new pages e click the Add Page toolbar button Result A new page is added after the last page to delete a page while select the page with Next Page or Prev Page in One Page mode e click t
161. External standard quantitation e Internal standard quantitation e Standard addition e Recovery calculations Quantitate uses peak data from standard runs to produce calibration curves which can then be used to evaluate the amount and concentration of components in a sample The Molecular Size Mol Size function determines the molecular size of components in a sample The function uses a molecular size curve prepared from one or more standards e p 389 13 The Analysis module 13 1 General information about the module Term definitions The table below lists definitions for some terminology that is used in this chapter Term Definition Amount This specifically refers to the injected amount In most cases the word amount is used as an abbreviation for concentration or amount Both concentration and injected amount can be used to produce the calibration curve When analyzing the sample both amount and concentration are calculated Calibration curve The relationship between amount and peak size of a component The relation ship can be shown as a curve and as a mathematical expression Level A known amount or concentration of a standard The levels are numbered 1 20 in decreasing or increasing order of concentration Molecular size curve The relationship between molecular size and retention volume for a num ber of components The relationship can be shown as a curve and as a mathematical expression
162. General system operations 3 7 How to set up a printer 3 7 Introduction How to set up a printer 03 0014 90 e p 66 How to set up a printer UNICORN uses the default printer and printer settings that are installed on your computer You can change your printer by changing the default Windows settings but you can also set up a printer in UNICORN for the current working session The table below describes how to set up a printer in UNICORN Step Action Select the File Printer Setup menu command in the UNICORN Manager module Result The Print Setup dialog box opens Select a printer from the Name drop down box Change all printer properties as necessary Click OK Note To save created reports electronically you can select to print the files in PDF format To be able to do this you must have a full version of Adobe Acrobat installed and select PDF Writer or Distiller in the Printer Setup Files and folders in UNICORN 4 4 Files and folders in UNICORN Introduction All UNICORN data is organized in files and folders Files and folders are handled like in any other Windows application with some exceptions This chapter describes how to work with UNICORN files and folders with the focus on the topics that are specific for UNICORN In this chapter This chapter contains these sections Topic See How to create folders How to open and preview files How to arrange and locate
163. Global or Personal folder and click OK Result The quantitation table is copied into the Quantitate_Sample procedure Note The procedure cannot be executed if a quantitation table has not been selected Time must have been selected as the X axis base unit Save the method with a new name Perform the run s Result The amount and concentration of the components in the samples will be printed automatically after each run ep 441 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update 13 5 3 How to perform automated update Introduction This section describes how to update quantitation tables automatically also in scouting runs See also 13 3 3 How to edit and update a quantitation table on page 422 How to perform The table below describes how to automatically update a quantitation table with automated update with the Replace the Replace option default option Step Action Open a method in the Method Editor e Click the Scouting tab in the Run Setup e Click the Clear All button to clear the scouting scheme e Double click each Quantitation_Type table cell and select the correct concentration level for the standards Click the Evaluation Procedures tab e Select the Update_Quantitation procedure Click the Import button to import the Update_Quantitation procedure if it isn t displayed on the list Result This procedure automatically integrates the firs
164. How to add pro cedures to the menu 03 0014 90 e p 386 printed documentation simultaneously for many result files e g for a number of scouting runs The table below describes how to create a procedure to batch run reports Step Action 1 Choose Procedures Record On to record a procedure 2 Choose File Report Result The Generate Report dialog box opens Choose a report format Click the Print button as the final instruction Choose Procedures Record Off Save the procedure Note A printing procedure can also be saved with a method to produce automatic prints at the end of a run Note When for example a batch run is performed the latest version of the procedure will be used However procedures that are saved with a method are not affected if the original procedure is edited at a later time You can add up to 15 created evaluation procedures to the Procedures menu in the Evaluation module The table below describes how to add procedures to the menu Step Action Select Procedures Menu Result The Edit Procedures Menu dialog box opens e Select the checkboxes of the procedures you want to display on the menu e Click OK Result The selected procedures are included on the Procedures menu Remove a procedure Open the Edit Procedures Menu dialog box and select the checkbox again to de select and remove a procedure from the menu 12 3 4 Introduction Evaluation 12 How
165. How to reduce noise 367 How to remove ghost peaks 367 How to differentiate 370 Simulated peak fractionation 372 How to create 373 Draw a straight curve between selected points 376 How to create a fraction histogram 377 Monitor signals stored as curves 463 Curve settings 463 Calibration curve models 521 Molecular size curve models 521 Curves pane in System Control Description 199 How to select curves to be monitored 199 How to display a vertical marker 199 How to set a reference point 200 How to change curve colors and styles 200 How to change scale of the Y axis 200 How to change scale of the X axis 201 How to zoom in regions of the pane 202 Reduce scale of zoom 202 How to select curve pressure units 202 How to select text alignment 202 How to display complete Logbook information 203 Delete files and folders 77 Delete Method blocks How to use the Delete Block dialog box 104 How to use the Block Delete Block command 105 How to delete unused blocks 105 Documentation Index epv Index How to view 270 Documentation tabs description 270 Result information 272 How to export 324 E Electronic signature How to sign a result 325 Evaluation Chromatogram window views 229 How to display chromatogram header information 229 How to display peak table information 230 Chromatogram window shortcut menu 231 How to optimize the chromatogram workspace 231 How to display a vertical marker 232 How to
166. Invalid Watch in structions 03 0014 90 ep 552 The illustration below shows peaks collected by the method in the example above Waste Peak_1 OutletF1 OutletF4 Peak_1 100 mAU OutletF 3 End_collect OutletF 1 In this example one or two absorbance peaks are collected through outlets F3 and F4 respectively with waste fractions collected through outlet valve F1 waste Each called block except End_collect resets the Watch condition so that the method reacts correctly to subsequent changes in the UV absorbance The design of a method of this kind with several Watch instructions for the same monitor is important The construction in the following three lines appears simpler but is incorrect 0 00 Watch_UV Greater_than 100 mAU Peak_2 0 00 Watch_UV Less_than 100 mAU End_collect 0 00 End_block Here the second Watch instruction will annul the first since a signal can only be watched for one condition at a time F 5 Introduction Recommendations Example instruc tion Method examples F Collection of three absorbance peaks This section contains an example of how to collect three absorbance peaks through outlets F3 FS and F7 with waste fractions through outlets F4 F6 and F8 The maximum number of peaks collected in this example is three due to the limited number of positions on the outlet valve Waste container needed The waste fractions between the peaks are collected throug
167. K Result Hatch marks are displayed as a background Note You can also right click in the Chromatogram window and select Hatch e p 239 10 How to view results 10 4 How to optimize the presentation of a chromatogram 10 4 4 How to change and fix the axes 10 4 4 How to change and fix the axes How to change The table below describes how to change and fix the Y axis and fix the Y axis Step Action Open a result file Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed Click the Y Axis tab Select the appropriate curve from the list Click the Fixed option Type the desired minimum and maximum values Click the All with this unit button if you want other curves with the same Y axis units as the current scaled curve to be similarly scaled Note The values will only be applied to existing curves They will not be applied to new curves created after this function was last used Click the appropriate Pressure unit MPa psi bar option to change Y axis units for pressure curves Note Default Pressure unit is From strategy which is the unit defined in the original run strategy e Click OK How to add a The table below describes how to add a second Y axis to the chromatogram second Y axis Step Action Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed Click the Y Axis tab Select the appropriate curve
168. ME REJECT_PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE 01 17 DEFAULT DEFAULT DEFAULT DEFAULT SUB 01 17 47 PEAK_INTEGRATE 47 4 REPORTI Chromatogiam Peaks UPDATE A PERSONAL Ext Std Chym DEFAULT DEFAULT AVERAGE ON ON Instruction m Parameter Bl C Curve operation aai Teva men E ee DEFAULT z Beplace C Export F ae RT Average or replace point Delete Dither RUN PROGRAM AVERAGE x fl C Test Update Extemnal Intemal quantitation table Chose How to perform It is possible to run both standards and samples in the same scouting run and automated update continuously update a previously created quantitation table with new values The in scouting runs l step 1 table below describes how to set up the evaluation procedures for the updates Step Action 1 Open the same method that was used to create the quantitation table from the standard runs and open the Run Setup Click the Evaluation Procedures tab 3 Select Update_Quantitation and click Quantitate Result The Quantitation table dialog box opens Select the quantitation table and click OK 5 Deselect the Update_Quantitation procedure on the Evaluation Proced ures tab Repeat steps 3 to 5 for the Quantitate_Sample procedure Note Make sure that both procedures are deselected after this is completed Otherwise they will be run twice 7 Proceed with the i
169. Molecular size table All necessary data required to determ ine the molecular size of one or several components in a sample The molecu lar size table contains the molecular size curve Peak size Used generally as a common term for peak area or peak height Peak table The result of a peak integration presented in tabular form The peak table can include for example height area and retention volume After the analysis the peak table contains the values for concentration amount and molecular size 03 0014 90 ep 390 The Analysis module 13 Term Definition Quantitation table All necessary data required to quantit ate one or several components in a sample The quantitation table con tains calibration curve s and peak identification settings Sample A sample with an unknown concentra tion of the component s of interest The concentration is determined by Quantitation For molecular size calculations the sample contains a component or sever al components of unknown molecular size Sample run A chromatographic sample run of a sample to be analyzed Spiking The addition of a known quantity of the component of interest to the sample prior to the sample preparation for the run Standard A defined concentration of one or several components The concentration does not have to be the same for all components in the standard One or several standards are used to produce a calibration curve For molecul
170. NICORN 1 Question Answer How should I use the Administration If you are an experienced adminis and Technical Manual trator of previous UNICORN ver sions you can read selected sections for reference e If this is your first experience of UNICORN we recommend that you study the manual in detail e p19 2 UNICORN concepts Introduction In this chapter 03 0014 90 ep20 UNICORN concepts This chapter contains e Definitions and descriptions of some of the specific concepts that are presented in this manual and in other UNICORN manuals e An overview of the UNICORN user interface e A Quick Start Guide that can be used as a shortcut for experienced users that want to start right away Note General concepts and common chromatography terminology are not explained here This chapter contains these sections Topic See Concept definitions The UNICORN user interface Quick Start Guide 2 3 2 1 Introduction Alarms Batch run BufferPrep Chromatogram Curves Method MethodQueue UNICORN concepts 2 Concept definitions This chapter contains explanations and definitions of a number of UNICORN concepts that are used in this manual The concepts are organized in alphabetical order Systems settings or method instructions specify acceptable limits for monitor signals during a separation run An Alarm dialog box will be displayed on the screen if the
171. NICORN Manager you can select if negative retentions should be displayed or not The default selection is that negative retention is not displayed The formula below is used to calculate Sigma n Sigma gt Xymax y i 1 A peak Where e nis the number of data points e x is the volume or time value Xymax 1s the volume or time value at the maximum amplitude value Apeak is the area of the peak Note The peak width for a Gaussian peak is 4 x Sigma The peak resolution is calculated with one of the following three algorithms 1 Vr2 Vra Wb2 Wo1 2 2 Veo Vra Sigma Sigma x 2 3 Vr2 Vra 2 x Who Wha 2 354 Where Vki Whi Sigma and Wj are the retention width Sigma and width at half height of the previous peak e Vp Wh2 Sigma and Wj are the retention width Sigma and width at half height of the current peak Note The Resolution algorithm variable in the Options dialog box in the UNICORN Manager determines which of the three algorithms is used If this variable has the value 1 2 or 3 then the algorithm with the corresponding number in the list above is used The default value is 3 The table below describes how to change the peak resolution algorithm in the UNICORN Manager Step Action 1 e Choose the Administration Options menu item Result The Options dialog box opens Capacity factor formula Evaluation fun
172. Noles Gradient BultePrep Columns Reference Curves ele Runi Run2 iu 3 4 so eC Js T eel A T Levelt Stay Level a Stats LeveL 2 E Sample sieja j Jz lt i I Display tooltip for extended scouting scheme cells Define Cear Al Add Insert Delete Series Edt Variatie Help 13 5 2 Instruction How to The Analysis module perform automated quantitation 13 The table below describes how to set up sample runs to perform automated quantitation Step Action e Select File Open in the Method Editor module e Select a method that has been used for standard runs in the Open dialog box See 13 5 1 How to set up for automated quantitation on page 439 e Click OK e Click the Scouting tab in the Run Setup e Click the Clear All button to clear the scouting scheme e Double click each Quantitation_Type table cell and select Sample for all sample runs Click the Evaluation Procedures tab e Select only the Quantitate_Sample procedure Click the Import button to import the Quantitate_Sample procedure if it isn t dis played on the list Result This procedure automatically integrates the first UV curve with default baseline settings and uses the selected quantitation table to quantitate the sample The amounts and concentrations are then printed Click the Quantitate button Result The Quantitation table dialog box opens Select the quantitation table from the
173. Peak A 70 560 sso 40 5 o 30 I 20 10 00 490 Exarrple result 001 Example resull 002 Exarnple result 003 File nome Select peak Select yawis Select pass Reterbon The list boxes Use the list boxes to select which peak to plot and the units of the x and y axes The command buttons The table below describes how to use the command buttons of the dialog box Command button Description Returns to the Data View dialog box Displays the data in 3 dimensional plot See How to use the 3D Data View dialog box below Prints the spreadsheet Copy to Clipboard Stores a figure for transfer to an external program Save Wizard Settings See How to save the Wizard Settings below Cancel Ends the Multifile Peak Compare wizard ep 297 11 How to edit results 11 8 How to import and compare different runs 11 8 1 How to use the Multifile Peak Compare wizard How to use the 2D Data View shortcut menu 03 0014 90 ep 298 Click the right mouse button in the plot area of the 2D Data View dialog box to open the shortcut menu See illustration below Undo Zoom A wide array of plot presentation options can be found on the shortcut menu Two of them are described below e Select Customization Dialog to open a dialog box which allows further customization of the graph Peak A Customization Nl Pot Subsets Ponts Avis Font Color fa Show Annotations e Select
174. Result The baseline is recalculated Note Select Baseline New Zero Baseline to replace the calculated baseline with a zero baseline 03 0014 90 ep 352 Evaluation 12 The Edit Peak The illustration below shows the Edit Peak Table dialog box Table dialog box Q Edi Peak Table iS x Ele Edt Bamine Integrate Pie Si Example tesult003 1_Uv Example iesit 003 1_UV 01 BASEM 0 100 200 300 wo s00 600 700 woo ma Retention min Peak start min Peak end min rea mAU min 17 13 17 79 19 47 21 65 22 97 25 52 med to deletea The table below describes how to delete a peak in the Edit Peak Table dialog box pea Step Action e Click the Edit peaks icon ma e Click the peak in the curve or in the peak table to select the peak e Right click and select Delete Peaks from the shortcut menu or e Select Edit Delete Peaks Result The peak is deleted and the remaining peaks are renumbered e p 353 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks How to add color The table below describes how to add a fill color and a pattern to a peak in the to a peak 03 0014 90 e p 354 Edit Peak Table dialog box Step Action e Click the Edit peaks icon M e Move the cursor over the peak you want to edit Result The cursor is changed into a larger arrow e Click to select the peak e Right click and select Fill Peak from the shortcut me
175. Standard Watch conditions Actions when a Watch condition is met How to enter set tings for Delta_Peak and Delta_Base The Delta_Peak setting 03 0014 90 ep 168 Condition Explanation Equal 1 Air detected Note To use the Watch_AirSensor instruction for air sensors the Alarm_AirSensor setting must be disabled The table below describes possible actions when a watch condition is met Instruction Effect Block name Calls the named block Pause Hold Pauses or holds the method Continues the method if paused or held Ends the current block and return to the point from which the block was called End_method Ends the method Ready Indicates that the next step in a MethodQueue may start Permanent settings Permanent settings for Delta_Peak and Delta_Base are entered with the WatchPar instruction for example WatchPar_UV WatchPar_Cond under System Settings in the System Control module see the Administration and Technical Manual Temporary settings Temporary settings that apply only for the duration of a given run can be entered in the Instructions field of the Instruction box in the Text Instructions editor Select Alarms amp Mon and then WatchPar The Delta_Peak setting helps the software to detect valleys peaks and peak maximum and to ignore noise in the chromatogram The Delta_Peak value should be set e large enough so that signal noise does not activate the condit
176. To use the automatic Calculate baseline function e To create a baseline based on a blank curve e To use a Zero baseline e To reuse an existing baseline The Calculate baseline instruction provides automatic calculation of the baseline In most cases the measurement is very accurate The calculation can be performed using the Morphological algorithm or the Classical algorithm A blank curve can be used as the baseline for peak integration e You can use a blank curve with the same chromatographic conditions as the corresponding sample or e You can subtract the blank run from the source curve and then perform peak integration on the resulting curve with the Calculate baseline instruction Note In addition to blank run curves it is also possible to select any curve from the current chromatogram as the baseline e g an edited baseline To use a Zero baseline means that there is no baseline subtraction at all To reuse an existing baseline for the selected curve is the default alternative whenever there is an existing baseline available The option Correlated baseline is selected if this is the case e p 329 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration 12 1 2 How to perform a peak integration How to performa The table below describes how to perform a basic peak integration peak integration 03 0014 90 e p 330 Step Action Open a result file in the Evalua
177. Update by Average may be used if you want to increase the precision of the calibration curve by performing several runs at each level The Replace option means that the old point representing the average of the old points at this level will be replaced with the new point shown in green The data for the old point can then not be recovered Update by Replace may be used to simplify the process of renewing the calibration curve before each analysis Instead of manually producing a new quantitation table you may renew an existing table by running all standard levels again and updating the table with Replace The old data will then be deleted The illustration below shows the Update Report dialog box r General User defaut Intemal standsed peak Date 4 11 2002 intemal standaed amount Last updated 4 11 2002 Reference peak Table Ext_Std_Ribo Nominal reference retention Injection volume Not used Update reference retention Absolute retention and Highest peak m dmum Retention Checked area Deviation Updated arca Averaged Compot eter Cheeta UmAbsat matt mata Ribonuclease A_ _ 0 0614 28 4703 8 87 40 7390 55 5730 5 1077 14 0643 40 8004 2 Out of limit 1 Features The list below describes some features of the dialog box e Components that will not be updated are shown in the column Updated area or Updated ratio if an internal standard is used with the text Out of limit e The column Avera
178. _Conditions_BP 0 00 Base SameAsMain R 0 00 BufferPrep_pH 7 000 BufferPrep_pH 0 00 Buffer alveAl A11 Buffer_Inlet 0 00 Flow 5 00 i Flow_rate ml min 0 00 Gradient 0 00 Start_ConcB B 0 00 base 0 00 End_Block 0 00 End_Block To extend the length of a block without performing any other operation set the breakpoint of the End_block instruction appropriately for example as in the illustration below Equilibration 0 00 Base SameAsMain 4 00 End Block 0 00 End Block WX How to view the accumulated method time or volume How to edit methods 6 The Log Format window in the Method Editor shows the accumulated method time or volume for the current method The accumulated time volume is an approximation and does not take into account time or volume for Watch blocks Wash commands or programmed Hold Also it does not compensate for splitter flow The table below describes how to view the accumulated method time or volume Action Select View Log Format or click the Log Format icon Result The Log Format dialog box is displayed Log Format Baze CY 1 000 8Column mi Any Block Flow_Rate Block Column_Pressure_Limit Block Start_Instuctions Block Alaim_Sample_PressureLimit Block Eluent_A_Inlet Block Eluent_B_Inlet Block Start_with_PumpWash_Purifier Block Start_Conc_B Block Column_Equilibration Block AutaZero_UV Block Flowthrough_Fractionation Block Direct_Injection Block Wash_Out_Unb
179. _____ ee eee Actual an ount added Apparent amount added 4m ount of spiked sample Amount of unspiked sample Example If 2 mg of the component of interest had been added to the sample and quantitation indicated an apparent amount added of 1 6 mg the recovery factor would then be 0 8 Reliability Below are some specific factors that determine if the recovery factor result is reliable e A spike amount that is of the same order of magnitude as the sample must be used to maximize the precision e It is assumed that the recovery is the same for both the sample and the spiked sample However if the recovery varies according to the amount of the component of interest the results are unreliable ep 407 13 The Analysis module 13 2 Quantitation overview 13 2 6 General reliability factors for the quantitation techniques 13 2 6 Reliability factors Further informa tion 03 0014 90 e p 408 General reliability factors for the quantitation techniques The following factors are valid for all quantitation techniques except for Standard addition Quantitation requires that the components of interest are completely resolved from all other components in the chromatogram Overlapping peaks will produce unreliable results The peak integration parameters baseline settings must be correctly selected The default settings will be satisfactory in many cases but the integration results have to be checked fo
180. a curve and store as new 245 How to change the size of fraction marks 246 03 0014 90 pii Index How to change the size of injection marks 246 How to change the size of logbook marks 246 How to print active chromatograms 247 How to add annotations 278 How to edit annotation text 278 How to rename 286 How to open several to compare 302 The command File Open to compare 302 How to open several with the File Open command 303 How to display several simultaneously 304 Commands to import curves from result files 305 How to import curves with File Open to compare 306 How to import curves with File Open 309 How to copy curves into a new 310 How to set a reference point 364 Classic algorithm Definition 340 Parameters 340 How to set 340 Shortest baseline segment 341 Slope limits 342 Noise window 344 Missing peaks 345 When to change the Max baseline level 346 How to set Max baseline level 346 Definition 493 How to measure baseline segments 495 How to measure noise level 496 How to measure the slope limit 496 Columns Column prompt in manual instructions 212 How to add a new 529 Normal column parameters 530 How to edit parameters 533 How to delete 533 How to export 533 How to import 534 Concentration levels Levels in quantitation definition 413 Conditional call Description 100 e piii Index Correlation Explanation 525 Curve fit models Linear 521 Linear through the origin 522 Quadratic
181. a run Up to 500 variables can be defined in a single method All variables are listed on the Variables tab of the Run Setup grouped according to the block in which they appear See 6 5 2 The Variables tab on page 125 Parameters defined as variables can be identified in two ways In the Text pane in Text instructions the parameter is given as the default value in parentheses followed by the variable name The illustration below shows an example of this E W 0 00 Block Wash_Out_Unbound_Sample ash_Out_Unbound_Sample 0 00 Base SameAsMain 2 Wash_column_with End_blo H 0 00 Block YolumeFractionation ps H 0 00 Block Linear_Gradient When the instruction is shown in the Instructions field of the Instruction box the VAR button beside the parameter field is displayed in capital letters that is VAR not Var The illustration below shows an example of the Instruction box where UV1 and UV2 are defined as variables and the UV3 position is fixed Variable values can be changed immediately before the start of a method run without using the Method Editor allowing one method to be used for runs under a variety of conditions How to change variable values Breakpoints or gradient lengths How to define new variables How to edit methods 6 To change default variable values you can either e edit the instruction in the Instruction box or e change the value in the Variables tab of Run Setup
182. able toL ora system to be controlled by other users Step Action e Select System Leave Control or e Click the Leave control of system icon Pe e4 Result The Leave Control of system dialog box opens Click the radio buttons to select to leave the system unlocked or locked Enter a password if the system is to be locked Click OK How to discon The table below describes how to disconnect from a system nect a system Action e Select System Disconnect or e Click the Disconnect from system icon PO Result If the system is in view mode e the system is disconnected If the system is in control mode e the Leave Control of System dialog box opens Select to leave the system locked or unlocked Click OK Result The system is disconnected 03 0014 90 ep 60 How to discon nect when quitting How to view or print a system summary General system operations 3 When you log off or quit from UNICORN you automatically disconnect all connected systems A Leave Control of System dialog box will be opened for each system that was connected Note If you disconnect from a system in control mode and re connect to it you may be connected in view mode Another user may have taken control in the meantime You can view and print a total summary of a selected system from the System Table Summary dialog box The table below describes how to view and print an i
183. able indicator D is removed ep 127 6 How to edit methods 6 5 Run Setup 6 5 3 The Scouting tab 6 5 3 The Scouting tab Introduction A scouting scheme is a series of runs where chosen variable values are varied You can define up to 99 runs in a scouting scheme When a method is run with scouting the method is automatically repeated for each selected run in the scouting scheme Typically scouting will vary one or more variables in a series of runs for example flow rate or elution gradient See 7 Scouting on page 175 for instructions on how to set up a scouting scheme and 9 4 How to perform a scouting run on page 216 Note The Scouting tab is available only if the method contains variables Example ve a The illustration below shows a scouting scheme for six flow rates and different pH scouting scheme yajues Reference Curves Evaluation Procedwes Method Infomation Stait Protocol Result Name Frac350 Variables Scouting Notes Questions Gradent BulfePiep Coimns I a a a pa o a ppe Flom Rate Flom Rate trimi 300 a10 320 330 Tomada cade this un cick the ighi mouse bulton ButterPrep BulterPrepsH 7 000 esoo esoo 6 700 a SS aE CE I Display tooltip for extended scouting scheme cells Defne Clear All Delete Insert Add Series Edit Vesiable Help Note The Edit Variable button on the Scouting tab opens the same Edit Variables dialog box that can be acces
184. ak identific click the desired peak in the curves window ation e click the Set as peak button e choose None in the Set As Peak dialog box e click OK replace or adda peak click a peak in the curves window identification e click the Set as peak button e choose a letter in the Set As Peak dialog box e click OK remove a row from the select the row table e click the Delete row button Note If you click Delete row without first selecting a row the first row A is deleted by default How to edit results 11 Step 4 How tose The illustration below displays the Peak Data Selection dialog box lect the Peak Data Peak Data Selection C Percent of total area Parcent of total paak aea Type of peak berets Peak endpant heights Fiaclion lube id Baseline haght T Sigma M Resolution x cock Hep Cee eo The table below describes how to select the peak data Step Action e In the Select Peak Data list select the peak characteristics on the list that you want to include in your comparisons e If available select the appropriate Scouting variables e Click the Next button and proceed to step 5 How to use the Data View dialog box below Note If Media life time study was chosen in the Operation dialog box when the wizard was started 2D Plot is selected in the Data View dialog box e p 295 11 How to edit results 11 8 How to import and compare differ
185. al informa tion How to improve quantitation How to perform External standard quantitation The Analysis module 13 External standard quantitation External standard quantitation is based on the use of a standard prepared in a number of concentration levels A run is performed for each concentration level and calibration curves are produced to show the relationship between amount and peak size for each component The calibration curves are used to quantitate the components in the sample Note The standard should contain known amounts of all the components that are to be quantitated in the sample External standard quantitation can be based on the use of a single standard concentration level but the calibration curve is then limited to a linear through the origin relationship The use of a number of different concentration levels of the standard broadens the range of the calibration curve It also allows the development of non linear calibration curves and improves precision Multiple runs at each level improve precision further The description in this section is based on the use of a standard e that contains two components e which is run at three different concentration levels The table below describes briefly how External standard quantitation is performed based on the use of a standard which contains two components and which is run at three different concentration levels Step Action Perform a run for eac
186. alog box opens If not proceed to step 2 e Select the baseline you want to edit from the list e Click OK Result The Edit Baseline dialog box opens e Click the Set Curve Points icon fe moo Result The cursor is changed into a cross Add a data point e Click the left mouse button to place a new baseline point in the chromatogram Result A new point is created marked by a green square The baseline curve is redrawn as a spline function based on the old and the new points The baseline is guided by the points but does not necessarily pass through them Delete a data point e Double click the data point or e Click the data point to select it and click the Delete button or e Right click the data point and select Delete Point from the shortcut menu Result The data point is deleted and the curve is redrawn Move a data point e Select the data point and drag it to a new position Result The baseline curve is redrawn Click OK Result The Save Edited Baseline dialog box opens ep 349 12 Evaluation 12 1 Peak integration 12 1 5 How to edit the baseline manually Step Action 7 e Confirm the location and type a new name if necessary e Click OK Result The new baseline is saved Edited baseline The illustration below is an example of a baseline before and after editing Before After How to draw a The table below describes how to force a straight baseli
187. an be saved as a layout It is possible to apply saved layouts to other chromatograms All saved layouts are user specific How to save a The table below describes how to save a layout layout Step Action Open a result file Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed Make the appropriate layout configuration within the various tabs View your changes Click OK if you want to return to the chromatogram window to see the applied affects of a given configuration Return to the Chromato gram Layout dialog box to perform further changes e Select the Layout Library tab e Click the Save current layout as button Result The Save Layout dialog box is displayed e Type a name for the layout e Ifyou want the current layout to be the new default layout select the Save as default option e Click OK Result The new name is added to the Saved layouts list e Click OK aoe to applya The table below describes how to apply a layout ayout Step Action 1 Select the Layout Library tab on the Chromatogram Layout dialog box 03 0014 90 ep 242 How to view results 10 2 e Select a layout from the Saved layouts list e Click the Apply selected layout button Result The layout is automatically applied to the active chroma togram window e Ifthe same layout is to be applied to all chromatograms on the Evaluation workspace select the Apply to all chromatogra
188. and BioProcess systems The model system For practical reasons the user documentation is based on a model system that consists of 03 0014 90 ep 14 Refer to other manuals Document struc ture Typographical representations Introducing UNICORN 1 e AKTAexplorer 100 e Strategy E100F400 e Frac 950 Note If you use another system you may find that the descriptions and instructions do not match your system on every point In that case you also need to refer to the user documentation for your specific chromatography system The User Reference Manual does not contain information about the installation procedure or network configuration You will find this information in the Administration and Technical Manual Sometimes you may find it more convenient to refer to the Getting Started with UNICORN guide for a linear step by step instruction how to perform a task Note When you install the UNICORN software you choose which manuals you wish to install You can also install the manuals after the program installation The manual is divided into chapters Each chapter starts with a brief overview that presents the contents and the headings for the sections that the chapter contains Most sections begin with an introduction that summarizes the content Some sections are divided into sub sections A section is divided into blocks of information with separating lines The blocks are identified by a label in the margin This makes
189. and temporary Chromatograms sccceeceeeeeeeeneeseeeeeeeneeeeeees 227 10 3 2 The chromatogram WINdOW wsiaswretvacisonaiene telecasts ce Soar tel aeacncag uated ce mad 229 10 4 How to optimize the presentation of a Chromatogram cssssssseeeeeeeesssseeeeeeeeeees 234 e piii Table of Contents 10 4 1 How to make changes in the Chromatogram Layout dialog boOxX 0004 235 10 4 2 The Curve tab and Curve Names tab cccccccseseeeseseeeeeeeeeeeeeesaeeeneeeneees 236 10 4 3 The Curve Style and Color ta becswinusavetaveencited etc iia tel cela inate mes 238 10 4 4 How to change and fix the ANS eves cccevewsi icone se eebadeva nse Vea ceiabe aes 240 10 4 5 How to save and apply a layOubecs vascacaaiveueieaseundiaci daidsoduexndaieieaoraweeiventeas 242 10 4 6 How to show part of a CUIVEs 21 ccaneancses plapiacidxe cwccsdgietasReuede tagatiadwessea ares 244 10 4 7 How to change the size of Fraction Injection and Logbook marksS 246 10 5 How to print active CHOMAlOSTAINS x crtssar rose titasiente data ae ee 247 10 6 How to create and print reports ccccccesssseseeeeeeeesssseeeeeeeeeeeessseeseeeeeeeseaeeeeeeenees 249 10 6 1 How to create and print a customized report cecceeceeeeeeeeeeeseeeeeeeeeeee eee 250 10 6 2 How to create and print a standard report ccccceccseeeeeeeeeeeeseeeeeeeeeeeeeees 264 10 6 3 How to edit an existing report Toritat cwreniatinre dee Need iit da
190. antitation 13 5 1 How to perform automated quantitation 13 5 2 How to perform automated update 13 5 3 13 5 1 Introduction Basic conditions for the quantita tion table How to prepare the quantitation table The Analysis module 13 How to set up for automated quantitation This section describes how to create a quantitation table for automated quantitation A quantitation table must be produced from standards before samples can be quantitated The list below describes the basic conditions for the quantitation table e The same method must be used for all standard and sample runs e Each level is an alias for a specific concentration of the standard e All runs with the same concentration must be assigned the same level e Level 1 must be selected for the standard with the highest or lowest concentration e The levels must be set in order of decreasing or increasing concentration of the standard The table below describes how to prepare the quantitation table for automated quantitation Step Action e Use the Method Wizard to create a method e Select Autosampler from the Injection Technique droplist in the Sample Injection dialog box Proceed with the following dialog boxes in the Method Wizard and click the Finish button on the last dialog box Result The Run Setup opens Click the Scouting tab See illustration below e Select the Quantitation_Type variable from the Scouti
191. any of the recipe values are unfeasible e Type a name in the dialog box e Click OK Result The new recipe is added to the recipe list Note It is recommended that restricted access be given to the right to edit global recipes The recipes are either globally available to all users or only personally available It is best not to edit the globally available recipes unless you save the changes under a new recipe name since other users may not appreciate the changes Buffer concentra Use buffer concentrations that are 2 4 times higher than the concentration that is Hon used in the normal preparation When BufferPrep is used the buffer will be diluted 2 10 times depending on the amount of acid base that has to be used to reach the desired pH value Up to five different buffering components can be selected To prevent a too high ionic strength the sum of the concentrations for all selected buffers should be between 0 03 M and 0 2 M typically 0 1 M 03 0014 90 ep 538 How to create and edit BufferPrep recipes E How to select the The useful pH range depends on the pKa value The table below describes how to pH range determine a pH range based on the pKa value Step Action 1 Choose Edit BufferPrep Recipes and click the New button 2 Click the Buffer substance button in the New Recipe dialog box Result The Define buffer substance dialog box opens Define Buffer Substance x Name Testsalt2 x i J Dete
192. ar size calculations the standard contains components of known molecular size Standard run A chromatographic standard run of a specific concentration level of a standard How to install the The table below describes how to install the Analysis module Analysis module Step Action Close all other applications Insert the installation CD in the CD drive Open My Computer ep 391 13 The Analysis module 13 1 General information about the module 03 0014 90 ep 392 Action Double click the CD drive icon Result The file window opens Double click Setup exe Follow the instructions on the screen Remove the CD after the installation is complete Note See the license agreement for information on the legal aspects of the installation The Analysis module in a network One or several computers in a network may have the Analysis module installed The module does not need to be installed on all network computers that run UNICORN All installations must be made in accordance with the license agreement 13 2 Introduction In this section The Analysis module 13 Quantitation overview Quantitation is used to determine the amount or concentration of components in a sample This section is an overview over quantitation in general and the four quantitation techniques that the Analysis module provides The section also contains information about the reliability of quantit
193. ar with the contents of those chapters before you begin with this chapter In this chapter This chapter contains these sections Topic See General information about the module 13 1 Quantitation overview 13 2 How to prepare for quantitation 13 3 How to quantitate the sample 13 4 Automated quantitation 13 5 How to measure molecular size 13 6 03 0014 90 p 388 13 1 Introduction Module functions Module menus Quantitate Molecular Size The Analysis module 13 General information about the module This section is an overview of the Analysis module including e Definitions of terminology that will be used in this chapter e A description of how to install the Analysis module e A description of the new procedure instructions that become available when the Analysis module is installed The Analysis module is an optional extra module that adds functionality to the regular UNICORN Evaluation module Basically the Analysis module is used e to determine the absolute quantity or concentration of a component e to determine the molecular size of a component The Analysis module is accessed in the Evaluation module After the installation two new Evaluation module menus are added e Quantitate e Mol Size Note The menus are only available when a result file is open in the Evaluation module The Quantitate function provides a wide range of techniques for quantitative analysis e
194. are Correction factors the measured pH deviation should be added to the old factors 03 0014 90 ep 544 Introduction In this appendix Method examples F Method examples This appendix contains practical method examples that can be applied in typical situations The examples cover three different topic groups e Watch instructions e Messages e Quality control Watch instructions allow the progress of a method run to be determined by the events during the method run for example start collecting fractions when the first peak elutes or equilibrate the column until the eluent conductivity has reached a given value This is facilitated by the Watch instructions The system strategy includes Watch instructions for each monitor defined in the system These instructions are used to survey method runs and instruct the system to call a specified block or an instruction when a particular monitor signal meets a given condition As long as the condition is not met the block is not activated Messages can be used in a method to provide information to the operator but also for interaction between the system and the operator A Quality control procedure in a method can be used to ensure that the quality of the results remain consistent in a series of runs This appendix contains the following sections Topic See Simple equilibration Equilibration with simple safeguard Equilibration with extra safeguard Collection o
195. arger drift in retention value Click Identification Settings Result The Identification Settings dialog box opens r Peak identification vj and Highest peak maximum 7 Absolute retention Identify peaks on Window width as Relative Absolute r Reference peak for relative retention Component z Window jp min Cancel Help See How to identify peaks within a window below Select Relative retention on the Identify peaks on droplist See Abso lute and Relative window width below Scroll down the Component menu and select the component to be used as the reference peak How to identify peaks within a window Absolute and Rel ative window width The Analysis module 13 Step Action 5 e Type the window width for the reference peak an absolute value Note Set the width fairly wide to accommodate a larger drift in the retention value Make sure that there are no other large peaks within the window e Click OK Result A column for the relative retention is added in the peak table Ret Ref The column displays the value of each component relative to the retention value of the reference component This reference component is marked Ref in the Window column The Window column shows the window width for each peak expressed as a per centage of its relative retention value Quantitate must be advised of how the peaks are to be identified if any of the windows inc
196. arget vol WINCORN LAB 21 ExampleResult GF001 AtAl 1 0000 1 6478 A243 1 5300 23716 ASAS 1 7800 09107 PAE ESE F Show al fractions F Show al columns Delete Delete at Brint Export cee te ep 283 11 How to edit results 11 5 How to pool fractions 03 0014 90 ep 284 Step e Click the Show all fractions checkbox to display the individual fractions instead of fraction ranges for the pools e Click the Show all columns checkbox to display all the information columns from the Pool table Possible actions in the Pooling Protocol To delete a single pool e select a pool and click the Delete button To clear the whole protocol e click the Delete all button To print the protocol on the default Windows printer e click the Print button to print the protocol on the default Windows printer To export the protocol e click the Export button to save the protocol in one of the following formats text txt Excel xls HTML htm XML xml Note The protocol is automatically saved for the user The pooling protocol will be available again when the user starts UNICORN the next time e Click the Close button to close the Pooling Protocol dialog box Result If the protocol was exported or only edited the dialog box will close If the protocol was printed a dialog box will open asking if you want to delete the list and start a new How to edit resu
197. asas varsandi cd waetiaiamaueaunda eave hunan 104 6 2 5 How to rename method DlOCKSa cutter vasa wna eeadae ote 106 6 2 6 How to find copy and move method DIOCKS ceccsecseseeeeeeeeeeeeeeneeeneees 107 6 2 7 How to import method IBIOCKS 3 5 axncsussuets veces tanecs uae etndantucouttouatssoeeemeneaenns 109 6 3 Method IMSETUGH ONS ccc sence alee vats ee late ae at aat aaaeeeaa aeaa anabi n ae 111 6 3 1 How to read method instructionS ssssssissrerernrrsrsrrnrrrrrrrrrrnrnrrnrsrsrrereena 112 6 3 2 How to add method instructionS ssssssnesrenennrrsnsrrrrrrrrrrrrrnrsrrnrersrererena 113 6 3 3 How to delete method InstructiONns ccccccecceecceeeeeeeeeeeeeeeeeeeeseeeeeseeeeeseees 114 6 3 4 How to change or move method inStrUCtiONnS ccccceecsseseeeeeeeeeeeeeeeneeeeeees 115 6 4 How to use method Varlables ricci cxcce toyaataserareriann wicinalandenndereaaaghavaumteneeanyenrsncs 118 Ghee ATTESTA T a EEE E TEET A EEEE 122 6 5 1 Overview of Run Setup ssssssssssrrsrssrrsrrsrrsrerrrrrsrtertrrtersrrtrrsrttrarrr trent trne 123 Bn EG Varia DIGS tabenan a a E a a ETa 125 6 5 3 Th Sco ting 1 is ares ete ln E EEE E 128 6 5 4 The Q estions tabes moricei iiep a E E E E e EEEa 129 6 5 5 The Gradient tabidir e a OEE aE a ANEA a EE Ria 133 6 5 6 The Notes tab irienn a aaa a r E E aa Aae r Eara ariaa 135 6 5 7 The Evaluation Procedures tab snssnsruierernrnrnsrsrrnrrrrrrrrrrsrnrrnrersreeree
198. ate button to create the backup file and store it in the selected location Note You can click the Information button to see which information files will be included in the backup file How to restore The table below describes how to restore the system data from a backup file located the system data example on a rescue diskette Action If the backup file is located on a diskette insert the diskette into the computer Choose Administration Create Restore Backup in the UNICORN Manager to display the Create Restore Backup dialog box Create Restore Backup x f Action C Create Restore C UNICORN Backup Browse Global Files Global BufferPrep Recipes Global Columns Global Evaluation Procedures Global Report Formats Personal Files System Files U System Setup ser Setup Information Restore Cancel Help __Pesoe Cancel Her 03 0014 90 e p64 General system operations 3 Step Action 3 e In the Action field select the Restore option e Click the Browse button to select the folder where the backup file is located Note Select A if the file is located on the diskette e In the Items field select which information to include from the backup file e Click the Restore button to restore the system definitions Note You can click the Information button to see which information files are included in the backup file e p65 3
199. ate00 t 1_CresiecCune How T a the In cases where you have created a curve and not selected the Spline through option curve throug you may want the curve to pass through some of the points that are outside the oints p created curve The table below describes how to force the curve through these points Step Action e Select the curve point immediately before the curve point you want to connect to e Click the Draw straight to next point button Result The curve is adjusted so that it is drawn as a straight line between the two points 03 0014 90 p 376 12 2 5 Introduction How to create a Fraction Histo gram Evaluation 12 How to use the Fraction Histogram The Fraction Histogram dialog box in the Evaluation module can be used to create a curve for the average fraction absorbance The table below describes how to create a Fraction Histogram curve Step Action Select Operations Fraction histogram Result The Fraction histogram dialog box opens e Select the desired UV curve Note The fractions curve should already be selected on the middle list If you have previous pooled fractions and created a pooled fraction curve select the desired fraction curve e Click OK Result The average fraction absorbance values are displayed as a new curve in the chromatogram e p 377 12 Evaluation 12 3 Automated evaluation procedures 12 3 Introduction In this secti
200. ated logout will not happen while a MethodQueue or a Scouting scheme is operating When you log on again after leaving the system locked with a process running or after an automated workstation lock you will be asked to unlock the system Log on to the system Result The System Unlock Confirmation dialog box opens Type your login password or the password that the system was locked with in the Password text box Click OK Note You can connect in view mode only without providing the password You can unlock a system that has been locked by another user if you have the correct password You may still be able to unlock a system even if you do not have the password Any user with Unlock locked systems authorization can override another user s lock by entering his or her own logon password However it is recommended that this authorization is limited to only a few users How to quit UNICORN General system operations 3 UNICORN will still be open after you have logged off To close the program you must log in again and quit UNICORN you cannot quit the program if you are not logged in The table below describes how to do this Step Action e Select the File Quit Program menu command in the UNICORN Man ager module or e Click the close icon in the top right hand corner of the program window Result A confirmation box opens Click Yes to confirm that you want to quit A Warning opens if y
201. ates a histogram from any non fraction curve source curve 1 and a fraction curve source curve 2_frac and stores the result in the target curve position If source curve 2 is not a fraction curve a run time error will occur The Y axis of the target curve position will be the same as that of the first source curve Performs a mathematical integration of the source curve and stores the res ult in a Result curve This instruction is not the same as Peak integrate which performs a real peak integra tion Pools fractions from the source curve and stores the result in the target curve position The fractions are pooled from the first selected fraction to the last selected fraction If the source curve is not a fraction curve or First or Last is not an existing identification a run time error will occur Multiplies the retention of the source curve by the Multiplication factor and stores the result in the target curve position Shifts the retention of the source curve by the Shift factor and stores the result in the target curve position Simulates Peak Fractionation ep 505 B Evaluation functions and instructions B 4 Procedure instructions 03 0014 90 e p 506 Instruction SMOOTH_AR SMOOTH_MA SMOOTH_MEDIAN SMOOTH_SG TDIV Description Smooths the source curve with an autoregressive filter and stores the result in the target curve position The Filter parameter decides the strength of the
202. ation This section contains these sub sections Topic See General information about quantitation 13 2 1 External standard quantitation 13 2 2 Internal standard quantitation 13 2 3 Standard addition quantitation 13 2 4 Recovery calculation 13 2 5 General reliability factors for the quantitation techniques 13 2 6 e p 393 13 The Analysis module 13 2 Quantitation overview 13 2 1 General information about quantitation 13 2 1 Introduction About quantita tion Quantitation steps 03 0014 90 ep 394 General information about quantitation This section is a brief presentation of the quantitation techniques that the Analysis module provides The section also contains an outline of the steps in a quantitation and the procedure instructions for quantitation that are added when the Analysis module is installed Most quantitation techniques use peak integration data from standards to produce calibration curves These curves show the relationship between the amount of the components of interest and the peak sizes at different concentration levels of the standard The relationship can be linear quadratic or point to point Quantitation is usually based on a number of test runs using a standard at several concentration levels The amount and concentration of the component s of interest in the sample are then determined from the peak size of the component using the calibration curve Note Quantitation
203. ation process The sample is spiked with a known amount of the component of interest The amount in the spiked sample is then determined from a calibration curve and is compared with the amount in an unspiked sample The recovery can only be determined for one component each time Analysis proced The table below describes the new procedure instructions for quantitation that eer eons become available when the Analysis module is installed Instruction Description QUANTITATE The instruction calculates the concen tration and amounts in the sample from a quantitation table Amount and concentration columns will be added to the peak table UPDATE The instruction updates a quantitation table with new data from one standard concentration level The default Limit value of 12 5 will be used The quantitation table will not be updated if the peak area or peak height of the new and the previ ous results differ more than the Limit value Note Either peak area or height is se lected for the Limit value Default values The DEFAULT value for the injection value will be taken from the injection volume reported by the Autosampler A 900 from the method DEFAULT can only be used when the injection is performed by the autosampler The DEFAULT value for the concentration level for the standard will be taken from the level entered in the QuantitationData instruction in the method 03 0014 90 ep 396 13 2 2 Gener
204. ault the block that is currently selected in the Text window is automatically selected in the dialog box Enter the new name in the New name field and click Rename e If needed repeat step 3 for other blocks e Click Close Note If the block you renamed is called in a Block or Watch instruc tion the block name in these instructions will be changed automat ically How to edit methods 6 6 2 6 How to find copy and move method blocks Introduction By using the Edit options in the Method Editor you can find copy and paste and move blocks within a method How to find text The table describes how to find text strings in the method text strings in the method text Action Choose Edit Find in the Method Editor or right click an instruction or a block in the Text window and select Find Result The Find dialog box is displayed Find what I Match whole word only f Pe H Cancel I Match case e A e I Search from top of document e Enter the text you want to search for search direction and case matching criteria e Click OK How to copy and The table describes how to copy a block paste a block Step Action e Right click the block you want to copy e Choose Copy e Right click the instruction line just above the point where you want the block to be pasted e Choose Paste Result A dialog box asks if you wish to rename the pasted block Click Yes to rename t
205. aved method files and result files You can also preview your result files to identify the correct file before you open it You open a method file in the UNICORN Manager module Click the file in the Methods window to select it and e choose File Open or e right click the file and choose Open from the short cut menu or e double click the file Result The file is opened for editing in the Method Editor module Note A method file cannot be opened on two workstations simultaneously You can open a result file in the UNICORN Manager module Click the file in the Results window to select it and e choose File Open or e right click the file and choose Open from the short cut menu or e double click the file Result The file is opened for editing in the Evaluation module The table below describes how to open a result file from the File Navigator in the Evaluation module Action Click the Files tab Step 1 e p69 4 Files and folders in UNICORN 4 2 How to open and preview files Quick View 2 e Locate and double click the result file Se Oe ooooa aeSS Recent Runs Files Find Example Result E ExampleResult GF ai Example Result001 ai Example Result002 ai Example Result003 ai Example Result004 ai ExampleResult GFOO1 L ExampleResult GF002 Result The result file opens Note The File Navigator is opens by default in the Evaluation module
206. average of the y value For the molecular size model Linear log Mw ex is the average of the logarithms of the molecular sizes Explained variance provides a measurement of how much of the variation in the data points xy pairs is due to the model The remaining variation can be attributed to noise i e random errors or to the fact that an inappropriate model has been selected This makes it possible to use the explained variance value for model selection e g to decide if a quadratic model fits the data better than a linear model This would be confirmed by a higher explained variance value e p 525 C Curve fit models and statistics C 2 Statistics Explained vari ance calculation Undefined value for explained vari ance 03 0014 90 e p 526 Note The explained variance is not calculated for curve models drawn through the origin The explained variance is equal to R adjusted for degrees of freedom The illustration below shows the mathematical model SSresduais n k D Explained variance 100x 1 SStetai n T D Where SSeaduais gt RY Residual Sum of Squares i 1 Sai gt x y Total Sum of Squares i l ej is the average of all y values 3 is a function value using the fitted model For example Ax Bx C en is the number of points xy pairs k is the number of x terms in the model For example 1 for Linear and 2 for Quadratic You can only obtai
207. ay the BuffPre_pH meter right click and select Properties Select BuffPre_pH on the Run Data Groups panel and click the OK button e Select Gradient in the Instructions list e Type 100 in the Target parameter box 0 in the Length parameter box and click Execute Result The gradient instruction is added Check the pH reading when it is stable at 100 See How to change the Correction factors below How to change If the readings described in the instruction above are acceptable at both 0 and en 100 the Correction factors do not need to be changed If the Correction factors do not produce an acceptable result they must be adjusted in the Method Editor module The table below describes how to change the Correction factors Action Choose Edit BufferPrep Recipes Result The BufferPrep Recipes dialog box opens Select the recipe from the Recipe droplist and click the Edit button Result The Edit Recipe dialog box opens ep 543 E How to create and edit BufferPrep recipes E 2 How to edit a BufferPrep recipe Action Click the Correction factors button Result The Correction Factors dialog box opens e Type the deviation at 0 and 100 Example If the pH is set to 7 0 and the actual pH is 7 1 the Correc tion factor is 0 1 If the actual pH is 6 9 the Correction factor is 0 1 e Click OK e Click the Save button or the Save as button to save the recipe Note If there already
208. ayed but only inserted into the logbook e Authorize i e the message will require a signature from the user before the user can interact with the system again e Select a sound on the Sound menu if desired e Click the Insert button Note If the Message instruction is inserted in a conditional block it will only be displayed if the conditions of the block for example a Watch is fulfilled Note All messages are erased when the system reaches the End status This also includes Authorize messages Set_Mark instructions are useful text messages They can be used e to insert manual notes for example when a problem occurs in a run e to highlight certain stages in a method Set_Marks differ from Messages in that they are inserted into the chromatogram at set points as well as into the logbook during a method run How to edit methods 6 Example of a The illustration below shows an example where Set_Marks are used to highlight Set_Mark the start and end of fractionation in a method Volume_Fractionation 0 00 Base SameAsMain _ 0 00 Set Mark Fractionation starts 0 00 Fractionation 18mm T ubeT yp EluateF rac O Etuate Frac_Size mi NextT ubejitEluateFrac_StanAt Volume 0 00 End_block 0 00 End Block W 0 00 Block Linear_Gradient 0 00 Block Gradient_Delay W 0 00 Block Fractionation Stop Fractionation Stop 0 00 Base SameAsMain 0 00 FractionationStop 0 00 Peak_FracStop Set_Mark F
209. baseline is created Choose Integrate Calculate Baseline to open the dialog box Evaluation 12 Test your paramet The best way to optimize the baseline is to change the baseline parameters step by er changes Shortest baseline segment step and then check the resulting baseline after each change When the desired effect is accomplished it is best to go back and try a parameter value in between the two last settings to avoid an unnecessarily low or high value How much the values should be changed depends on the cause of the peak integration problem The table below is a general guideline Baseline parameter Recommended initial change Shortest baseline segment 20 50 Max baseline level Usually not necessary to adjust Slope limit 25 50 Note If necessary click the Default button to restore the default values If a too high Shortest baseline segment value is set short curve segments between peaks in the middle of the chromatogram are not identified as baseline segments The calculated baseline does not follow the source curve see below ep 341 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm The Shortest baseline segment value is decreased by 50 in this example Slope limit A changed Slope limit will often improve the baseline calculation The Slope limit sets the maximum slope of the curve to define when a peak is recognized A too high Sl
210. bed below will only affect the selected part of the curve e p 361 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks Step Action If desired change the integration parameters Reject peaks e Choose Integrate Settings Result The Reject Peaks dialog box opens e Change the settings as desired and click OK Skim peaks e Choose Integrate Peak Skim Result The Peak Skim dialog box opens e Select the Skim Peaks checkbox and type a ratio e Click OK e Choose Integrate Peak Integrate Result The selected part of the curve is peak integrated based on the changed parameters 03 0014 90 ep 362 Evaluation 12 12 1 8 Measurements Introduction It is possible to determine the coordinates of any point on a curve and to obtain values for retention and peak height This is a useful tool for many other functions such as for measuring the parameters used in baseline calculations Measurement op Coordinates can be obtained in two ways tions e Through direct measurement e From peak table data How to make dir The table below describes how to make direct measurements in a chromatogram ect measurements Step Action Right click in the chromatogram and select Marker Result A vertical line is set in the chromatogram A text box in the top left corner of the chromatogram displays the X axis and Y axis values of the curve at the
211. box Evaluation 12 Morphological al The parameters for the Morphological algorithm are gorithm paramet ers e Structure width e Noise window e Minimum distance between points Structure width Structure width determines the length of the straight line that follows the chromatogram The default value is set at the widest peak in the chromatogram multiplied by 1 5 The illustration below is an example of how a morphological baseline follows the peaks at the different levels in the curve ep 337 12 Evaluation 12 1 Peak integration 12 1 3 How to optimize the baseline with a morphological algorithm The correct struc ture width settings Noise window 03 0014 90 p 338 Too low settings Too low Structure width settings can result in a baseline that reaches too high up in the peaks of the curve Sometime a wider peak is not recognized because it contains a cluster of smaller peaks The Structure width is then set to a value according to the largest width of the identified narrower peaks and must be increased Too high settings Too high Structure width settings mean that narrower peaks especially in fluctuating curves are not properly followed This happens when an artifact in a curve is identified as the widest peak by the morphological algorithm and then is used to set the default Structure width value The illustration below is an example of baselines using the default morphological algor
212. build a complete procedure step by step The procedure instructions are described in B 4 Procedure instructions on page 504 The table below describes how to create a new procedure with instructions Step Action Choose Procedures Edit New Result The Procedure Editor opens in Edit mode e Select an instruction from the Instruction list e Type the appropriate parameters in the Parameter field e Click Insert Evaluation 12 Step Action Repeat step 2 until the procedure is complete Choose File Save Type a procedure name and click OK Click the Close button in the Procedure Editor ep 381 12 Evaluation 12 3 Automated evaluation procedures 12 3 2 How to edit a procedure 12 3 2 Introduction How to edit a procedure Descriptions of the procedure in structions 03 0014 90 e p 382 How to edit a procedure Evaluation operations are represented by instructions in the Procedure Editor dialog box The instructions can be modified to suit other specific evaluation needs and be saved for later use This section describes how to use the Procedure Editor to edit a procedure The table below describes how to edit an existing procedure Step Action Select Procedures Edit Open Result The Open Procedure dialog box opens Select the procedure from the list and click OK Result The Procedure Editor opens in Edit Mode Select an instruction in the procedure window
213. ccess to the modules described in this chapter Only the available modules will open when the program is started In this section This section contains these sub sections Topic See UNICORN Manager 2 2 1 The Method Editor module 2 2 2 The System Control module 2 2 3 The Evaluation module 2 2 4 Search functions 225 Help functions and manuals 2 2 6 Snapshots 22 7 03 0014 90 ep 24 2 2 1 UNICORN Manager UNICORN concepts 2 Introduction The UNICORN Manager is mainly used for file and folder administration The UNICORN The module is divided into two windows the Methods window and the Results Manager windows SP UNTLURN Manager mis w Adneetrstor Teos widow Heo Prce Facer UXICCRH Dena DKE Method Fic UNDCCRN Dena SOB Method Fie UNDOC RN Dena SEB Method Fie window See the illustration below 4 30 2003 BC Lt 2 26 2003 48 24 BOF IES Zi 4 Eang t Res RICI Evange Res RIC Jeme t Resak Erang Resukoct Exave eResu EFCOL Prey Foy User Fold Scouting Foor Result Ae Resik e Result Fie Resi Fie Resik Fie Resik Fie 2 2 2003 03 23 226D 18 25 AZO 4s AZ MOE SS ANODE S2 ASCO 52 ASIDE O14 HISILO MIS a ia eT The Methods win The Methods window contains all the saved methods MethodQueues and all the dow folders containing methods that are available to the user See the illustration below i Methods c WDelaut Prev Fokler User Folder User Folder User Folder
214. ch MethocBate C Otha Note If the column is changed you will be asked if the linear flow rate or the default flow rate should be used If the linear flow rate cannot be used due to the max flow rate of the system or new column you will be advised that the max flow rate will be used instead e p 163 6 How to edit methods 6 6 How to use selected method instructions 6 6 7 Gradients and eluent concentrations 6 6 7 Introduction Parameters of the Gradient Example of a Gradient instruc tion How to form a step gradient in struction Breakpoints for gradients 03 0014 90 ep 164 Gradients and eluent concentrations Gradient instructions are given in the Text Instructions editor of the Method Editor This type of instruction defines gradients and immediate changes in eluent concentration The table below shows the two parameters of the Gradient instruction Parameter Description Final eluent composition expressed in eluent B Duration of the gradient The starting point for the Gradient is always the current eluent composition The instruction can be read as follows form a Gradient to reach Target after Length Example of instruction 10 00 Gradient 50 B 20 base The example instruction above forms a gradient to 50 B Target starting at breakpoint 10 with duration 20 method base units Length The example instruction will finish at breakpoint 30 If the current
215. cific pH by setting correction factors The table below describes how to fine tune the recipe with correction factors o N n A Ow N lt 2 ge 10 Action In System Control select Manual Other Select BufferPrep Recipe and Recipe Name Click the Execute button e Set the pH in the instruction BufferPrep_pH in group Pump e Click the Execute button e Set the flow rate to be used during the run in the Flow instruction e Click the Execute button Check the pH reading when stable Allow at least 30 ml of eluent to pass through before expecting a steady pH reading e Change to 100 B by setting the Gradient instruction in Manu al Pump to 100 for Target and 0 for Length e Click the Execute button Check the pH reading when stable at 100 B e Ifthe readings are acceptable at both 0 and 100 the correc tion factors do not need to be changed e Ifthe readings are not acceptable click the Corr Factors button in the BufferPrep tab in the method Enter the deviation at 0 and 100 e g if the pH is set to 7 0 and the actual pH is 7 1 enter 0 1 Enter 0 1 if the pH is 6 9 Note If correction factors already exist the measured pH deviation should be added to the old factors Save the method Note When changing the correction factors for the recipe selected in the method the recipe with the same name on the list of all BufferPrep recipes is not affected The changes will only apply in the sp
216. columns for a method not selec ted from a tem plate Column definition Update paramet ers Pump Methodbase instruction 03 0014 90 ep 156 The table below describes how to select columns for a method not selected from a template Action In the Instruction box of the Text instruction dialog box mark the Other Base instruction e Select the required column from the drop down list for the Column parameter e Click the Var button to define the Column parameter as a vari able This is an optional but recommended step that will make it easy to change the column selection for different runs e Enter a variable name and click OK e Click Yes to confirm A column definition can be chosen and defined as a variable even if the base for the block is set to volume or time Parameters in the column definition will then be used for linear flow rate and column performance calculations Recommendation A selected column definition applies locally within the block for which it is selected and is not transferred to other blocks We strongly recommend that the column definition be selected for the main block If you want parameters for example flow pressure and averaging time to be updated when you change the column you must define these as variables Volume or column volume base is calculated from the flow rate of the SystemPump or the SamplePump selected with the instruction Pump Methodbase If no P
217. counters e Select the warning to display the parameters Counters show the remaining time or number of operations before the next maintenance warning See the illustration below Maintenance manager a xj Information Warning Info Warning a System Air sensor Auto Sampler Frac 950 pH Cond Pump Pump P 950 a Uv Ay Lamp check Valvel Valve 2 Valve 3 Valve 4 Valve 5 Valveb Lamp check arning frequency Every 6 months or every 500 method runs whatever occurs o Next time out 2002 10 11 No of method runs completed 0 Les q oo 03 0014 90 p 466 System maintenance and error reporting 15 How to reset the The table below describes how to reset the maintenance warning counters counters Step Action e Select System Maintenance in the System Control module to open the Maintenance manager dialog box e Click the Warning tab e Select the warning you want to reset on the component list e Choose Warning Edit or e Right click and select Edit on the shortcut menu Result The Maintenance manager dialog box changes into edit mode and the text boxes are activated e Type new text if necessary e Click the Reset button Result The Reset parameters dialog box opens Reset parameters t xj No of method runs completed 0 EEN Next time out 2002 10 11 Reset OK Cancel Help e
218. cribes e some of the contents of the run documentation e how to view and print the run documentation e how to save the method from the run as a new method How to view and The table below describes how to view and print the run documentation print the run docu mentation e Choose View Documentation in the Evaluation module e Click the view Documentation icon Result The Documentation dialog box opens See further information about some of the tabs below e Click the Print button Result The Print dialog box opens e Select the documentation items you want to print and click OK The tabs of the The table below describes the contents of some of the Run Documentation tabs Documentation dialog box l Documentation tab Contents Variables The Variables tab lists the parameters that were used during the method run Scouting The Scouting tab displays the whole scouting scheme with the values for the current result file displayed in yellow cells 03 0014 90 ep 270 How to view results 10 Documentation tab Calibration Logbook Evaluation Log Method Information Result Information Contents The Notes tab displays the notes that you have made at various times during the method run You are also able to type new comments on the Evaluation Notes sub tab Note Click the Find button to search for a specific text string in the Notes The Calibration tab disp
219. cription Solution Stand alone installation Reinstall the strategy as described in If you receive the error message the Administration and technical Strategy file error in a stand alone manual Install selected software installation the strategy file is prob components after the initial installa ably corrupt Hons Network installation Make sure that the computer is logged In a network installation the error onto the network before UNICORN message Strategy file error may ap S started so that the strategy file on pear if you try to create a method for the server disk is accessible a system not physically connected to the computer 03 0014 90 p 478 A 2 In this section Unable to access certain UNICORN func tions Connection prob lems UNICORN access Troubleshooting A This section describes how to solve the following UNICORN access problems e Unable to access certain UNICORN functions e Connection problems Connections are not available System is not available Error message in a network installation You cannot control the system e Run data Connection in System Control displays a NO 1 NO 2 or NO 3 The table below describes an access problem and its solution Problem description Solution UNICORN functions to which you do Choose Administration User Setup in not have access appear grey in the the UNICORN Manager to change the menu and cann
220. ct the Evaluation Procedures tab and click the Rename button Result The Rename dialog box is displayed e Select a procedure from the list and change the name in the Re name item to field e Click Rename Repeat step 2 until you have renamed all procedures required Click the Close button How to edit an The table describes how to edit evaluation procedures in a specific method evaluation proced ure Step Action Select a procedure on the Evaluation Procedures tab and click the Edit button Result The Procedure Editor dialog box is displayed with information about the selected procedure Enter the new parameter values in the appropriate place of the Parameters field and click the Replace button Result The selected instruction in the evaluation procedure is up dated in accordance with the new parameters assigned to it If needed insert new instructions after the currently selected proced ure instruction Do the following 1 Select an instruction type and instruction in the Instructions field 2 Enter the appropriate parameter values in the Parameters field 3 Click the Insert button Result The new instruction is added to the evaluation procedure To remove an instruction from the evaluation procedure select it and click the Delete button 03 0014 90 ep 138 How to edit methods 6 Step Action Select File Save as to save the edited procedure with a new name Click the Close button Selec
221. ct the component to be used Evaluate the amount of a component in the sample How to prepare The table below briefly describes how to prepare for the quantitation for the Standard addition quantita i tion Step Action Perform a sample run with the unspiked sample and a run with the spiked sample Open one of the two result files Use File 0pen Curves to copy the second curve to the opened result file 3 Peak integrate the sample curves to produce the peak tables for the unspiked and the spiked samples Note The sample curves must use the same X axis base unit Time 5 is the recommended unit for highest reliability e Check that the integrations are correct e Optimize the peak integration if necessary Select File Save to save the peak table 03 0014 90 ep 432 How to select the component and identify the sample peaks The Analysis module 13 The table below describes how to select the component to be used for the standard addition and how to identify the sample peaks Step 1 Action Select Quantitate Standard addition Result The Standard Addition dialog box opens 1319GLewel 1 to 4004 1_UV1_280nenO1 PEAK 1BELevel to 4004 1_UV1_ 200m PEAK frg id106Level 1 to 40M4 UV 220m0 PEAKI id 16Level 1 to 4004 1 UVI 200E PEAKI iR Cancel Hee Select the chromatogram that contains the peak table for the un spiked sample in the Source chromatogram droplist
222. ction on The criteria for peak identification Example RETEN TION Order number The sequential order number of the peak Example 1 03 0014 90 ep 558 Method examples F Parameter Description Peak table parameter The peak table parameter that will be tested by the control instruction Example RETENTION Less than Values less than the parameter value will be out of the acceptable range Example 10 Greater than Values greater than the parameter value will be out of the acceptable range Example 11 The action the system will take when the controlled value is out of the acceptable range Example PAUSE Message text Free text message that is displayed when the con trolled value is out of the acceptable range Example Retention out of range Note All values must be included before the instruction can be inserted How to add the The table below describes how to add the quality control procedure to a method quality control procedure to a f method Step Action e Open the method in the Method Editor e Click the Run Setup icon E Result The Run Setup for the method opens e Select the Evaluation Procedures tab e Click the Import button Result The Import dialog box opens e Select the quality control procedure you created and saved Ex ample QC_test in the Select field e Click the Import button Result The quality control procedure is added to the available evaluation procedu
223. ctions Introduction You can use the Text Instructions editor in the Method Editor to build your method step by step You can also use the editor to modify instructions in methods created by wizards or based on templates Advanced editing facilities are available when you work directly in the Text Instructions editor This section is a very brief description of this process See 6 How to edit methods on page 92 for detailed instructions Note Each method is written for a specific strategy The function of the method cannot be guaranteed on systems having other strategies When do I use Use Text Instructions when you want Text Instructions f to change selected instructions in the method for example the outlet valve position e to add blocks or instructions for example Watch instructions e to change method instructions to adapt to non standard system configurations e to create new methods for applications not covered by the supplied templates or wizards How to edit Text Open the Text Instructions editor by following the steps in the table below Instructions Step Action Select the Method Editor module and click the Text Instructions icon e Click the Customise Panes icon and select Text and Instruction Box e Click OK Select instructions in the Instruction box in the lower part of the Method Editor and use the Insert Change Replace or Delete buttons All text entries are shown in the Text pane Applicab
224. ctions and instructions B droplist e Select the desired algorithm number described as described in Peak resolution algorithms above in the Resolution algorithm M Global J Run UNICORN in single application mode r Evaluation I7 Show negative retentions Asymmetry Ratio at Resolution algorithm fi 0 0 100 of peak height IV Start message MV Sequence check 7 Sequence paste r OPC Tl Logon Logoff security P AllUsers IT Take Control HDA File cache path HDA Memory cache limit jo HDA File cache limit 10 TEMP Browse e Click OK changed Cancel Help Result The dialog box closes and the peak resolution algorithm is Note You must repeat the peak integrations after the change to update the values based on the new algorithm The formula below is used to calculate the Capacity factor VRO E Where e Vp retention volume k e V total liquid volume ep 501 B Evaluation functions and instructions B 3 Peak table column components Kav formula Asymmetry for mula 03 0014 90 e p 502 The formula below is used to calculate Kav Ve Vo kw Vo Vo Where e Vp retention volume e Vo void volume e Vo column volume The formula below is used to calculate the Asymmetry Asymmetry B A Where e Aisa partial peak width measured at a percentage of the peak height for the leadi
225. d ep 549 F Method examples F 3 Equilibration with extra safeguard Stage Description Equilibration of the column is continued until the conductivity value is stable allowed to vary by max 2 mS cm over a period of at least 5 minutes If this condition is not met within 10 column volumes the method is again paused Note At each pause the operator can decide whether to continue or abort the run 03 0014 90 ep 550 Method examples F F 4 Collection of absorbance peaks Introduction This section contains an example of how to collect absorbance peaks through outlets F3 and F4 Example instruc This is an example instruction as it would be presented in the Text window ai 0 00 Block ELUTION Elution 0 00 Base SameAsMain 0 00 Gradient 100 0 20 00 base 0 00 Watch UV1 Greater Than 100 mAU Peak_1 Peak_1 00 Base SameAsMain 00 OutletValve F3 Oo OO Oo 00 Watch UV1 Less Than Or Valley 100 mAU Waste Waste 00 Base SameAsMain 00 OutletValve WasteF1 Oo O Oo 00 Watch _UV1 Greater Than 100 mAU Peak 2 Peak 2 0 00 Base SameAsMain 0 00 OutletValve F4 0 00 Watch _UV1 Less Than 100 mAU End collect End_collect 0 00 Base SameAsMain 0 00 OutletValve WasteFl 0 00 End Block 0 00 End Block 0 00 End block 0 00 End Block 20 00 End Block e p 551 F Method examples F 4 Collection of absorbance peaks Illustration This is what hap pens
226. d menu or e press the Delete button in the Instruction box or e press the Delete key on your keyboard End_Block instruction If you delete the End_Block instruction the block will end at the last instruction in the block If a gradient is currently being formed the gradient will continue into the next block An instruction that has been deleted can only be recovered by re inserting the instruction If you want to suspend execution of an instruction temporarily for example during development work you can replace the breakpoint with a value after the End_Block or End_Method instruction 6 3 4 How to edit methods 6 How to change or move method instructions How to change an The table below describes how to change an instruction in the Text pane of the Text instruction Effects of the Change button and the Replace button on break points Instructions Editor Step Action Select the instruction the Instruction box or select a new instruction in the Instruction Box Click the Change button or the Replace button Note These buttons are equivalent unless changes are made to the breakpoint or the length of a gradient See below Result The instruction with its current parameters is displayed in Make the required changes to the breakpoint or parameters The table below describes the difference in function between the Change button and the Replace button when you cha
227. d Loghook text can be set to the following alignment options e Vertical e Horizontal e Fly Over which sets text labels as hidden text that appears only when the cursor is carefully positioned over a fraction mark The table below describes how to change the color and style of a curve Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed e Select the curve you want to change from the list e Select the desired color and style e Click OK The table below describes how to display and filter logbook curve information e Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed How to view results 10 Step Action e Click the Curve tab e Select the logbook curve e Click the Curve Style and Color tab e Click the Filter button in the Logbook text alignment field Result The Filter Logbook dialog box is displayed e Select the type of logbook information you want to show e Set the maximum block depth to show e Click OK How to displaya The table below describes how to display a hatched background in the hatched back chromatogram window ground Step Action Open a result file e Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed e Click the Curve Style and Color tab e Select the Hatch box e If desired select the Apply to all chromatograms box and click O
228. d definition 3 Peak identification 4 Calibration curve and quantitation table creation The table below describes how to input the standard data in the Evaluation module Step Action 1 Select Quantitate Edit Quantitation Table New on the menu bar Result The New Quantitation Table dialog box opens with the name of the active chromatogram displayed in the Source chromatogram field e p411 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step Action e Double click a result file in the Select peak table list if you want to select a source chromatogram from another result file Standaeds expressed in Ura damani Vohuso unt e C Corcertraion Ing voima fD C Amount unit Jabel feo Concentration unit mg ml If desired the standard can be expressed in Concentration instead of in Amount e Click the Concentration checkbox and edit the injection volume in the Inj volume field Note The software will always calculate both amount and concen tration for the sample e Highlight the standard peak table of level 1 on the Peak table s list and click the Select button Note This should be the table for the highest or lowest concentration of the standard Result The peak table is added to the Level Peak table s list e The level is automatically copied onto the list if it already was set in the method If so continue with step 4
229. d to be within the specified interval from the absolute 0 level UV_RESPONSE_INTERVALS Sets the level intervals for the UV_RE SPONSE_TEST 03 0014 90 ep 518 Evaluation functions and instructions B Instruction Description UV_RESPONSE_TEST The amplitudes for the 0 and 100 levels are calculated and the difference between the values are calculated The results of 1 Curve2_Difference Curve1_Difference and 2 Curve2_Difference Curve3_Differ ence are calculated The calculated points are checked if they are outside the defined limits from the 50 level ep 519 C Curve fit models and statistics Introduction In this appendix 03 0014 90 e p 520 Curve fit models and statistics The Analysis module optional is used to produce calibration curves and molecular size curves for analytical purposes The quality of the curve fit model determines the accuracy of the curves This appendix describes e The available curve fit models e The statistical measurements in the Analysis module This appendix contains these sections Topic See Curve fit models Statistics C2 C 1 Calibration curve models Molecular size curve models Statistics The Linear model Curve fit models and statistics C Curve fit models The Analysis module provides a comprehensive range of curve fit models The following models are available for calibration curves Linear Linear t
230. dentification window The software uses this window to search for peaks on other levels and in the sample runs A peak found in the window is assumed to be the component of interest You can change the limits by dragging a limit cursor line Both cursor lines move symmetrically so that the limits center on the component peak The window should be set wide enough to include peaks on the other levels despite minor variations in retention volumes However the window should also be narrow enough to exclude unwanted peaks that will interfere with the quantitation Instruction The table below describes how to adjust the window width for the best results Step Action Drag the cursor lines to set the window to a suitable width e Use the Show curve for level menu to display all levels and check that the width is suitable the window width is the same on all levels e Click the lower green or black triangle to display the actual reten tion for a peak Repeat steps 1 and 2 for all selected peaks Note Overlapping windows are not allowed If necessary click the Identification settings button to edit the settings See How to adjust the identification settings below this table e Click the OK button to accept the default identification settings Result The Quantitation table dialog box opens Continue to Step 4 How to create a calibration curve and a quantitation table below this table e p415 13 The Analysis m
231. dialog box and the corresponding text in the logbook are both color coded in red Warning texts are color coded in orange both in the dialog box and in the logbook The text in the logbook is changed into black when the Alarm or Warning is acknowledged Note The Alarms are not active unless the mode is set to Enabled Alarms and warning messages are displayed on all stations with a connection to the concerned system This is regardless of the activity that is currently performed in UNICORN and regardless of the identity and access rights of the current user Alarms and warnings can only be acknowledged from the station that is connected in control mode ep 461 14 System settings 14 2 Alarms The hysteresis set The hysteresis setting not available for AKTAdesign systems for a warning ting 03 0014 90 e p 462 determines to which extent the signal can oscillate up or down from the warning limit threshold without re activating a warning After the signal has activated a warning the warning will not be repeated as long as the signal remains within a window defined by the hysteresis setting above and below the warning limit This prevents repeated warnings from noisy or oscillating signals close to the warning boundary Warning limit Hysteresis x2 WARNING WARNING Note Hysteresis is only relevant for warnings since an alarm puts the system into Pause mode at the first alarm 14 3 Introductio
232. dialog box opens The dialog box displays a suggested name and location for the peak table Confirm the name and location and click OK The baseline can be adjusted graphically see also 12 1 5 How to edit the baseline manually on page 348 in the Edit Peak Table dialog box The table below describes this Action e Click the Set Curve Points icon Result The cursor is changed into a cross ep 351 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks Step Action 2 Perform the operations below as desired e Click to insert a new data point e Double click on a data point or right click the point and select Delete Point from the short cut menu to delete the point e Click a data point and drag the point to a new position to move the baseline Note Accept negative peaks must be selected before the peak integ ration if you want to be able to drag a data point to move the baseline above the curve How to calculate The baseline can be recalculated in the Edit Peak Table dialog box The table below a new baseline describes how to do this Step Action e Select Baseline New Calculate or e Right click and select New Calculate from the shortcut menu Result The Settings dialog box opens e Select an algorithm Morphological is default e Adjust the Baseline parameters as desired or e Click the Default Values button for the default values e Click OK
233. e Result file defined in File name and stores it in the Resulting peak table If is entered as File name the current Result file will be used The File name parameter may include a path from the current users root folder B Export The table below contains a list of instructions for export operations Instruction EXPORT_CURVE_AIA EXPORT_CURVE_ASCII EXPORT_CURVE_WKS Description Exports the curve in AIA format Exports the Source curve to the file defined in Export to File in ASCII format If is entered as File name the cur rent Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question In the part of the source curve limited by the Left limit and Right limit every lt n gt sample is exported Exports the source curve to the file defined in Export to File in WKS format If is entered as File name the cur rent Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question In the part of the source curve limited by Left limit and Right limit every lt n gt sample is exported e p 509 B Evaluation functions and instructions B 4 Procedure instructions 03 0014 90 ep 510 Instruction EXPORT_EVAL_LOG_ASCII EXPORT_EVAL_LOG_WKS EXPORT_EVAL_LOG_XLS EXPORT_METHOD_ASCII
234. e The name for the default format will automatically be changed to DEFAULT e Click OK e p 263 10 How to view results 10 6 How to create and print reports 10 6 2 How to create and print a standard report 10 6 2 How to create and print a standard report oe a a You can only select a number of pre formatted items when you create a Standard Standard report report format If you want to edit the layout in detail you must create a Customized report format See 10 6 1 How to create and print a customized report on page 250 The table below describes how to create and save a Standard report format Step Action Open a result file e Select File Report or e Click the Report icon Result The Generate Report dialog box opens Click the New button Result The Create New Report Format dialog box opens e Select Standard format and click OK Result The Create Standard Report Format dialog box opens The illustration below shows the Create Standard Report Format dialog box with the Header tab selected Create Standard Report Format x Evaluation log Frac 950 Contents Header Method Documentation Chromatogram The selected items will be included in the report M Select Items Current user Select All Run user Current date amp time Clear All Run date amp time Report title Result file name Method file name Page number Frames Report title O K O K K K O K
235. e It must have similar chemical properties to the component s of interest To be able to compensate for losses during the sample preparation all the standard concentration levels must be subjected to the same sample preparation procedure as the samples Note If there are several components of interest they must all be chemically similar The table below describes briefly how Internal standard quantitation is performed Add an additional component the internal standard in the same concentration to all the standards and to the sample Perform a run for each standard and the sample The Analysis module 13 Step Action Integrate the curves to produce a peak table for all standard runs and for the sample Result Each curve contains a peak from the internal standard Changes in the size of the internal standard peak indicate changes in the system See illustration below mau 250 200 Standard 150 100 3levels Sample Internal standard 15 0 15 5 16 0 16 5 17 0 min ep 401 13 The Analysis module 13 2 Quantitation overview Area ntemal Standard Action e Plot all peak sizes relative to the size of the internal standard peak to produce a calibration curve for each component The standard peak area relative to the internal standard peak area is used to produce a point on the calibration curve See illustration below 200 Standard Internal standard One of th
236. e Select File Copy to External or e Right click and select Copy to External from the shortcut menu Result the Copy to External dialog box opens Select the destination drive and folder Click the Save button The function Copy from External can be used to import files and folders e Ifthe files were saved using the function Copy to External they will automatically be decompressed e Copied method files must be connected to the same type of system they originally were created for This is part of the Copy from External procedure e Method files that have been copied in and connected are displayed in the designated folder in the Methods window The table below describes how to use the function Copy from External Action Select a destination folder in the Methods or the Results window e Select File Copy from External or e Right click and select Copy from External Note Do not select a file icon Result The Copy from External dialog box opens Select the files you want to copy Click Save Result e Result files are copied into the designated folder in the Results window e If method files were selected the Method System Connection dialog box opens Files and folders in UNICORN 4 How to connect a The table below describes how to connect a method to a system method to a sys tem How to rename files and folders How to delete files and folders Select
237. e a report from the UNICORN Manager 470 Gradient Effects of Change and Replace on gradient length 116 Gradients Instruction parameters 164 Step gradient instruction 164 Gradient breakpoints 164 Text instructions 165 Define length as a variable 165 ep vii Index Instant Run How to start 191 Internal standard quantitation Suitable components 400 How to perform 400 Reliability 403 L Linear flow rates Description 163 Log on and log off routines How to start the program 46 How to log on 46 Log off alternatives 47 Log off and set a password for a running process 47 Unlock the system 48 Quit UNICORN after log off 49 Logbook How to display an overlay in the Curves pane in System Control 203 How to display an overlay in the chromatogram window 232 How to filter the information 238 Logbook pane Description 205 Autoscroll function 205 How to filter the contents 205 Search function 206 M Maintenance How to view maintenance information 465 How to set up a maintenance warning 466 How to view warning parameters 466 How to reset warning counters 467 How to use the Generate Report Wizard from the UNICORN Manager 470 How to use the Generate Report Wizard from the System Control 473 Manual direct commands 03 0014 90 ep viii Buttons in System Control 208 Manual instructions in System Control During a method run 212 Column prompt 212 Functions of buttons 213 How to save results manually
238. e and displays the Manual instruction dialog box The Evaluation icon opens the Open Result dialog box Select a result file and click OK to start the Evaluation module The MethodQueue icon opens the MethodQueue Editor The Existing MethodQueue icon opens the Running MethodQueue dialog box to display MethodQueues in progress 03 0014 90 ep 26 Limited access to the UNICORN Manager UNICORN concepts 2 Some user groups may be defined to have only a limited access to the UNICORN Manager functions The available functions in the limited version are e Log off e Change User Attributes e Change Password e Quit Program e Help There is also a Cancel button which minimizes the dialog box The illustration below shows the limited access version of the UNICORN Manager UNICORN Manager 7 x UNICORN Logon Information Eric is logged on Logoff N Change User Attributes Help Change Password Cancel Note For more information about how to change passwords and user attributes please refer to 3 4 How to change your passwords and user attributes on page 56 For more information about how to log off and quit the program please refer to 3 1 Log on routines and log off routines on page 46 Guit Program e p27 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 2 The Method Editor module 2 2 2 The Method Editor module Introduction The Method Editor module provides co
239. e and select the appropriate option to e maximize e restore or e hide the pane ep 195 9 How to perform method runs 9 2 How to monitor a method run 9 2 2 The Run Data pane 9 2 2 The Run Data pane Description The Run Data pane displays the current values for selected run parameters The update interval is defined in the system strategy The figure below displays an example of the Run Data pane How to change The appearance of the pane can be changed so that it includes more or fewer data the appearance of displays The table below describes how this is done the pane In System Control select View Properties or right click on the pane and select Properties on the menu Result The Properties dialog box is displayed Select the Run Data Groups tab and if desirable do one or more of the following e Select an available group to be displayed in the list to the left e Edit an available group Select the group from the list on the left and click the Edit Group button Modify the included readings in the list to the right and click OK e Create a new group Click the New group button and select the readings that you want to view from the list Enter a name for the group and click OK e Delete a group Click the Delete Group button and select a group in the Delete Layout dialog box click OK and confirm the deletion 1 2 3 Select the run data parameters that you want to display in the list to the rig
240. e can be presented graphically or in a spreadsheet Step 1 How to se The table below describes how to select the operation lect the Operation Step Action In the Evaluation module e choose File Multifile Peak Compare Start Wizard or e click the Multifile Peak Compare toolbar icon Result The Multifile Peak Compare wizard entry dialog box is dis played Click the Next button to display the Operation dialog box Select e one of the available operations see descriptions of the operations below this table e a retention unit If you select Batch quantitate e Select a quantitation table in the Select quantitation table field If you select Batch Mw determination e Select a molecular size table in the Select mol size table field Click the Next button to proceed to the Data Selection dialog box 03 0014 90 ep 288 The Operation dialog box How to edit results 11 The illustration below displays the Operation dialog box Select operation omose peak dnd Compare scouting runs Media ite jme study Batch quantitate C Batch Mw determination Select retention unk Cm C Co Co Cr The operation op The table below is a brief description of the operation options tions Operation Compare peak data Compare scouting runs Media life time study Batch quantitate Description This option is used to compare differ ent results This option
241. e currently active window is affected How to filter The files in the Method window can be filtered to show only methods for selected Method files systems You can also limit the displayed files by using standard Windows wildcard characters The title bar of the Method window indicates if a filter has been activated The table below describes how to activate a filter Step Action e Select View Filter or e Right click and select Filter from the shortcut menu Result The Filter dialog box opens Click the check boxes for the systems for which you want to show files Enter a file name specification if necessary Click OK ep 73 4 Files and folders in UNICORN 4 3 How to arrange and locate your files How to find files 03 0014 90 e p74 The table below describes how to perform a search for files Step 1 Action Click either the Methods or Results window and e Select the File Find menu command or e Right click and select Find from the shortcut menu Result The Find files dialog box opens Co i x Systern Pah SE CA Melo 1 ta Atty Getdin Ml CN Wea WE Herod lies z fa Aray Getaision M3 cA Mela Fi ba Exchoeer text 1 Demorysiem ci Desa T Dete hem i001 te ae Add search criteria to the dialog box for example e Type a name in the Name field e Select a file type from the Type drop down box e Select if the search should incl
242. e method see 9 1 How to start a method run on page 190 Result The Start Protocol will display the scouting scheme as defined in the method assuming that the Scouting box is selected on the Start Protocol tab of the Run Setup Check through the settings for the scouting scheme in the Scouting tab and if required do the following e Change the scouting variable values e Right click the top of the Run column to toggle the run status between Run and Excluded 3 Work through the rest of the Start Protocol Click the Start button Results of a scout The results of a scouting run are saved in a special scouting folder as defined in mg tun the Results tab of the Start Protocol Within the folder each run is saved in a separate result file named according to the usual naming rules see 6 5 12 The result name tab on page 147 If the Start Protocol is displayed for each run in a scouting scheme you are able to change the result file name during scheme execution How to maane e At any time during a run you can click the View Documentation icon in System phe ee Control and change the scouting variables on the Scouting tab for runs which have not yet been started EJ e Settings for the run that is currently in progress cannot be changed e You can add more scouting runs to the scheme as long as the last run has not been started e You can use this feature to adjust variables for scouting even if the start prot
243. e or accept the default e Select the Multiply amplitude check box e Type the multiply value 1 e Click OK Result The mirror image of the original curve is displayed in the active chromatogram window 03 0014 90 ep 316 How to edit results 11 Step Action Shift the mirror image curve downwards Shift the mirror image curve downwards for an improved presenta tion e Choose Operations Shift Result The Shift dialog box is displayed e Select the curve to be shifted in the Source chromatogram list e Select the same curve number in the Target chromatogram list box as in step 2 e Select the Shift amplitude check box since the shift is to be made along the Y axis e Type a shift value e Click OK The illustration below shows the original curve and the mirror image displayed wo 330 If you want to display other curves in the active chromatogram window e choose Edit Chromatogram Layout to open the Chromatogram Layout dialog box e select the curves that you want to display e click OK ep3l7 11 How to edit results 11 9 How to import and export results 11 9 How to import and export results Introduction Curves and data can be imported and exported in different formats This section describes how to import and export results In this section This section contains these topics Topic See How to export results 11 9 2 03 0014 90 ep 318 1
244. e or by Replace The same selection ap plies to all components See explanations for the options below this table Select each component table rows in turn and check that the new point falls within acceptable limits Click the Statistics after update button Result The Statistics after update dialog box opens e Use the statistical data to check the curve model Note The old non updated calibration curve is still shown but the statistics apply to the data after the update If the new point is red the statistics shown will be those for the old curve e Click OK to close the Statistics after update dialog box Repeat steps 2 4 for each component Click OK Result The Update report dialog box opens This report provides a summary of the proposed update so that you can assess its viability See illustration below e Click the Print button for a print out of the Update report and or e Click Save or Save as to save the updated table The Average option means that the average area value is calculated from the old point representing the average of the old points at this level together with the new point The green point represents the new average value and not the position of the point from the new peak table e p 425 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Update by Re place The Update Re port dialog box 03 0014 90 ep 426
245. e position for collecting the first peak When the UV reading goes down to 4 75 mAU the outlet valve switches to the next position to separate the waste fraction from the collected peak fraction This process is repeated twice for the next two peaks so that when the UV reading rises above 5 mAU the position switches to collect the peak fraction and the position switches again to collect the waste fraction when the UV reading falls again 03 0014 90 ep 554 F 6 When to use a message How to add a Message instruc tion Protecting a meth od run with a message Method examples F Messages Messages are used to inform the operator of the progress of the run Messages can also be used for interaction between the operator and the system when necessary A message can be for information in a screen only or it can require a signature before the user can control the system The messages are all added to the logbook text This appendix describes how to add a message to a method The appendix also gives two examples of how a message can be used The table below describes how to add a Message instruction to the method Step Action e Select Other in the Instructions field of the Instruction box e Select Message in the instructions list Type a message in the Message text box in the Parameters field Select one of the display options on the Mode menu e Screen i e only a text message is displayed e No
246. e runs However if losses during sample preparation are constant between the two runs they may be accounted for by spiking the sample prior to the sample preparation e A spike amount which is of the same order of magnitude as the sample must be used to maximize precision e All the runs must be performed consecutively to reduce systematic errors and thereby maximize precision e p 405 13 The Analysis module 13 2 Quantitation overview 13 2 5 Recovery calculation 13 2 5 General informa tion The recovery factor How to perform Recovery calcula tion 03 0014 90 ep 406 Recovery calculation Recovery calculation is used to determine losses that can occur during the sample preparation process Recovery can also be used to determine the recovery factor of a preparative purification or a chromatographic process The recovery factor can only be determined for a single component A calibration curve is produced using a concentration series of an external standard The calibration range must cover the amount in both the sample and the spiked sample Two runs are performed one with the sample and a second with the sample that was spiked prior to the sample preparation with a known amount of the component of interest Quantitation of the data from the two sample runs allows the recovery factor of the sample preparation to be calculated Note The recovery is measured as the recovery for the sample preparation not for th
247. e separation during the chromatographic analysis The recovery factor can be used to manually compensate for losses during sample preparation The apparent amount in a sample is divided by the recovery factor to obtain the corrected amount The table below describes briefly how Recovery calculation is performed Step Action Perform a run with each level of the standard Peak integrate the curves to produce a peak table for each level Use the data from the peak tables to produce a calibration curve Note This is the same process that is used in the External standard quantitation Spike a portion of the sample with a known amount of the compon ent of interest prior to the sample preparation Run both the spiked and an unspiked sample Peak integrate both samples to produce peak tables for the unspiked sample and the spiked sample The Analysis module 13 Step Action The amounts for unspiked and spiked sample are calculated from the calibration curve The difference between these amounts provides the apparent amount of the addition See illustration below mau Sample unspiked 240 200 150 100 16 5 Sample with addition Area corresponding spiked to the sample Area corresponding to the addition The ratio of this apparent amount compared to the amount actually added to the sample determines the recovery of the system Apparent amount added Rei factor
248. e system will be paused until the message is acknowledged and signed No other interaction with the system is available to the user The operator will see a screen with a reminder to inject the sample before the method run proceeds The illustration below shows the selected message instruction in the Instruction box and the parameters for the message described above Instructions M Parameters Message K Pump Var Load sample loop C Flowpath Mode C AlarmstMon Var Authorize x Sound c Fie Default sound Y C Watch Pause Other Ready z The illustration below shows the text instruction for the message described above E 0 00 Block Sample_Injection_ Sample_Injection_ 0 00 Base SameAsMain Message Load sample loop Authorize Default sound 0 00 Pause INFINITE Minutes 0 00 InjectionValve Inject E 0 00 Block Sample_Injection Sample_Injection Note The message instruction must be followed immediately by the Pause instruction as shown above Method examples F F 7 Quality control procedure Introduction When a series of runs is performed irregularities in samples or in system or column performance can produce errors that will make the results inaccurate A quality control procedure can be added to a method to be used for a test run during the series of runs The control procedure can ensure that the results remain within acceptable limits If the
249. e the baseline parameters This section describes how to optimize the baseline with a morphological algorithm The Morphological algorithm can be described as a line that follows the chromatogram parallel to the X axis Data points for the baseline are created whenever the line touches the curve and the points are joined at the end to create a baseline The Morphological algorithm gives the best result in curves with drifting baseline and peak clusters The morphological baseline follows the curve faithfully and a curve with a baseline at a more even level can be created by subtracting the morphological baseline The Morphological algorithm does not work well if there are negative peaks or if quantitative data from negative peaks are important in the run Note The Morphological algorithm is the default baseline setting The table below describes how to choose a Morphological algorithm and define baseline settings Step Action Select Integrate Peak Integrate Result The Integrate dialog box opens Click the Baseline settings button in the Integrate dialog box Result The Settings dialog box opens e Select the Morphological algorithm e Change the Baseline parameters if necessary See more information about the parameters below this table e Click OK Note The same settings can be edited in the Calculate Baseline dialog box when a new baseline is created Choose Integrate Calculate Baseline to open the dialog
250. e the selected question e Click the Delete all button to delete all questions 03 0014 90 ep 132 How to edit methods 6 6 5 5 The Gradient tab Introduction The Gradient tab provides a graphical overview of the block structure and eluent gradient in the current method The description of this tab can also serve as a description of the Gradient pane of the Text Instructions Note For scouting runs click Run X to see the gradient for each run Illustration The illustration below shows an example of a Gradient tab Reference Curves Evaluation Procedures Method Information Stat Protocol Ss ResukName Frac 350 Variables Scouting Notes Questions Gradient Bulteep Cokmns Gradiert Block name 0 00 Main How to zoom in The table below describes how to zoom in on a selected area of the Gradient tab on a selected re gion Step Action e Press and hold the left mouse button and drag a rectangle on the screen to select the area you want to zoom in on e Release the mouse button Result The display is now zoomed in on the selected area Repeat the process for further magnification of selected areas How toreduce the To reduce the scale of the zoom function right click the tab and choose either scale of the zoom s function e Undo Zoom to reverse each zoom in action a step at a time or e Reset Zoom to reverse all of the zoom in actions to the default scale setting
251. e user has access to all files and sub folders in the selected folders Only selected folders will be visible in the methods or results panels of the UNICORN Manager module Note All users should have access to the Failed folder on each local station in a network installation This will ensure that users can access results that were saved in the Failed folder in case of a network communication error The Instructions dialog page is used to define the manual instructions and system sounds that are available to the user as well as which monitors the user is allowed to calibrate Click the check box for each selected instruction sound or monitor The level of access to UNICORN functions for each user is determined by the Access group that the user is assigned to The access authorizations can be edited for each group normally by the systems administrator Refer to the Technical and Administration Manual if you need to edit an Access group Note User access can be limited to only some UNICORN modules If that is the case the unavailable modules will not be displayed E g if the UNICORN Manager is unavailable you will only have access to a dialog box with the basic functions to change limited user attributes passwords and to log out and quit the program e p 55 3 General system operations 3 4 How to change your passwords and user attributes 3 4 How to change your passwords and user attributes Introduction Every user can change
252. e window See the dow illustration below amp Evaluation ExampleResutt GF001 1 V UNICORN Local ViTWikios Wesull VExampleResull GFOO1 EampleResult GFOO1 res Fe Edt View Integrate Operations Procedures Window Help ax Os 3E ONH x Vartables A Recent Rurs Fi Fred Colunn Superdex_75_MR_10 30 rere Enabled vial e i TE x Toots fang beanie sect Exampieheauit OFOO1 1 VVI 21an G01 BASEM paid tif n AN ee ee E T 7 I Wie SS toy Neel pa ste yp se _ jah nan gn ai2 632 pl oo so 10 15 0 20 250 300 350 30 60 s00 min Deere sitet Ocot Peet Heb No Peak name Retention min Area mAU min Height mau 1 foo 156 0 15 73 734 0150 705 676 Bovin Serwm 17 59 1327 5542 1200 002 alfa lactog 20 20 389 1587 325 176 Cytochrome t 25 21 219 3104 228 572 Vitamin Bi2 36 49 205 4120 168 451 Cytsdine 0 2 51 6568 48 742 nanty Toolbar icons in The table below describes the toolbar icons in the module the Evaluation module Icon Function ag The New icon opens an empty chromatogram The Open icon displays all available result files and result folders in Lay the Open Result dialog box The Open Curves to Compare icon opens the Open Curves to Compare dialog box which is used to select and open curves for comparison The Save icon saves the edited result file Zs The Print icon opens the Print Chromatograms dialog box ep35 2 UNICORN concepts
253. eak IdEnUfCAallONis yore cruise diate 366 12 2 2 How to find slope ValUCS cris vcard tetareansunters tua tOoinade felecGenridermerenteteddesmotaliades 369 12 2 3 How to simulate a peak fractionation cccccccsessseseceeeeeeeeeeeeeeeaeeeneeeneees 372 12 2 4 How to create CUIVESiassicesvctuamsaldeitvs bute sa ceainnels onde Soe t rts ttt rst tt Ers E tr rE EEEn rer Ennn Ene 373 12 2 5 How to use the Fraction Histogram ssssessssssrssreersrrrrrsrrrrrrrrererrsrrsrrene 377 12 3 Automated evaluation ProCe dUres cccccssssseeseeeeesssssseeeeeeeeeesssceeeeeeeeeeseeeeeeeenees 378 12 3 1 How to create a new PrOCeECUre cceecccccseecseceeesseeeseseeeeeeesseeseesaeeeneeegeees 379 12 3 2 How to edit a procedure ssssssssserssrsrrrrrrtrsrrtrrsrtersrttrrstttrttrttrerr nrt tere 382 12 3 3 How to run a procedure sssssrssserssrrtrtrrttrsrt rrtt rttr artt rttr ttrt EEE rnnr EnEn aes 384 12 3 4 How to rename and remove procedureS sssssrssresrsrrrrrsrrtrrrrrersrrsrrsrrere 387 13 The Analysis MOGU wicicsesccticcdicccriscsuactesasdrccrvesevsendeduoudsandubeus bowtdcundnunaedavsdeveadecacmededias 388 13 1 General information about the MOdUIe cccccceceseeseeeeeseeeeeeeeseeeeeeeeeeneeeneees 389 13 2 Quantitation SOV GIN IC Wie has fescsecd otc tas seas ts acecenttaninus sansausssoudacedsuneanssauseuetinatnteonseeasitnsocs 393 13 2 1 General information about Quantitation ccccececeeeseeeeeee
254. ecific method ep 145 6 How to edit methods 6 5 Run Setup 6 5 11 The Method Information tab 6 5 11 Introduction The Information sub tab The Signatures sub tab The Method Dura tion sub tab 03 0014 90 ep 146 The Method Information tab The Method Information tab displays information about the method This tab is for information only and cannot be edited There are three sub tabs on this tab Information Signatures and Method duration The Information sub tab displays e method information such as method name creation date creator and date of last change e target system e strategy information such as strategy name date and size The Strategy Notes button displays what systems programs and file versions the strategy is designed for The Signatures sub tab has five information fields for all signatures The table below describes the content of each field Field Description Date of the signature Short description explaining the meaning behind the signature Full name of the user who signed the method UserName User name of the user who signed the method Position Position of the user The Method Duration sub tab presents e the estimated total time e the estimated buffer volume required for the method If the method includes a scouting scheme click the Run x button to see values for the different scouting runs 6 5 12 Introduction Illustratio
255. ed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters are OFF then no method is exported If Main is ON then the main method is included and if Blocks is ON then all blocks are in cluded in the exported file EXPORT_METHOD_XLS Exports a method to the file defined in Export to file in XLS format If is entered as File name the current Result file will be used If 2 is entered fol lowed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters are OFF then no method is exported If Main is ON then the main method is included and if Blocks is ON then all blocks are in cluded in the exported file EXPORT_MULTI_CURVES_ASCII Exports multiple curves previously defined with EXPORT_SEL_CURVES in structions in ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered fol lowed by text e g Enter a file name as File name a full search path must be entered in answer to the question ep5l1l B Evaluation functions and instructions B 4 Procedure instructions Instruction EXPORT_MULTI_CURVES_WKS EXPORT_MULTI_CURVES_XLS EXPORT_NORMALISE_RETENTION EXPORT_PEAKTABLE_ASCII EXPORT_PEAKTABLE_WKS Description Exports multiple curves previously defined with EXPORT_SEL_ CURVES in
256. edures tab other wise they will be run twice Click the Scouting tab e Select the Update_Average procedure for the second and all follow ing runs at each standard level concentration Note The Update_Quantitation procedure Update by Replace should still be used for the first run at each level ep 447 13 The Analysis module 13 6 How to measure molecular size 13 6 Introduction In this section 03 0014 90 ep 448 How to measure molecular size The molecular size of components in a sample can be determined by size exclusion chromatography A molecular size calibration curve must first be created with components of known molecular size The retention is inversely related to the molecular size This section describes how to measure the molecular size This section contains these sub sections Topic See Overview of molecular size determination 13 6 1 How to determine the molecular size 13 6 2 The Analysis module 13 13 6 1 Overview of molecular size determination How to createa The table below is a brief description of how to create a molecular size curve molecular size curve Step Action 1 Perform a run with one or more standards to create a standard curve Note The standards should contain a number of components of known molecular size and these should extend beyond the size limits that are expected in the test sample Peak integrate the standard curve to pr
257. eeeeeeeeeeeaeeeaeeees 394 13 2 2 External standard QuantitatlOny a s ccxecsceesaeaecdsvesesceceds a sauaevddds oaetingencautes 397 13 2 3 Internal standard Guan titaTlOnancescvecsvipiinterensataccoytiaseterawee tence catoned 400 13 2 4 Standard addition Quant itatlOnanicrese cic csc apie sane aacaveaeruvee nade 404 Sve Recovery lt CalCUlANONssasmanands aretiran aaneen eds neonain ner a Lateacuumaeeies 406 13 2 6 General reliability factors for the quantitation techniqueS cccceeeeeeeees 408 13 3 How to prepare for Quantitation cccccceesssseseeeeeesssssseeeeeeeseeesseeeeeeeeseseeneeeeeeeees 409 13 3 1 Preparations before QUANTITATION sah ivseniues cir tulak as es Cancel avee eee 410 13 3 2 How to create a quantitation Ta DIG cic varisatunccaestaneutbwatitisyacastadleinicwinnavven 411 13 3 3 How to edit and update a quantitation table ccccceceeeeeeeeeeeeeeeaeeeneeees 422 13 4 How to quantitate the Sample ccccceesessseeeeeeeesssseeeeeeesseessseeeeeeeeesesneaeeeeeenees 428 13 4 1 External and internal standard quantitation ccccceccseeeseeeeeeeeeeaeenseenes 429 13 4 2 Standard addition QUaNTLAT ON wisdicctesictwaiersdresradeshantanteractaasebbiniealeunniasees 432 13 4 3 How to calculate the recovery factor ss ssserssrerrrrrerrsrrtrrsrrrrsrrrrrerrrrne 435 13 5 Automated CNANTTANONsecicclis coo ass lice tide eels Rieter eee eee 438 13 5 1 How to set up for automated quan
258. eeeeeeeeeeeeeaees 75 o How to Create a method naiiai enn araa dianae daan dharar aaa asana iia iaaa h aadd adda aonana annda 79 5 1 How to use the Method Wizard sssssssrsssssrsrrerrsrrtrrsrrrrsrrtrrsrttrrrrersrrtrrnrerrn 80 5 2 How to use the Method Templatesicsisse vesescedeecteevewcdauicsvthan ial iersaeacesteteesacesatnemeses 85 5 3 How to use Text MSTFUCLIONS 2uncncdetyyor aati aris uaadies tains Cental eeniaenauets 88 5 4 How to sign the MELO cvs audevectsarentslosvinueiaadentaasettedunt daar atisecuxatentimwinauteasanders 91 6 HOW to edit MetHOUS cswsssescnnteinioncinscesdeheetvibiie us veiesecenivk sa iadetien iin canteen aeeeile 92 6 1 The Method Editor INCH AG Os ccsceticetesdsses ce ceveccetercunieruetescs ceows coneseaverncvedeseseumcasveteseomted 93 Table of Contents 6 1 1 Method Editor module innsies dnt on eceorumtu es ecatenteiaetonaathsqanieourelacneioutiiudecaes 94 6 1 2 Text Instructions CCilOr etre sens tats sacvaoskaeatab eta oeesesuas vise tebae ater euh Re eunanteeeta ne 95 6 2 M thod DIGG KS iiss a a ct eee raaa a aa a ae a ae a ee eects 97 6 2 1 How to view method blocCkS e nsnenennsnsnnnnsrsrerrrrnrsrrnrsrsrrrrrrnrrrrnrsrsrrererrene 98 6 2 2 How to call method DlOCKS Aemcacewsarnenanaasarraeadihadewueuvadnitunteiinraiean weet 100 6 2 3 How to add method bloCkS esnensnnnnsnsrererrernnnrrsrsrrnrrrrrrrrrrsrnrrnrersreerrena 101 6 2 4 How to delete method blocks si aserasrswegesnsune
259. egrate menu Evaluation 12 The table below describes how to move the drop lines to adjust the peak area in the Edit Peak Table dialog box Step Action e Click the Edit peaks icon Ka e Click the peak in the curve or in the peak table to select the peak Result Two vertical bars become superimposed over the drop lines that delimit the selected peak The area between the bars is filled with a yellow fill pattern Drag the bars to define the new limits for the selected peak Result The drop lines are moved and the peak areas are automatic ally recalculated Note A drop line can never be moved beyond another drop line or beyond a point where the peak meets the baseline The table below describes how to use the zoom function in the Edit Peak Table dialog box Action e Click the Zoom icon Result The cursor is changed into a magnifying glass e Press and hold the left mouse button e Drag the cursor over the area you want to zoom in on e Release the mouse button Result The area is enlarged Right click and select Reset zoom to restore the full view If needed you can use the selections on the Integrate menu to perform a peak integration in the Edit Peak Table dialog box This is useful for example if you want to re integrate the curve using different settings or integrate only part of a curve with different settings See 12 1 7 How to integrate part of a curve and how
260. eight Description Retention value at peak start and peak end time or volume base A and G in the diagram above Name of the peak Peak area as a percent of the total area under the curve above the baseline Time or volume base Note This value can differ in time and volume base if the flow rate is not constant throughout the method Peak area as a percent of the sum of all integrated peaks Note This value can differ in time and volume base if the flow rate is not constant throughout the method Peak resolution See definition below this table Retention at the peak maximum time or volume base C in the diagram above Standard deviation for a Gaussian shaped peak See definition below this table Identifies the criteria for peak start and peak end as either the baseline intersec tion or dropline to the baseline or skim line Difference in retention between the peak end and peak start time or volume base G A in the diagram above Calculated by taking the maximum height of the peak above the baseline then determining the peak width at half this value above the baseline Time or volume base B D in the dia gram above where BD bisects CF ep 499 B Evaluation functions and instructions B 3 Peak table column components Sigma formula Peak resolution al gorithms How to change the peak resolu tion algorithm 03 0014 90 e p 500 Note In the Options dialog box in the U
261. elect one of the available models in the Curve model field Linear Linear logMw Theoretically this is the best choice Quadratic Quadratic logMw Point to point Point to point logMw e p 453 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size Molecular size Statistics How to open an existing table With the exception for the two point to point models the molecular size curves can be expressed as mathematical expressions The expressions and related items can be viewed in the Statistics dialog box e Click the More button in the Statistics field of the Molecular size table to open the dialog box The expression is shown at the top of the window followed by the values for the constant that it contains Statistical reference values e The correlation value only for linear models should be as close to 1 00 as possible e The explained variance value should be as close to 100 as possible Note Explained variance values are usually high A value of 90 indicates a very poor model The table below describes how to open an already existing molecular size table for editing in the Evaluation module Step Action Select Mol Size Edit Mol Size Table Open Result The Open molecular size table dialog box opens e Select a molecular size table from the Molecular size table s list Note By default the list will show the molecular size
262. elow describes how to sign the method Action Choose File Sign Method in the Method Editor Result The Sign the Method dialog box is displayed Click the Signing tab and do the following e Select a user in the User drop down list box In most instances you will want to use the current user shown on the list e Inthe Meaning field provide a short text description explaining the meaning behind the signature for example Method now fully tested and approved e Type your signature password in the Password field If desired select the Lock box to lock the method permanently from further changes by other users e If needed view a list of all signatures associated with the current method on the View Signatures tab e Click OK on either the Signing or View Signatures tab e p91 6 How to edit methods 6 How to edit methods Introduction This chapter describes the complete facilities for editing methods in UNICORN For many applications suitable methods can be created by changing the default values in one of the wizard generated methods supplied with UNICORN Use the more advanced editing facilities described here when you want e to change selected instructions in the method for example change the outlet valve position e to add blocks and instructions e to change method instructions to adapt to non standard system configurations In this chapter This chapter contains these sections Topic
263. eluent concentration is greater than 50 the gradient will be negative A step gradient is an immediate change in eluent composition To form a step gradient set the Length parameter to 0 in the Gradient instruction Example of instruction 10 00 Gradient 50 B O base The example instruction above forms a step from the current eluent composition to 50 B at breakpoint 10 The method continues with 50 B The breakpoint for a Gradient instruction defines the time or volume according to method base for the start of the gradient A gradient with a non zero duration occupies time and volume in the method and breakpoints for other instructions may be set to occur before the gradient is completed For most instructions the instruction is simply carried out at the requested breakpoint while the gradient is forming Instructions that affect gradients Gradients with variable length Instruction after a gradient How to edit methods 6 The table below describes the instructions that affect the gradient Instruction Effect A new gradient will start at the requested breakpoint Any remaining duration of the previous gradient is ignored The eluent flow rate will change at the requested breakpoint If the current base is volume or column volume the duration of the gradient will be changed If the method base is time the volume of the gradient will be changed End_Method The whole method will stop interruptin
264. em An integrity check means that a new checksum calculation is performed for the same files in their folders This new calculated value is compared with the checksum value obtained during installation The results of the comparison are presented in the report and any deviations are included e Click the Next button Result The Generate report dialog box is displayed Go to step 3 below Step 3 How to The table below describes how to generate and save the report generate and save the report Action By default the report is saved in the folder Unicorn Reports If you want to save the report in another location select a folder in the tree structure You also have these options e Click the Preview button to open the report in Notepad e Click the Print button to print the report without any preview Click the Finish button to generate and save the report ep 475 A Troubleshooting A Troubleshooting Introduction This appendix describes different problems which may arise in UNICORN and how to solve the problems In this appendix This appendix contains these sections Topic See Logon UNICORN access Methods and method runs Evaluation AKTAdesign system specific problems AS 03 0014 90 ep 476 Troubleshooting A A 1 Logon In this section This section describes how to solve the following log on problems e Unable to log on to UNICORN e Error message
265. em description Red instructions instructions with a red dot in a method are syntax errors and may be due to the following e The method was connected to the wrong system that is the strategy of the system is incompatible with the method e The method instructions do not correspond to the components you have chosen for your system Check your system components under Ad ministration System Setup in the UNICORN Manager e The Copy function was used instead of Copy from external when a meth od was imported from a diskette e The wrong system may have been selected in the Save As dialog box in the Method Editor e You may also have templates not intended for your system which might be the case for custom de signed systems e The systems strategy has been up dated with a new strategy that dif fers in the instruction set Solution There are several actions that you can take Check that the method has been connected to the correct system in either of these ways inthe System Method Connection dialog box when you use the Copy from external dialog box in the Save As dialog box in Method Editor If the system is custom designed open the Method Editor select the red instruction and either delete it or replace it with a corresponding instruction if available from the Instruction box Repeat this for all red instructions before saving the method e p 485 A Troubleshooting A 3 Method
266. ent runs 11 8 1 How to use the Multifile Peak Compare wizard Step 5 How to The Data View dialog box presents a comparison of the chosen data for the use the Data View designated peak comparisons The illustration below shows the dialog box dialog box 23 01 22 81 9730 2001 22 97 Mean L2G Std Dev QWs Rel Std Dev ase 19 62 19 36 9730 2001 19 47 Mean IIAP Std Dev aE Rel Std Dev ait 25 59 25 36 649 4573 30 2001 25 52 484 0643 mrem nE Ai eas an I jnchide Wizard Settings 20 Fra 30 Prot Print Save Spreadsheet Save Weard Settings The table below describes how to use the command buttons of the dialog box Command button Function Displays the data in 2 dimensional plot See How to use the 2D Data View dialog box below Displays the data in 3 dimensional plot See How to use the 3D Data View dialog box below Print Prints the spreadsheet Save Spreadsheet Allows you to save the data in different formats e Excel xls e Tabbed text txt e FarPoint spread ss3 Save Wizard Settings See How to save the Wizard Settings below Cancel Ends the Multifile Peak Compare wizard 03 0014 90 ep 296 How to use the 2D Data View How to edit results 11 The 2D Data View dialog box presents a two dimensional plot of a selected peak See also How to use the 2D Data View shortcut menu below The illustration below shows the dialog box
267. equired parameter in the Parameters field e Click the VAR button Enter a new variable name in the dialog box and click OK Note Variables can also be renamed in the Edit Variables dialog box in the Method Editor See 6 5 2 The Variables tab on page 126 for more information How to edit methods 6 How to remove a_ The table below describes how to remove a variable by converting it into a fixed variable valie Step Action In the Text pane of Text instructions select the instruction with the variable you want to remove Result The parameters for the instruction are shown in the Instruction box e Locate the required parameter in the Parameters field e Click the VAR button e Click the Clear button to delete the variable e Click OK Result The VAR button changes to Var to confirm that the variable is removed Note Variables can also be deleted in the Edit Variables dialog box in the Method Editor See 6 5 2 The Variables tab on page 126 for more information e p 121 6 How to edit methods 6 5 Run Setup 6 5 Run Setup Introduction Run Setup is a part of the Method Editor It has several tabs for defining method properties This section describes how to use the tabs and the information displayed on the tabs In this section This section contains these topics Topic See Overview of Run Setup 6 5 1 The Variables tab 6 5 2 The Scouting tab 6 5 3 The Questions tab 6 5 4 The G
268. er The table below describes how to attach a file Step Action The Attachments dialog box is displayed Files Result Method System log Global loa Baseline example 1 1 Baseline exampl r System information IV Computer amp operating system information Tn AKTA hardware informator IV Integrity check e Depending on the character of the file to be attached select the appropriate tab Result Method System log or Global log e Attach a file Click the Add button Select a file in the dialog box and click the Attach or OK button Result The selected file is added to the tab in the Attachments dialog box Note To remove a file select the checkbox and click the Delete button 03 0014 90 ep 474 System maintenance and error reporting 15 Step Action To include more information in the report select the appropriate check boxes in the System information field By default all options are checked Computer amp operating system information A summary of the computer and operating system information for example type of processor processor speed RAM hard disk capacity and printer AKTA hardware information A summary of the specific AKTAdesign hardware for example the instrument and PROM version for every instrument that is connected Integrity check When UNICORN is installed a checksum calculation is performed on the stationary files dll and exe for the syst
269. er determines the highest acceptable signal level for the baseline ep 493 B Evaluation functions and instructions B 2 Baseline calculation theory Baseline paramet ers Baseline paramet ers illustration 03 0014 90 ep 494 The baseline parameters can be illustrated as a rectangular box that the source curve has to fit into in order to be identified as a baseline segment where e The length of the box corresponds to the Shortest baseline segment e The height of the box corresponds to the maximum level of noise on the baseline segments This is referred to as the Noise window e The box is allowed to be tilted with a maximum slope corresponding to the Slope limit e The box is not allowed to move up above the Max baseline level The illustrations below shows the baseline parameters graphically Shortest baseline segment C_ Noise Window Max baseline level Slope limit Baseline segment identification Baseline points Classic al gorithm Baseline drawing How to measure the baseline seg ment Classic al gorithm Evaluation functions and instructions B The table below describes the baseline segment identification process Description The box is virtually moved along the source curve in steps of one third of the Shortest baseline segment length to look for baseline segments A baseline segment is found whenever the currently examined part of the source
270. erPrep TOCI PG vides dcosaianssa waico i aaiguucreratis wetaaora nua 537 E 2 How to edit a BufferPrep FECIDC ic siwacorstiancatiatind wiscetaeraddeheniactasaxhenierelercarenbands nas 542 F Method OXami ples icics ccccccevicaccssccnceswesecehecccnastessascaazseecseeasteustechansaavesuaxdsaususeewedonsaedsaaneiieens 545 Fede SS IBIS equilibration eia e e a a tues nce weak EE 546 F 2 Equilibration with simple Sates U ard uses sewn twarevie eo dtneetyvinis vent axwnvaeatytaaded 548 03 0014 90 epvi Table of Contents F 3 Equilibration with extra Sate Quand vcctenrcnmsted wiecatertedarrunsmsGaaacrammnyadercatenthadsnt 549 F 4 Collection of absorbance Peaks 5 2 c coscdsecoiacadavas ansanedvsyes tuandew snes ataakasstaaceondseete x 551 F 5 Collection of three absorbance peakS sessssssrsrssrrsrrrrrsrerrsrrrrrererrrnrrrrererrrnn 553 FO Messages me i rE aie ater EETA EE AEA A E AAT a vans RTE te tues 555 F 7 Quality control DrOCeGUley siccenxecraascsachoxtartcvadsrnnuaccnadacbeaneneaeusainsevaatooavarvinands 557 e pvii Table of Contents 03 0014 90 ep viii Introduction In this chapter Introducing UNICORN 1 Introducing UNICORN This chapter contains e A general overview of the UNICORN system e Information about the user documentation for UNICORN and how to use it This chapter contains these sections Topic See About UNICORN About this manual About the UNICORN user documentation 13
271. ered to be stable In other words it defines the permitted variation for the Stable_Baseline condition For this condition to be activated the signal may not vary by more than the Delta_Base value up or down over the time interval specified in the Stable_Baseline condition in the Watch instruction Note The Delta_Base setting affects the Stable_Baseline condition only ep 169 6 How to edit methods 6 7 Standard Watch conditions The condition Watch Stable_Baseline 03 0014 90 ep170 The condition Watch Stable_Baseline is met if the signal does not deviate by more than Delta_Base from the baseline during the time interval specified for the watch The baseline value is determined by the signal at the start of the watch If the condition is not met a new interval is started with a new baseline value defined by the signal level at the start of the new interval The illustration below shows an example of this WATCH WATCH Stable_baseline interval Stable_baseline interval condition not met How to edit methods 6 6 8 How to save or delete a method template How to save a You can save a method that you have created yourself as a template if you have T asatem Edit global lists authorization see the Administration and Technical Manual Recommendation The templates for each system are common for all users Be restrictive in saving methods as templates We recommend that only methods that are useful
272. erform chromatographic runs with an appropriate standard with components of known molecular size The standard should contain components of sizes that extend over the range that is expected in the sample If you are using many components it may be better to split them into two or more standard runs e Peak integrate the curves to produce peak tables The standard curves must all use the same X axis base unit during the integration Volume is the recommended unit for molecular size determination e Save the results This dialog box is used to select the peaks that will be used to produce the molecular size curve Each curve and its peak table name is color coded All the available peaks for all the curves are listed together in the Retention Mol size table Triangles show that a peak has been selected The name of its source peak table is shown above the curve window This is useful when you wish to know which peak has been selected of two closely spaced peaks from different peak tables The illustration below shows the Molecular size table dialog box Molecular size table asa_2000_del x Peak table id 231 wizard size exechssion001 10_UV1_215nm 01 PEAK1 Double click on a peak to select it Mol size kDa C Linear Linear log Mw GQuedietic B Quadratic log Mw Poirg to point Point to point flog Mw 67 000 r Stalistics 35 000 Explained variance 98 800 12 400 Correlation 0 9952 1 355 0 243 Tog Mal
273. ers to start it with Updates a Quantitation table with new data from one standard concentration level The default Limit value of 12 5 will be used e p517 B Evaluation functions and instructions B 4 Procedure instructions Test instructions The Instruction field also contains a group of test instructions These instructions are only available for the UNICORN software development team Instruction Description AUTOSAMPLER_PEAK_INTERVALS Sets the area intervals for the AUTO SAMPLER_PEAK_TEST AUTOSAMPLER_PEAK_TEST Locates the first peak in the peak table Compares the area of the peak in the peak table with the specified maximum and minimum areas GRADIENT_TEST_INTERVALS Sets the level intervals for the GRADI ENT_TEST GRADIENT_TEST The theoretical straight line between the 0 and 100 levels are calcu lated The deviation between the curve and the ideal straight line is compared in both directions from the center pos ition 50 until the deviation exceeds the defined maximum deviation The calculated deviation points arechecked against the defined limits STEP_RESPONSE_INTERVALS Sets the level intervals for the STEP_RESPONSE_TEST STEP_RESPONSE_TEST The relative amplitude is calculated at the specified retentions The 0 and 100 amplitudes are used for refer ence The calculated relative amp litudes are checked against the spe cified error margins The 0 level amplitude is verifie
274. es how to log on to UNICORN Action e Select Tools Logon in the UNICORN Manager module or e Click the Logon Logoff icon in the UNICORN Manager module gt i Result the Logon dialog box is displayed Note You do not have to perform this step if you start up UNICORN When you start UNICORN the Logon dialog box is automatically displayed The four program modules Log off after you are finished Processes can run after log off General system operations 3 Click OK The program has four modules When you start the program and log on you work in the UNICORN Manager module UNICORN also automatically opens the Method Editor the System Control and the Evaluation modules These modules are minimized until you activate them Up to four System Control module windows may open if UNICORN was set up to control more than one system at the installation Note If the access rights are limited to only some modules the other modules will not open Always log off when you leave the computer to prevent others from accidentally changing or deleting your files or disturbing your UNICORN runs There are two ways to log off in the UNICORN Manager e Select Tools Logoff or e Click the Logon Logoff icon gt i Note In case your access to the UNICORN Manager is restricted you will still be able to log off The process will continue even if you log off while a separation run is in progress You can
275. esult Name tab will be the suggested result name but you can change this at the start of the run By default result files are stored in the home folder of the user who starts the run The table below describes how to change the folder where the result file will be stored Action Step If the run contains information that is not important you can save disk space by selecting the No result check box thereby storing the result in the Temporary folder named Manual Runs where only the latest 10 result files are saved If not go to step 2 Click the Browse button e Double click the required folder icon e Click the OK button Scouting results will be saved in a special folder as specified by the result file path To select a folder type a name for the folder in the Scouting subdirectory field Each time the scouting method is run a new folder will be created with the name and a serial number entering IEXSC will create folders IEXSC001 IEXSC002 etc 6 5 13 Introduction How to set up fractionation Manual runs Total number of tubes How to edit methods 6 The Frac 950 tab The Frac 950 tab is used for defining options for Frac 950 The user can choose rack type and fractionation order The table describes how to set up the fractionation Action Select rack type from the Rack drop down list Select the order for fractionation by using the Fraction order radio buttons
276. ew How to change the sorting order 03 0014 90 ep72 How to arrange and locate your files This section describes how to arrange the way the files are displayed in your UNICORN workspace and how to locate files through a search You can choose how the files and folders are displayed in the UNICORN Manager windows The options are the standard Windows alternatives e Details e List e Large icons e Small icons If you want to change the view you either e Select View and the option that you want or e Right click and select View and the option that you want from the shortcut menu The files can be sorted in a different order when a window is displayed in detailed view The table below shows the options Sorted by Order Alphabetical order or reverse alphabet ical order Alphabetical order or reverse alphabet ical order Method window only Smallest or largest files first Alphabetical order of file extension type Modified Most recently modified files first Created Most recent creation dates first Select one of the methods below to change the sorting order e Select View Sort and the option that you want or Files and folders in UNICORN 4 e Right click and select Sort and the option that you want from the short cut menu or e Click the column header for the option that you want to sort by a second click on the same header will reverse the order Note Only th
277. f absorbance peaks Collection of three absorbance peaks Messages Quality control procedure E7 e p 545 F Method examples F 1 Simple equilibration F 1 Simple equilibration Introduction This section contains an example of how a Watch instruction for simple equilibration can be inserted into a method Example instruc This is an example instruction as it would be presented in the Text pane tion 0 00 Block EQUILIBRATE Equilibrate 0 00 Base SameAsMain 0 00 Watch Cond Less than 5 mS cm CONTINUE 0 00 Hold 0 10 Watch UV1 Less than 100 mAU CONTINUE 0 10 Hold 0 10 End Block If you are not using KTA instruments If you are not using KTA instruments a delay should be added after the Hold Pause instruction so that the following instruction will not be executed simultaneously with the Hold Pause instruction This is what hap The table below describes what happens in the above example pens Description The Watch is started on the conductivity signal and the method is then put on Hold Continue is issued and Watch_cond is turned off automatically when the Watch_cond condition is fulfilled Method execution continues issuing a Watch_UV command Again the method is put on Hold until the Watch condition is fulfilled Note Even though the line Watch Cond Less_than 5 mS cm Continue is in the method placed before Hold the method is put on hold first and then continued only a
278. file e Click the Print button to print the snapshot How to select The retention time and amplitude of any peak can be viewed directly in a peak peak table data table after an integration This data and more is selected in the Chromatogram Layout dialog box The table below describes how to select peak table data Step Action Click the Chromatogram Layout icon SE Result The Chromatogram Layout dialog box opens Click the Peak Table tab e Select the checkboxes on the Select peak table columns list for all items that you want to display in the table e Click OK 03 0014 90 ep 364 Evaluation 12 12 2 Other evaluations Introduction This section describes how the results can be used for other types of evaluations In this section This section contains these sub sections Topic See How to find slope values 12 2 2 How to simulate peak fractionation 12 2 3 How to use the Fraction Histogram 12 2 5 Peak purity and peak identification 12 2 1 e p 365 12 Evaluation 12 2 Other evaluations 12 2 1 Peak purity and peak identification 12 2 1 Introduction Peak purity Peak purity and peak identification Ratios between UV curves measured at different wavelengths give useful information about peak purity or peak identity The absorbance ratio can be used to check peak purity If the peak is pure the absorbance spectra are the same over the whole peak and t
279. file is opened and viewed a default layout is applied to display all the original curves The default layout can be changed by the user see 10 4 5 How to save and apply a layout on page 242 Information for each curve Each curve is automatically assigned a default color and style with default information about each curve displayed in the key above the curves This information includes e result file name e chromatogram name e curve name Choose the Y axis scale Each curve has a correspondingly colored Y axis To choose the appropriate Y axis scale e click on the Y axis until the desired scale is displayed or e click on the name of the curve Run curves short When viewing curves in the Evaluation module you can access a menu that provides cut menu Optimizing the workspace a quick alternative to menu commands Right click the run curves view to display the menu shown in the picture below Undo Zoom Add Text Edit Text Mode fil Peak Integrate Shift Offset Hatch v Marker Set Marker Ref Point Reset Marker Ref Point Snapshot Copy to Clipboard Copy to Metafile Base Type gt i Properties The chromatogram window can be minimized and maximized using ordinary Windows commands The table below describes extra features to optimize the workspace Use the command if you want Window Arrange icons to arrange icons of minimized windows
280. for all users be saved as templates The table below describes how to save a method as a template Action Choose File Save as Template in the Method Editor Result The Save as Template dialog box is displayed Enter a name for the template in the Name field or choose an existing template name from the Templates list that shows the available templates within the chosen system e Select the system for which the template is intended in the For system field e Select the appropriate technique on the Technique list e Click OK Result The method is saved as a template How to deletea The table below describes how to delete a template template Step Action Choose Edit Delete template in the Method Editor e Select the system and the template that you want to delete e Click the OK button and the Yes button to confirm e p 171 6 How to edit methods 6 9 How to print a method 6 9 How to print a method Instruction You can print a copy of the method including items from the method documentation in Run Setup and the Text Instructions editor The table below describes how to print a method Step Action In the Method Editor select File Print or click the print icon Result The Print dialog box is displayed showing the available items from the Method Editor O Frac 950 Variables O Scouting E M Test Method C Block List C Current Expansion O Exclude Unused B
281. form 404 Reliability 405 How to identify sample peaks 433 How to select the component 433 Start Protocol 03 0014 90 ep xviii How to use 191 Statistics Correlation 525 Explained variance 525 Stock solutions Description 143 Strategy How to display the strategy instructions 90 System Control module Description 32 Overview 194 How to select the displayed panes 194 How to customize the panes 195 Toolbar buttons 208 Manual instructions during a method run 212 Manual instructions 212 How to save manual results 214 Alarms and warnings 215 System Control status bar Description 210 Watch status 211 System Control Toolbar Manual command buttons availability 208 Manual command buttons functions 208 Windows buttons 209 System Access buttons 210 System data How to backup 63 How to restore backup data 64 System operation Automated workstation lock or logoff 48 Unlock the system 48 Unlock system locked by other users 48 How to connect to a system 58 Connection modes 59 How to leave control of the system 60 How to disconnect a system 60 System settings System summary 61 How to change default settings 459 How to assign a new value 460 Index e p xix Index T Temporary chromatogram Description 227 Text instructions Message instruction 160 Set_Mark instruction 161 Hold instructions 162 Pause instruction 162 Hold_until instruction 162 Linear flow rates 163 How to form a step gradie
282. fter the conductivity has reached a level less than 5 mS cm This is because Hold is an instruction that will be executed at its breakpoint while Continue is not an instruction but rather an action for the Watch instruction 03 0014 90 ep 546 Method examples F Evaluation of the This method works satisfactorily although one drawback is that it might never method end and thus consume all of the buffer if the conditions for some reason are unfulfilled See appendices F 2 Equilibration with simple safeguard on page 548 and F 3 Equilibration with extra safeguard on page 549 e p 547 F Method examples F 2 Equilibration with simple safeguard F 2 Introduction Example instruc tion This is what hap pens 03 0014 90 ep 548 Equilibration with simple safeguard This section contains an example of how a Watch instruction for simple safeguard can be inserted into a method This is an example instruction as it would be presented in the Text pane 0 00 Block EQUILIBRATE Equilibrate 0 00 Base SameAsMain 0 00 Watch UV1 Less than 100 mAU END BLOCK 5 00 Watch Off UV1 5 00 Message The Condition was never reached Screen No sound 5 00 End Block This is what happens in the above example The column is equilibrated until the UV has reached a level below 100 mAU or until the column has been equilibrated with five column volumes of buffer whichever condition is met first In this way
283. g the gradient End_Block The gradient formation will continue uninterrupted unless a new Gradient instruction is issued in the next block For example this means that a block can be called conditionally during gradient formation without interrupting the gradient For many purposes it can be useful to define the length of the gradient as a variable When this is done breakpoints for instructions issued during or after the gradient in the same block are automatically shifted in proportion to the length of the gradient with the same functionality as Change in the Text Instructions editor Any instruction that you want to insert after a gradient should be placed after the combined breakpoint and gradient length since gradients function over time e p165 6 How to edit methods 6 7 Standard Watch conditions 6 7 Introduction When is a Watch active How to insert a Watch instruction 03 0014 90 e p 166 Standard Watch conditions Watch instructions allow the progress of a method run to be determined by the events during the method run for example start collecting fractions when the first peak eluates or equilibrate the column until the eluent conductivity has reached a given value This is facilitated by the Watch instructions The system strategy includes Watch instructions for each monitor defined in the system These instructions are used to survey method runs and instruct the system to call a s
284. ge e Select the number of copies e Click OK Note You can also print the report from the Generate Report dialog box How to save the The table below describes how to save the finished report as a PDF file report in PDF format Step Action e Click the UNICORN Manager icon on the Windows taskbar Result The UNICORN Manager opens e Choose File Printer Setup Result The Print Setup dialog box opens Select an Adobe Acrobat printer from the Printer Name list e g Acrobat Distiller e Click the Properties button and edit the document properties if needed e Select the appropriate paper size and orientation e Click OK e Click the Evaluation icon on the Windows taskbar Result The Evaluation module opens e Print the report as described in How to print the report Result The report is created as a PDF file and saved in the location specified in your Acrobat settings Note You must have a full installation of Adobe Acrobat or a suitable printer driver to be able to do this 03 0014 90 ep 262 How to view results 10 How to save the The table below describes how to save the finished report format report format Step Action e Choose File Save or e Click the Save icon Result The Save Report Format dialog box opens e Type a name for the format e Select if you want to save the format for global use e Select if you want to save the format as default Not
285. ge 222 for detailed instructions on how to locate files and set up File Navigator preferences e p 221 10 How to view results 10 2 How to use the File Navigator 10 2 How to use the File Navigator Introduction The File Navigator can be used to locate and open result files in the Evaluation module Recent runs are also listed based on the user preferences How to open the To open the File Navigator File Navigator e Click the Evaluation module icon in the Windows task bar e Select View File Navigator Result The File Navigator opens in the Evaluation module The File Navigator can be resized and dragged to other positions in the module How to open files The table below describes how to use the Files list to locate and open a result file from the Files list Step Action e Click the Files tab Result The Files list opens in the File Navigator The list is identical to the Results window in the UNICORN Manager and shows all user available folders and files ix Recent Runs Files Find 320030924 920030925 920030930 920031001 320031002 920031007 920031016 20031017 Example files manual runs System M4 uti 002 ju 003 e Navigate to the desired folder e Double click the desired result file Result The result file opens in the Evaluation module 03 0014 90 ep 222 How to use Find to search for files How to view results 10 The Find fu
286. ged replicates shows the number of points used to calculate the average area value After each update by Average the number is increased by one After an update by Replace the number will be one e Nominal reference retention shows the retention for the reference peak in level 1 1 e Update reference retention shows the retention for the reference peak in the new peak table How to rename a quantitation table How to delete a quantitation table The Analysis module 13 The table below describes how to rename an existing quantitation table Step Action Select Quantitate Edit Quantitation Table Rename Result The Rename quantitation table dialog box opens e Select Personal to display the quantitation tables that are restricted to your own user ID if needed e Select the quantitation table you wish to rename on the Quantita tion table s list e Click in the Quantitation table name text box and type a new name e Click the Rename button e Click the Close button Note You must have Edit global list s rights to be able to rename a global quantitation table The table below describes how to delete an existing quantitation table Action Select Quantitate Edit Quantitation Table Delete Result The Delete quantitation table dialog box opens e Select Personal to display the quantitation tables that are restricted to your own user ID if needed e Select the quantitation table you wish to
287. h information about the active Watch instruction e p211 03 0014 90 9 How to perform method runs 9 3 Manual system control 9 3 2 Manual instructions 9 3 2 Introduction Manual instruc tions during a method run The manual in structions dialog box Column protec tion e p212 Manual instructions The chromatography system can be controlled with manual instructions issued from the Manual menu in the System Control module The available instruction options are dependent on the strategy Manual instructions can be issued while a method is running A manual setting applies until the next method instruction of the same type is executed Example A manual Flow instruction will set the flow rate until the next Flow instruction in the method is executed Manual instructions that you issue during a method are recorded in the logbook for the method run The Manual menu in System Control opens a dialog box similar to the Instruction box in the Method Editor The name of the connected system is displayed on the title bar of the dialog box See an example in the illustration below E ES oe lt lt lt _ Parameters C Pump oss ae lcd ooo o Cette BulfeiVaivedl C Alams amp Mon Purprlet ea C PumpBinlet Frac SampleValve wae ee C Other FlowOirection x pera nra Help T Auto update If this is checked the parameter fields wil be updated during method run Note
288. h standard level Integrate the curves to produce a peak table for each run mA Standard 3 levels ep 397 13 The Analysis module 13 2 Quantitation overview 13 2 2 External standard quantitation Step Action Use the peak tables from the standard runs to produce a calibration curve for each component This curve shows the relationship between amount and peak size Below is a calibration curve for one of the components Area Amount Perform a run with the sample and peak integrate the curve Identify the components of interest by the peak identification settings from the sample peak table Use the peak size s to calculate the concentration and amount from the calibration curve s Illustration how The illustration below describes how the calibration curve is used to determine the to use the calibra mount based on the sample peak area tion curve Amount 03 0014 90 e p 398 The Analysis module 13 Reliability External standard quantitation normally produces accurate results and is fairly simple The following reliability factors are specific to the technique e Precision is limited by changes that may take place between the runs for example column degradation and mobile phase variations e There is no compensation for losses of sample during the sample preparation process prior to analysis ep 399 13 The Analysis module 13 2 Quantitation overview 13 2 3 Internal
289. h the outlet valve positions F4 F6 and F8 so ensure that the tubing from these positions is lead to a suitably large container Condition for UV threshold The UV threshold for collecting the waste fraction must be below the threshold for collecting the peak fraction so that the waste condition will not be fulfilled simultaneously or immediately after peak collection This is an example instruction as it would be presented in the Text window 0 00 Block Eluate_Fractionation Eluate_Fractionation 0 00 Base SameAsMain 0 00 Watch UV1 Greater Than 5 mAU Peak Peak 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch _UV1 Less Than Or Valley 4 75 mAU Waste Waste 00 Base SameAsMain 00 OutletValve Feed Oo O Oo 00 Watch _UV1 Greater Than 5 mAU Peaki Peak1 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch UV1 Less Than Or Valley 4 75 mau wastel Wastel 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV1 Greater Than 5 mAU Peak2 e p 553 F Method examples F 5 Collection of three absorbance peaks Peak2 0 00 Base SameAsMain 0 00 OutletValve Feed 0 00 Watch_UV1 Less Than Or Valley 4 75 mAU Waste2 0 00 End_block 0 00 End_block 0 00 End_block 0 00 End block 0 00 End_block 0 00 End_block This is what hap The table below describes what happens in the above example pens Description When the UV reaches 5 mAU or more the outlet valve is switched to th
290. hange the Fonts e Click OK Result The method object is inserted onto the page e p 257 10 How to view results 10 6 How to create and print reports 10 6 1 How to create and print a customized report How to add docu The table below describes how to add documentation to the report mentation How to add the Evaluation Log 03 0014 90 e p 258 Step Action e Click the Documentation icon e Press and hold the left mouse button on the report page and drag out a box to the size of the item Release the button Result The Setup Documentation dialog box opens Select the items to be included in the report e Select All includes all items in the report e Clear All removes all selections e If desired change the Fonts e Select if the documentation should start on a new page e If Select All Logbook or Run summarySelect All or Logbook was selected make the necessary changes to the Base and Logbook filter settings e Click OK Result The selected documentation items are inserted into the report The table below describes how to add the Evaluation Log to the report Step Action e Click the Evaluation Log icon E e Press and hold the left mouse button on the report page and drag out a box to the size of the item Release the mouse button Result The Setup Evaluation Log dialog box opens e If desired change the Fonts e Select if the
291. hat the runs will be performed automatically without operator intervention All runs the Scouting start protocol will be displayed at the beginning of each run in the scouting scheme 03 0014 90 ep 152 How to edit methods 6 6 5 15 How to export the values in the Run Setup Instruction You can easily export the values in the Run Setup to a file and save it in ASCII format This is useful when you want to enable others to read the methods without having access to UNICORN on their computers The table below describes how to export the values in the Run Setup and save them to a file Step Action In the Text instructions Editor or the Run Setup select File Export Run Setup Result The Export Run Setup dialog box is displayed Select parts to include in the export Method Information Variables Questions Evaluation Procedures Start Protocol Result Name Method Notes iv M M v M M M ie Cancel Help e Select the boxes to select the parts of Run Setup that you want to export e Click the Export button Result The Export dialog box is displayed e Type a file name and select the target drive and folder e Click the Save button ep 153 6 How to edit methods 6 6 How to use selected method instructions 6 6 How to use selected method instructions Introduction This section provides recommendations for how to use some common programming features in UNICORN
292. he Curves tab Note The curves in the list are those for which Store is set to On in the system settings together with any reference curves defined in the method In the Display curves list select the curves you want to display If you want all curves to be displayed click the Select All button If you do not want any curves to be displayed click the Clear All button Click OK The table below describes how to display a vertical marker line Action Right click the Curves pane and select Marker Drag the marker line with the mouse Result Where the line bisects the curve the X axis and Y axis values are displayed at the top right corner of the pane e p 199 9 How to perform method runs 9 2 How to monitor a method run 9 2 3 The Curves pane Note Right click and select Snapshot to record the marker position values See 2 2 7 Snapshots on page 41 for more information about the Snapshot function How to set a refer When the vertical marker is displayed you can set a reference point to display ence pomt curve data The table describes how to set a reference point Step Action e Display a Marker in the Curves pane e Right click and select Set Marker Ref Point to define a reference point for the marker position 2 When the marker is moved from the reference point the X axis and Y axis values for the new position are displayed together with e the new position in relation to the positi
293. he Delete Page toolbar button and confirm the deletion to delete a page in Two select the page with Next Page or Prev Page Page mode e click an object on the page e click the Delete Page toolbar button and confirm the deletion How to change The page layout is changed in the Page Setup dialog box The table below describes the page layout how to set up the page layout Step Action Double click anywhere on the report page in the Report Editor not on an object Result The Page Setup dialog box opens e Type new values for the Margins if necessary e Select the appropriate Settings and Unit Note An extra Header tab will appear if you de select the option to have the same header on all pages The First Header tab is used for the first page header only and the Header tab is used for all sub sequent pages e Click the First Header tab e Select all the items you want to include in the header from the Select Items list e Click the Font button to change the font for all items if necessary e Type header text in the Free text box and click the Font button to alter the default font if necessary e Type the report title in the Report title box and click the Font button to alter the default font if necessary 03 0014 90 ep 252 How to view results 10 Step Action e Select the Logo check box and click the Browse button if you want to locate and select a logo image file e Select the
294. he Properties dialog box is displayed e Select the X axis tab Select the appropriate base Time or Volume Note Curves are collected in time and recalculated for display in volume Thus the resolution of the two bases may appear slightly different Select the appropriate Axis scale e Total will show the curves as far as they have come in the run e Window allows you to set the portion of the total pane to be dis played either in minutes or ml depending on the selected base e Adjust retention zero to injection sets the retention value to zero at the point of the first injection e Click OK How to switch e Click the legend of the X axis between time and volume units or e right click and select Base Type to switch the display between time and volume units The run is controlled according to the time volume base defined in the current block regardless of the base in the curves display ep 201 9 How to perform method runs 9 2 How to monitor a method run 9 2 3 The Curves pane How to zoomin The table below describes how to zoom in on a selected region of the curve pane the Curves pane Step Action e Press and hold the left mouse button and drag a rectangle out on the screen to encompass the area to be viewed e Release the mouse button Result The display is now zoomed in on the selected area Repeat the process for further magnification of selected areas How to zoom out
295. he Report icon 5 Result The Generate Report dialog box opens e Select a Standard report format to edit e Click the Edit button Result The Edit Report Format dialog box opens e Select Standard format and click OK Result The Edit Standard Report Format dialog box opens Click the appropriate tabs and select the check boxes for each item that you want to include in the report format Note See 10 6 2 How to create and print a standard report on page 264 for more information e p 267 10 How to view results 10 6 How to create and print reports 10 6 3 How to edit an existing report format Create Standard Report Format x Header Method Documentation Chromatogram Evaluation log Frac 950 Contents This is the information included in this report format Method Main Method Blocks Documentation Variables Method Notes Evaluation Notes Chromatogram All chromatograms Print Preview Save As Cancel Help Pint __ Preview Senet Her e Click the Save As button Result The Save Report Format dialog box opens e Type a name in the Report format name text box Select the Save as global format check box to make the format available to other users Select the Save as default report format check box if desired The format is saved as DEFAULT e Click OK Result The Generate Report dialog box opens again The new report format
296. he block before insertion or No to insert the copied block directly Result The pasted block is inserted with the same breakpoint value as the block or instruction selected for point of insertion ep 107 6 How to edit methods 6 2 Method blocks 6 2 6 How to find copy and move method blocks How to move a The table describes how to move a block block e Right click the block you want to move e Choose Cut e Right click the instruction line just above the point where you want the block to be pasted e Choose Paste Result The block is now removed from its original breakpoint and pasted at the new breakpoint The pasted block is inserted with the same breakpoint value as the block or instruction selected for point of insertion 03 0014 90 ep 108 How to edit methods 6 6 2 7 How to import method blocks Introduction You can import method blocks from other method files You can also use this function to copy blocks within a method In the latter case it is important to note that it is the saved version of the method that will be copied not changes that have been made after you last saved the method The block is imported exactly as it appears in the source method If the base of the imported block is defined as SameAsMain the block will inherit the main base in the new method regardless of the base in the source method Also the imported block is inserted with the same breakpoint value as
297. he illustration below Note Manuals can be added after the UNICORN installation See the Administration and Technical manual for more information How to open a manual To open a manual e choose Help Manuals in the UNICORN Manager module Result The Manuals dialog box is opened x Select a Manual and press OK UNICORN Online User Manual UNICORN Basic Components El Explorer Explorer Installation Guide 18 1139 59 aa Explorer Making Your First Run 18 1140 78 ab Explorer System Manual 18 1139 58 aa J FPLC Optional Configurations E Purifier UNICORN OK Cancel Help e Select the manual and click the OK button Note Some manuals are only available in PDF format In each dialog box there is a Help button If you press that button either of the following will be displayed e A message box with relevant information for example the dialog box options e The Help file with relevant information displayed in the right pane UNICORN concepts 2 2 2 1 Snapshots Introduction A Snapshot provides information about a method run at a certain point in time It contains information about the values of all the variables at the selected point Snapshot functionality is available in e the Method Editor where Snapshot instructions can be inserted in a method to be recorded during the method run e the Evaluation module where you can take Snapshots from a result file using the Marker e
298. he ratios should therefore remain constant The peak is probably not pure if the absorbance ratio is not the same over the whole peak The illustration below shows a simulated chromatogram of two co eluting components with differing absorbance spectra and a small difference in retention time Co eluting peaks AU ae Ratio a A B 3 08 25 OF 06 A270nm ae A255 nm 03 Ratio Az70nm A255 nm 02 01 0 0 0 1 2 3 4 Time units Peak identification The absorbance ratio can be used for peak identification Different compounds have a specific ratio between absorbancies at different wavelengths The illustration below shows a simulated chromatogram of two components with differences in their absorbance spectra Separated peaks gt Ratio 3 Ratio A270 nm A255 nm essoocoooces So Kwa D Wow Oo gt Time units How to divide the Both curves must have a baseline close to zero AU before they can be divided This curves 03 0014 90 e p 366 is achieved with baseline subtraction The table below describes how to subtract the baseline from an earlier integration and divide the curves Step Action 1 Create a baseline for each UV curve How to filter the result curve Evaluation 12 Step Action Select Operations Subtract Result The Subtract dialog box opens e Select the UV curve in the first list of curves e Select its baseline in the second list of c
299. hromatogram is automatically saved from one evaluation session to the next but is not saved within the result files Curves can be copied into Temporary and comparisons or evaluations can be performed This is particularly useful if you do not want to clutter up your original chromatograms with a large number of curves It can also be used to keep blank run curves or curves to compare when you open different result files The table below describes how to copy curves into Temporary Step Action Open a result file Select Edit Copy Curves Result The Copy Curve dialog box is displayed Select a source chromatogram and a curve to be copied in the Source Chromatogram fields Select Temporary as the target chromatogram and a position for the new curve in the Target Chromatogram fields Click the Copy button Result The curve is copied into the Temporary chromatogram Click the Close button The table below describes how to clear the contents of a temporary chromatogram Step Action Open the relevant result file e p 227 10 How to view results 10 3 Basic presentation of chromatograms 10 3 1 Introduction and temporary chromatograms Step Action 2 e Select Edit Clear Temporary Chromatogram e Click the Yes button to confirm 03 0014 90 ep 228 10 3 2 Main views How to view header informa tion How to view results 10 The chromatogram window The chromatogram window is d
300. hrough origin Quadratic Quadratic through origin Point to point Note The average peak size for all points at a specific level is used to calculate the calibration curve The following curve fit models are available for molecular size curves Linear Linear log Mw Quadratic Quadratic log Mw Point to point Point to point log Mw The Analysis module provides values for the appropriate constants that are used in each curve equation for all models except for the point to point models It also provides statistical data that you can use to assess the quality of fit of the curve to the data Click the More button in the Statistics field of the Quantitation table or Mol size table dialog boxes to view the applied model statistics The table below describes the features of the Linear curve fit model Feature Description Equation y Ax B Mathematical model The constants A and B are determined by linear least squares regression Minimum number of required points 2 at least 4 points recommended Measuring range for the calibration Within the highest and lowest values curve for the points e p 521 C Curve fit models and statistics C 1 Curve fit models The Linear through origin model 03 0014 90 e p 522 Note A variant of this model is available for the production of a molecular size curve This uses the logarithm of the molecular size as the x value in the expression
301. ht 4 Click OK to view the selected items in the Run Data pane The name of the selected layout replaces the default layout name Run Data 03 0014 90 e p 196 How to change text color or text background How to set the pressure units How to view and select manual in structions How to perform method runs 9 The table describes how to change the text color or background in the displayed reading boxes Step Action Right click on the pane and select Properties Result The Properties dialog box is displayed Select the Run Data Color tab Click the Text or Background buttons Select a new color and click OK Result The color change is displayed in the test field Make further adjustments to the colors as appropriate Click OK to apply the changes If the Pressure reading box is displayed in the Run Data pane you can set the displayed units The table below describes how this is done Select Set Unit and the appropriate unit MPa bar or psi Result The selected unit is displayed Some strategies directly link specific manual instructions to the reading boxes in the Run Data pane This is indicated by a double arrow gt gt A particular reading box can have one or more instructions attached to it In cases where there is more than one instruction one of the instructions is the main instruction There are two ways to view the manual instructions Option 1 e
302. ias for a specific concentration of the standard The list below describes how the levels are applied Level 1 should be selected for the standard with the highest or lowest concentration The levels must be set in consecutive order of changing concentration of the standard All runs with the same concentration must be given the same level If many small irrelevant peaks are detected it may be an advantage to re integrate after adjusting the Reject peaks criteria The number of largest peaks to detect has a default value of 20 and it may be helpful if this is set to a smaller value 13 3 2 Introduction How quantitation tables are created Four process steps Step 1 How to input the standard data The Analysis module 13 How to create a quantitation table The quantitation table contains all the necessary data such as the calibration curves that are needed to quantitate one or several components in a sample This section describes how the quantitation table is created Quantitation tables are created in the same way for both external standard quantitation and for recovery calculations They both use absolute values of standard peak data For quantitation with internal standard the peak sizes relative to the size of the internal standard peak are used to create a calibration curve The creation of the quantitation table can be divided into four steps 1 Standard data input 2 Component selection an
303. iate values in the Value cells for each pKa and dpKa dT parameter Note All values must fall within the stated Range limits Up to three values can be entered for each buffering component When the component has less than three pKa values the other values should be set to zero A dpKa dT value of zero means that the pKa does not change with temperature e Type the number of acidic protons for the buffer substance in the form that it is actually weighed in Example The number is 2 for NaH PO 4 1 for Na HPO and 0 for Tris e Type the charge of the completely de protonated ion This will be a negative value for an acid and zero for a base Example The value is 3 for NaH PO and 0 for Tris e Click OK Result The new buffer substance is added to the list of available substances Before you can define a new salt you must ensure that the new salt is inert i e a salt with no buffering properties The table below describes how to define a new salt Step Action Choose Edit BufferPrep Recipes and click the New button How to create and edit BufferPrep recipes Step 3 ik Action Click the Salt button in the New Recipe dialog box Result The Define salt dialog box opens Define Salt x Name NaCl3 hd E Delete __ Parameter Value _ Charge of Anion Charge of Cation OK Cancel Help Click the New button Result The New component dialog box opens T
304. ic variables or underscore characters these characters will automatically be replaced by spaces Points and underscores are not allowed in the result names ep 147 03 0014 90 6 How to edit methods 6 5 Run Setup 6 5 12 The Result Name tab Serial numbers and unique identi fiers Batch ID for each test run Specify result name as change able How to save the result files in a dif ferent folder How to save scouting results ep 148 If the result file folder already contains files with the same file name base the serial number is changed automatically For scouting runs the 3 digit serial number will be the number of the executed run column in the scouting scheme A unique identifier can also be generated automatically in addition to the serial number The identifier is a string of numbers inserted between the result file name and the three digit serial number e Select Add unique identifier to result name in the Result name field UNICORN will automatically issue a Batch ID to each method run This ID is displayed before the Base in the logbook and can be used to identify individual runs See illustration in 9 2 5 The Logbook pane on page 205 If Changeable batch ID is selected another ID string can be typed in the Start Protocol The result name can be specified as changeable in the Start Protocol see 6 5 14 The Start Protocol tab on page 152 In that case the information you supply on the R
305. ider a part of the baseline Use the marker box on the chromatogram to measure the length of the segment Choose Integrate Calculate Baseline and insert this value as the Shortest baseline segment value e p 495 B Evaluation functions and instructions B 2 Baseline calculation theory How to measure Curve coordinates can also be used to measure noise levels on the source curve noise level Classic The table below describes how to do this algorithm Use the Zoom function to focus on a part of the curve that is repres entative for the baseline noise e Calculate the noise range as the difference between the max and min values e Add an extra 20 e Choose Integrate Calculate Baseline and insert this value as the Noise window value How to measure The table below describes how to measure the slope at any part of the curve the slope limit Classic al gorithm Description Select Operations Differentiate in the Evaluation module Result The Differentiate dialog box opens e Select the desired source curve e Select the First order calculation option e Click OK Result The differentiated curve will appear in the active chromato gram Select an appropriate Y axis scale right click and select Marker to measure the Y axis values for the differentiated curve with the curve coordinates function Result The Y axis value is interpreted as the UV curve slope at the selected retention
306. ie OS id148Quaniitate001 1_Flow 10 id14 Quantitate001 1_Temp 17 id1 48Quantitate001 1_UV1_280em 01 BASEM ero UV1_Z20rm ar SIMPF Fraction size f m Hin vadth fp min Start slope 92782 mAU mn End slope 83 5038 mAU min Select the Source Chromatogram and the curve the simulated peak fractionation is to be generated for If necessary select a Destination Curve and type a new Curve name Type new values in the Parameters text boxes Click OK Result The simulated peak fraction curve is displayed on the chro matogram 03 0014 90 ep 372 Evaluation 12 12 2 4 How to create curves Introduction You can draw a curve of your own in the Evaluation module This section describes how this is done Note The right to create and rename curves is defined in the user access rights and may be restricted How to create The table below describes how to set up a chromatogram window to create a curve curves step 1 in the Evaluation module Step Action 1 Open a result file 2 Select Operations Create Curve Result The Create Curve dialog box opens Create Curve x Select help curvefs Y axis scale id148Quantitate001 1_UV1_280r g idi 48QuantitateO01 1_U 2_Onm Minimum 53 9230 03 id148Quantitate001 1_UV3_Onm 04 id 48Quantitate001 1_Cond Maximum fass 3870 05 id 48Quantitate001 1_Cond DE id 48Quantitate001 1_Cone 08 id148Quantitate001 1_Pressure
307. ignal falls below a specified value Slope_Greater_Than The rate of change of the signal ex ceeds a specified value expressed in monitor units minute for example mAU min Slope_Less_Than The rate of change of the signal falls below a specified value expressed in monitor units minute for example mAU min Less_Than_Or_Valley The signal falls below a specified value or a valley is detected A valley is de tected only after a Peak_Max has been detected and the valley is defined by a local minimum followed by an in crease to 102 of the local minimum value plus the Delta_Peak value see below Peak_Max The signal falls to a specified fraction of the most recent peak maximum minus the Delta_Peak value Factor 1 detects peak maximum Stable_Baseline The signal is stable within the limits of the Delta_Base value for the period specified by the minutes parameter Note For slope values use the Differentiate function in the Evaluation module to measure the slope of the test chromatogram The Simulate Peak Fractionation technique can also be used to find the slope values Watch conditions Two Watch conditions are available for systems with air sensors although they for air sensors and may be handled differently depending on the system The table below describes AuxIn he the conditions and their explanations Condition Explanation Equal 0 No air detected e p 167 6 How to edit methods 6 7
308. ile defined in Export to file in XLS format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_PEAKTABLE_XML Exports the peak table in Peak table source to the file defined in Export to file in XML format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question EXPORT_SEL_CURVES Selects a curve for subsequent export using the EXPORT_MULTI CURVES_ instruction The curve is cut according to the right and left cut limit and the number of points to be exported may be set by the Export parameter for ex ample every fifth point EXPORT_DOC_400_ASCII Exports the documentation in the current result file in ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters to this function are OFF then no documenta tion is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file ep 513 B Evaluation functions and instruc
309. ilibrate a column and another block contains instructions to load a sample etc In the Text pane of the Method Editor the method is shown as a list of blocks denoted by the blue square symbols Note that a block can also contain sub blocks The figure below shows the text instructions in blocks Base CV 53 09 ml HiLoad_26 10_Q_Sepharose_HP W 0 00 Block Flow_Rate 0 00 Block Column_Pressure_Limit W 0 00 Block Start_Instructions W 0 00 Block Alarm_Sample_Pressurelimit W 0 00 Block Eluent_A_Inlet W 0 00 Block Eluent_B_Inlet 0 00 Block Start_with_ PumpWash_Purifier 0 00 Block Start_Conc_B W 0 00 Block Column_Equilibration 0 00 Block AutaZero_UV AutoZero_UV 0 00 Base SameAsMain 0 00 AutoZeroUV 0 00 End_Block W 0 00 Block Flowthrough Fractionation Flowthrough_Fractionation 0 00 Base SameAsMain 0 00 Fractionation 18mm Flowthrough_TubeT ype 2 Flowthrough_FracSize ml NextT ube itFlowthrough_StartAt Volume 0 00 End_block 0 00 Block Direct_Injection Ditect_Injection 0 00 Base SameAsMain The table below describes how to view or hide the instructions If you want then to view the instructions click the symbol or double click the block name to hide the instructions click the symbol or double click the block name The Block pane The Gradient pane How to edit methods 6 The organization of blocks in the method is shown graphically in
310. ill display the rack layout that was used in the run e Click OK Result The Frac 950 data is inserted into the report ep 259 10 How to view results 10 6 How to create and print reports 10 6 1 How to create and print a customized report How to move and The table below describes how to select move and resize objects freely resize objects freely If you want then to select a single object click the Select icon click the object of interest to select several ob e click the Select icon jects e press and hold the lt Ctrl gt key while you click the objects to move the selected click on the objects hold down the left mouse button object s and drag the object s to the new position to resize the selected click one of the object border anchors either in the object s corners or in the middle of a border and drag the box to the new size Note Some Text objects cannot be resized Alignment toolbar Objects can be placed in exact positions and sized in relation to other objects The icon functions table below describes the function of the Alignment toolbar icons in the Report Editor Tool Function bar icon Align left Matches the left alignment of all selected objects to that of the highlighted object Align right Matches the right alignment of all selected objects to that of the highlighted object Align top Matches the top alignment of all selec
311. ill then calculate default baseline parameters for each new curve Calculates a baseline from the curve crvSrc using a morphological method DEFAULT can be selected in the Baseline parameters which will then calculate default baseline parameters for each new curve The baseline is stored in curve crvDst Clears the peak table in Peak table source from the computer memory Copies a peak table from Peak table source to Resulting peak table Controls the baseline behavior in sub sequent baseline calculations If ONOFF is ON then the baseline can be drawn above the curve and negative peaks can be detected by PEAK_INTEGRATE If ONOFF is OFF then the baseline is never drawn above the curve Performs a peak integration on the source curve and stores the resulting peak table in Resulting peak table It is assumed that the baseline is subtrac ted Specifies which part of the source curve that will be integrated Peaks between retention Left limit and Right limit will be detected if the ONOFF parameter is set to ON If ONOFF is set to OFF the whole curve will be used for integration ep 507 B Evaluation functions and instructions B 4 Procedure instructions Instruction REJECT_PEAKS SET_COLUMN_HEIGHT SET_COLUMN_VO SET_COLUMN_VT SET_SKIM_SIZE_RATIO WINDOW_PEAK_INTEGRATE Description Any combination of conditions is al lowed If all parameters are OFF then every detected peak is included in the
312. illustration below shortcut menu Viewing Style gt Font Size Numeric Precision Include Data Labels Grid Lines gt Show Bounding Box Rotation Animation Rotation Increment Ls Rotation Detail gt Plotting Method Maximize Customization Dialog Export Dialog Help The 3D Data View shortcut menu differs some from the 2D Data View shortcut menu and allows the figure to be viewed by animated rotation The shortcut menu displays different plot presentation options e Select Customization Dialog to open a dialog box that allows further customization of the graph Peak A Customization General More Font Color Style Main Title Show Annotations Peak A A Grid Lines a G Both C Y C XC None A ic Precision Miewing Style Numeric 2 Cc o Color i 2 Monochrome Monochrome Symbols K Eon size iN Show Bounding B C Lage Med Small pol a While Rotatinc Always C Never Cancel Apply Help Original Export Maximize e Select Export Dialog to export the view Note You can also click the Export button from the Customization dialog box 03 0014 90 p 300 How to edit results 11 How to save the The wizard settings can be saved from either of these dialog boxes Wizard Settings e The Data View dialog box e The 2D Data View dialog box e The 3D Data View dial
313. in the original method these will not affect the method in the MethodQueue To avoid confusion between different versions of method files make sure that MethodQueue definitions always contain updated methods To implement changes in a MethodQueue method do one of the following e Edit the method in the MethodQueue folder or e Edit the original method then use the MethodQueue editor to update the MethodQueue and replace the old method with the changed version The table below describes how to edit an existing MethodQueue Step Action 1 Right click the selected MethodQueue folder icon in the UNICORN Manager and select Edit from the displayed menu Result The MethodQueue Editor dialog box is displayed Start MethodQueve AsSoonAsPossitle Attime 0545 PM j D z si Add System AD300 ExamplePeakFrac Delete System Example5 Insert Row Before Inset Row After Delete Row Move Row Up Double click on a cell to change s content Save Saye As Jan Core Hep ep 187 8 MethodQueues 8 2 How to edit a MethodQueue 03 0014 90 ep 188 Step Action Select a table row to edit and do the following as required Double click the System cell and select a new method from the Method for row dialog box Double click the Condition cell and edit the delay time for the method Click the Add System button to add a new system to the queue and use it for a MethodQueue step Click
314. ines and the peak is split Note The area under each new peak will not be the same if the symmetry of the original peak was not perfect e p 355 12 Evaluation 12 1 Peak integration 12 1 6 How to edit the peaks How to join peaks Itis possible to join the areas of adjacent peaks if they are separated by a drop line The table below describes how to join adjacent peaks in the Edit Peak Table dialog box Step Action e Click the Edit peaks icon lig e Click the peak in the curve or in the peak table to select the peak e Right click and select Join Left or Join Right from the shortcut menu or e Select Edit Join Left or Edit Join Right Result The original intervening drop line is removed and all peaks are renumbered How to add peak The table below describes how to add names in the Edit Peak Table dialog box to names identify the peaks Step Action e Click the Edit peaks icon lig e Click the peak in the curve or in the peak table to select the peak e Right click and select Peak Name from the shortcut menu or e Choose Edit Peak name or e Double click the peak in the peak table or the curve Result The Edit Peak Name dialog box opens The number and re tention of the selected peak is displayed Type a name in the Peak name textbox and click OK 03 0014 90 ep 356 How to adjust peak areas with drop lines How to use the zoom function The Int
315. informa tion Disadvantages How to erform Standard addition quantitation 03 0014 90 ep 404 Standard addition quantitation Standard addition quantitation is a simple way to obtain measurements of amount in your sample concentration is not calculated It requires only a first sample run and a second sample run which has been spiked with a known amount of the component of interest The technique is straight forward and relatively fast when you are running only a few samples Standard addition can be useful when you want to use the internal standard technique but do not have a suitable internal standard The disadvantages of Standard addition quantitation are e its limited precision compared to the external and internal standard techniques e its lack of ability to measure more than one component The table below describes briefly how Standard addition quantitation is performed Step Action Perform a sample run Perform a second run with a sample that has been spiked with a known quantity of the component of interest prior to the sample preparation See illustration below mau Sample unspiked 240 200 150 100 Sample with Standard addition Area corresponding spiked to the sample Area corresponding to the added amount 15 0 15 5 16 0 16 5 17 0 min The Analysis module 13 Step Action Perform peak integration on both curves in the Evaluation module to produce a
316. ing runs 444 Automated update with Average for scouting runs 447 Baseline Calculation options 329 The Calculate function 329 Reuse existing 329 How to edit manually 349 How to adjust the baseline graphically 351 Definition of a segment 493 Parameters 494 Batch run How to perform 384 BatchID Logbook illustration 205 Blank curve Index epi Index Calculate baseline based on 329 BufferPrep Description 143 Stock solutions 143 How to create a method 144 How to adjust the correction factors 145 How to create a recipe 537 Buffer concentration 538 How to select the pH range 539 How to define a new buffer substance 539 How to define a new salt 540 How to edit a recipe 542 How to verify correction factors 542 How to change correction factors 543 C Calibration curve How to update 425 Chromatogram Layout Curve tab 236 Default curve names 236 How to choose curve name appearance 236 The Curve Style and Color tab description 238 Chromatogram window How to display header information 229 Shortcut menu 231 How to optimize the workspace 231 How to display a vertical marker 232 How to display the Logbook overlay 232 Chromatograms Description 227 Temporary chromatogram 227 How to make layout changes general 235 How to change and fix the Y axis 240 How to add a second Y axis 240 How to change and fix the X axis 241 How to save a layout 242 How to apply a layout 242 How to cut
317. ion modes The table below describes how to connect a chromatography system that is locally connected to your computer Step Action Open a System Control module Note Each UNICORN installation may have up to four System Control modules The number of modules are selected when the software is installed e Select the System Connect menu command or e Click the Connect to system toolbar icon rA Result The System Connect dialog box opens Select the system you want to connect Click OK Each computer workstation may have up to four chromatography systems connected locally In a network installation you may connect a system that is physically connected to another computer the local station Your system is then a remote station The local station that is connected to the chromatography system must be logged on to the network and the UNICORN drivers must be running However the connection will work even if the UNICORN program is not running on the local station General system operations 3 Network logon Ensure that your workstation is logged on to the network before you start a chromatography system that is directly connected to the station You can operate a local system without logging on to the network but there are several disadvantages to this e Files stored on network drives are not accessible e Changes made to global files e g user settings files will apply only locally and
318. ions and e small enough so that the condition is activated close to the valley or peak As a general guideline set the value to 2 3 times the noise level and 5 10 of the smallest expected peak height If you set a too high value you can prevent a new peak from being detected after a local minimum Use of the Delta_Peak setting The Delta_Base setting How to edit methods 6 The Delta_Peak setting e sets the threshold for signal increase after a local minimum that will be interpreted as a valley for the Less_Than_Or_Valley condition A valley and a new peak are detected when the signal increases to 102 of the local minimum plus the Delta_Peak value Note A valley is detected only after a Peak_Max has been detected Example If there is a local minimum at 0 05 AU and a Delta_Peak of 0 01 AU a valley will be detected at 1 02 x 0 05 0 01 0 111 AU sets the threshold for signal decrease after a local maximum that will activate the Peak_Max condition Peak_Max is detected when the signal falls to the specified fraction of the most recent peak maximum minus the Delta_Peak value The figure below illustrates the Delta_Peak setting where Peak_Max is detected when the signal falls by Delta_Peak from a local maximum if the Peak_Max factor is set to 1 oie ae Peak max Delta_Peak I detected No valley en ae detected The Delta_Base setting helps the software to determine when the baseline is consid
319. ious Row Next Row Cancel Help Note Use the Previous Row and Next Row buttons to select other methods for editing e Click the Wait radio button select the number of hours and minutes that the method is to be delayed and click OK Result The execution of the MethodQueue will be held for the selected number of hours and minutes and then resume e Click the Save button to save the method Result The Save MethodQueue dialog box opens e Type a file name and click the Save button How to set up If you have more than one system available the System column will not be displayed MethodQueueson 4 first in the MethodQueue Editor The table below describes how to set up a several systems MethodQueue for several systems Step Action 1 e Click the MethodQueue icon e Click the Add System button and select a system for the first MethodQueue step from the Add System dialog box 03 0014 90 ep 184 MethodQueues 8 Step Action 2 e Repeat this for each system when you want to use a different system in the MethodQueue Result Another system column will be added for each additional system Relative timing of The setting of the Condition dialog box reached by double clicking a Condition cell steps Unattended opera tion of the Meth odQueue in the MethodQueue Editor dialog box determines the relative timing of the steps of a MethodQueue If successive methods are run
320. it 3 Rename e Type anew variable name in the New name text box e Click the Rename button Result The variable is renamed Delete e Click the Delete button e Confirm that you want to delete the variable Result The variable is deleted How to change a Detail variables are only shown if the Show details checkbox is selected on the variable into a de Variables tab The table below describes how to set up a detail variable tail variable Step Action e Click the Edit Variable button on the Run Setup Variables tab or e Choose the Edit Variable Method Editor menu option Result The Edit Variables dialog box opens The variables are listed alphabetically Select the variable to be changed e Select the Set visible in details only checkbox e Click the Close button Result The variable is marked by the detail indicator D 03 0014 90 ep 126 How to edit methods 6 How to changea The table below describes how to change a detail variable into a regular variable detail variable into a regular variable Step Action e Click the Edit Variable button on the Run Setup Variables tab or e Choose the Edit Variable Method Editor menu option Result The Edit Variables dialog box opens The variables are listed alphabetically Select the variable to be changed e De select the Set visible in details only checkbox e Click the Close button Result The detail vari
321. it the scouting variables list to include e Procedure e Vial_Number e Injection_volume e Sample_ID e Quantitation_Type Note The Procedure variable will appear at the beginning of the list of variables even though the Evaluate instruction is inserted at the end of the method Set up all the standard runs in the scouting scheme e Select the Update_Quantitation procedure e Ensure that Quantitation_Type is set to the correct standard level for each run Result The quantitation table will now be updated with new values after each run Since the runs will be performed with the Replace the default selection option you can only perform one run at each level Proceed with the instructions on how to set up the scouting runs for the samples in step 4 see table below Note The quantitation table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value The Limit value is defined either for peak area or height How to perform The table below describes how to set up the scouting runs for the samples automated update in scouting runs step 4 Step Action Select the Quantitate_Sample procedure Select Sample in the variable Quantitation_Type for all the sample runs Result All samples will be run automatically and the amount and concentration of the components of interest will be printed after each run Note The result files will include a
322. itation procedure e Click the Quantitate button Result The Quantitation table dialog box opens Select the quantitation table from the Global or Personal folder and click OK Result The quantitation table is copied into the Update_Quantitation procedure Click the Edit button on the Evaluation Procedures tab Result The Procedure Editor dialog box opens See illustration below Select the existing UPDATE instruction N n A Ow N lt A ge Use the scroll bar in the Parameter field to locate the Average or re place point droplist e Select the AVERAGE option e Click the Replace button to the right of the scroll bar Select File Close in the Procedure Editor dialog box to return to the Run Setup ik Save the method and perform the runs Result The quantitation table will be updated automatically after each run New average values will be calculated from the old points together with the new points Note The quantitation table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value The Limit value is defined either for peak area or height ep 443 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update The Procedure r c The Procedure Editor dialog box is illustrated below Editor dialog box amp Procedure Editor Update_Quantitation px Fie Control Help Big es BASE TI
323. ithm settings A and a morphological algorithm with an increased Structure width value B mapa iOa I UNI 2Sa mara 10a 1 UWI DISAM BASEC Tape 104 t_UV1_2 ISAM got BASEME a 13 302 Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The solution to this is to increase the Noise window parameter However this can result in peak limits too high up on the peak slopes Note You can also use the Reject peaks function in the Integrate dialog box to reduce the number of peaks based on the total number of accepted peaks or the minimum peak height Evaluation 12 Minimum distance The Minimum distance between points is a measure of the distance between the data between points points used to generate a baseline The largest number of data points is produced at the slopes of the curves If you increase the Minimum distance between points value fewer points will be collected on the slopes The illustration below is an example of a baseline A that is created with the Minimum distance between points parameter set at a low value The number of data points is reduced when the Minimum distance between points parameter is set to a higher value B e p 339 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm 12 1 4 Introduction What is the Clas sic algorithm
324. ivided into four main views e File Navigator e header information e curves e peak and pool tables The displayed areas for the views can be adjusted by dragging the borders with the mouse cursor between the views The picture below shows an example of the window with all views present Evolustion ExampleResutt GF001 1 V UNICORN Local VTWiklas result WExomplefesull GFOO1 ExampleRlasult GFOO1 res jF Edt View Integrate Operations Procedures Window Help 8x OSSA 3E ONN x Variables A RecertRurs Fiet Find Colunn Superdex_75_MR_10 30 rere Enabled vial e 3 Enrol s Pudu beanae Ean pihent OFI I lorem G01 SASEN ample FOOLS WI Riian 1200 to 3 i 7 aH pad nif n TA lg ee re a ss Afi A an JAJI IAL 1532 BII Waes t CE ia mhd meet ye ae aee ee ee e ob sb 100 150 20 330 300 350 0 o 200 min 1H a Bete Cocear Oaoa Peet C Re No Peak name Retention min Area mAU min Height mAU 15 73 734 0150 709 676 Bovin Serw alfa lactog Cytochrome t Vitamin B12 Cytidine 0 17 59 20 20 25 21 36 49 40 23 1327 5542 389 1587 219 3104 205 4120 51 6568 1200 002 325 176 228 572 168 451 48 742 You can display header information at the top of a chromatogram with details on variables scouting variables questions and or notes Header information cannot be displayed for imported chromatograms The table below describes how to display header
325. k server and network communication has been lost The result file is saved in the Failed folder on the local station The table below describes a problem and its solution Problem description If the Method System Connection dialog box keeps appearing you have some method s which is not connected to a system Reason Most likely you have impor ted some method s with the command File Copy from External in the UNICORN Manager Solution Connect the method s to the appropri ate system e p 483 A Troubleshooting A 3 Methods and method runs The Method Edit The table below describes a problem and its solution or window does not fit on the BA screen Problem description Solution The Method Editor window does not e The display screen resolution may fit the screen and has scroll bars be set to 1024x768x65536 with Reason The incorrect font size might Large fonts You need to install Beansialied the Small fonts This requires that you have the Windows 2000 or Windows XP CD ROM that was shipped with your Compaq com puter e Insert the CD ROM and follow the directions on the screen Note Always install the latest service pack after you have installed something from the Windows 2000 XP CD ROM 03 0014 90 ep 484 There are red in structions in a method Troubleshooting A The table below describes some solutions to syntax error problems Probl
326. k the Curve model radio button for the best curve model e Linear recommended e Linear through origin e Quadratic e Quadratic through origin e Point to point Result The curve is displayed in the Calibration curve window Each component level is labelled with crosses If more than one run has been performed for any level all points in that level will be shown The average of these points is calculated and this value is used to produce the calibration curve Repeat steps 3 and 4 for all the remaining components Result The quantitation table is complete with a calibration curve for each component e Save the quantitation table and click Close or e Click the Save as button Result The Save quantitation table dialog box opens Note The Save button is used to save updates in an existing quant itation table However this will overwrite the original table You might prefer to use Save as and create a new name for the edited table to preserve the original e Specify if the table is to be globally accessible to any user or re stricted to your personal user ID The default is global e Type a name in the Quantitation table name field e Click the OK button ep 419 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table How to select an Internal Standard 03 0014 90 e p 420 The table below describes how to select an internal standard in the Qua
327. ks of interest and use these values Evaluation 12 Illustration Slope The illustration below shows a measurement of the slope limit after differentiation value measure en ment Bjidisblevel 1 to 4002 1 v UNICORN Local fil Niklas result Unicorn4001 1 lid1S6Level 1 to 4001 id186Level 1 to 4002 res Of x id186Level 1 to 400 UV1_280nm lt id19SLevel 1 to 4002 1_Inject id186Level 1 to 4002 1_UV1_280nm o1 BASEM id 13OLevel 1 bo 4002 1_UV1_230nmGOLGASEM idtGOLevel 1 to 9002 1_UVt_260nmG01 161 9 47 min 1 595 mAU ep 371 12 Evaluation 12 2 Other evaluations 12 2 3 How to simulate a peak fractionation 12 2 3 How to simulate a peak fractionation Introduction You can create a curve that simulates a peak fractionation to test the outcome before the actual peak fractionation is run This section describes how this is done How to simulate The table below describes how to simulate a peak fractionation in the Evaluation a peak fractiona nodule tion Step Action Choose Operations Simulate Peak Fractionation Result The Simulate Peak Fractionation dialog box opens Simulate Peak Fractionation x Source chromatogram Target chromatogram 01 161480 usntitate001 1_ UV1 230r 1 v 1 8 5 02 id148Quantitate001 1_UV2_Oom O3 id 48Quantitate001 1_UV3_Qom 04 id148Quaniitate091 1_Cond 05 idi 48Quantitate001 1_Cond O8 i6148Qusntitate001 1_Cone OR 1d148Quaniitate001 1_Pressu
328. l manuals i e experienced users of previous versions of UNICORN The instructions assume that all installations were made according to the instructions that the model system is used and is connected The table below describes the easiest way to create a method run the system and generate a printed chromatogram The procedure is based on an Instant run Step Action Click the Instant run icon in the UNICORN Manager module Ee Result The Instant run dialog box opens e Select Wizard e Select a system if necessary e Click the Run button Result The Method Wizard opens in the System Control module Go through all selections on the Method Wizard pages Click the Next button to proceed from page to page Click the Run button on the last page Result The start protocol opens Verify the method on the Variables page and change values as re quired Click the Next button to proceed through several pages Select Print_Chromatogram in the Evaluation procedures page Result A printout will automatically be generated after the run Click the Start button on the last page Result The run starts General system operations 3 3 General system operations Introduction This chapter describes how to start the program assign user properties and set up the system Refer to the Administration and Technical Manual for installation and network configuration instructions In this chapter This cha
329. lays the system calibrations and when and by whom they were made The Logbook tab displays what happened during a method run You can view information concerning alarms the method manual changes during the run errors etc Note Click the Find button to search for a specific text string in the Logbook The Evaluation Log lists all of the evaluation opera tions that you have performed for the result file dur ing all sessions including at the end of the method The Method Information tab displays information about the method such as the method name the target system and the date of the last change Information about the strategy includes name date and size There is also a sub tab for Signatures The Frac 950 tab displays the setup parameters for the fraction collector provided it is included in the strategy See The Result Information tab in this section e p271 10 How to view results 10 7 Run documentation The ea Inform The Result Information tab displays information about the result file such as ation ta e the result file name e the system that was used e the last date it was changed Information about the strategy includes name date and size The Run Summary sub tab is a summary of the run expressed in volume or time per block There is also a sub tab for Signatures and a sub tab where all Snapshots that have been taken during the run are displayed Documentation x Frac 950 Variables
330. le variables can be edited for each selection The illustration below shows the Instruction box 03 0014 90 p 88 Instructions can be organized in blocks How to save the new method How to create a method 5 Individual text instructions can be grouped in blocks of instructions marked by blue square symbols for a specific functional use e g to load a sample to equilibrate a column etc A block may contain other blocks or individual instructions This is an example of text instructions in the Text pane E Main 0 0 Alarm _Pressure Enabled 10 00 MPa 0 00 MPa 0 00 Wavelength 265 nm 254 nm 280 nm 0 00 ColumnPosition Position Bypass 0 00 OulletValve WasteF1 W 0 00 Block PREPARE W 0 00 Block LINGRAD W 0 00 Block STEPGRAD STEPGRAD 0 00 Base SameAsMain 0 00 Gradient 95 00 XB 0 00 base 20 00 Gradient 70 00 XB 0 00 base 40 00 Gradient 30 00 2B 0 00 base 60 00 Gradient 5 00 XB 0 00 base 80 00 Gradient 0 00 B 0 00 base 100 00 End_block 0 00 End_method A new method is untitled and must be saved under a method name before it can be used The table below describes how to save a new method Step Action Click the Save Method toolbar or choose File Save e If required save the method in a folder other than the default home folder e Enter a Method name for the method The total path can be up to 256 characters long The method name must be unique for
331. leave the process locked and set a password to protect it from interference The table below describes how to log off and set a password for a running process Step Action e Select Tools Logoff in the UNICORN Manager module or e Click the Logoff icon Result A confirmation box opens Click Yes to confirm that you want to log off Result The Leave Control of system dialog box opens Click the Locked radio button ep47 3 General system operations 3 1 Log on routines and log off routines Unlocked Log off Automated work station lock or logout How to log on and unlock the system Systems locked by other users 03 0014 90 e p48 Click OK It is not recommended that you log off and leave a running system unlocked This means that the run is in progress without a user that is responsible for the process The system administrator may set an automatic workstation lock or log off after a specified time for a user If there are no keyboard entries or mouse movements within the time limit the workstation will be locked or logged off Note A locked workstation can be activated again only by the previous user if the regular log in password is entered If another user wants to log on and use the workstation the previous user can be logged off without entering the correct password The previous user s files will be closed and the new user will only have access to his own files Autom
332. length 20 gt _ Grradient_length 30 Replace How to move an Move an instruction within the same breakpoint instruction 4 Select the instruction in the Text pane of the Text Instructions Editor and drag it to its new location to change the order of instructions within the same breakpoint in a block Move an instruction to another breakpoint The table below describes how to move an instruction to another breakpoint Step Action e Select the instruction in the Text pane of the Text Instructions Editor e Choose Edit Cut 2 Select the instruction line just above the point where you want the cut instruction to be pasted Choose Edit Paste Result The instruction is now removed from its original breakpoint and pasted at the new breakpoint The pasted instruction is inserted with the same breakpoint value as the instruction selected for point of insertion e p117 6 How to edit methods 6 4 How to use method variables 6 4 Introduction Identifying vari ables When to change variable values 03 0014 90 ep 118 How to use method variables Method variables can be used to edit suitable methods Variables can be assigned to most instruction parameters including breakpoints Variables also form the foundation for automatic method scouting Each parameter defined as a variable is also assigned a default value which is used if no changes are made to variable values at the start of
333. les before the method run starts e The First run only button Select this to display the start protocol before the first run only The settings entered in the Start Protocol for the first run will apply throughout the run and the scouting series will be performed automatically without user intervention e The All runs button Select this to display the Start Protocol before each run in the scheme This gives the operator an opportunity to change variable values or fill the sample loop before each run Note The operator must then click the Start button before each run Scouting 7 How to set up The table below describes how to set up series series Step Action Select a cell on the Scouting tab and click the Series button Result The Insert Series dialog box is displayed In the Insert Series dialog box type the selected series values within the specified range limits separated by commas and click OK Result A new set of runs is inserted on the Scouting tab with the values provided How to delete or Scouting variables can be deleted or renamed in the scouting scheme in the same rename scouting way as in the Variables tab The table below describes how to delete or rename a variables variable in the Scouting tab Step Action Click the Edit Variable button on the Scouting tab Result The Edit Variables dialog box opens The variables are listed alphabetically Select the variable
334. lick the Integrate button Result The Peak Selection dialog box is displayed Note If the results from the automatic peak integration is not satis factory you must cancel the wizard and perform the integration manually See 12 1 2 on page 330 The Peak Selec The illustration below displays the Peak Selection dialog box tion dialog box a M Curve idl 49Quanttate001 1_UV1_280rm Peak Table id148Quanttate001 1_UV1_280rm 01 PEAK 7 66 73 5676 6 33 25 7677 9 74 79 2309 11 01 173 1411 409 779 ep 291 11 How to edit results 11 8 How to import and compare different runs 11 8 1 How to use the Multifile Peak Compare wizard How to adjust im proper peak integ rations Step 3 How to se lect the peaks 03 0014 90 ep 292 Dialog box description The dialog box displays the following properties for the first of the chosen curves e The integrated peak and the associated peak table e The Peak identification settings table Its purpose is to identify the peak parameter to be used in the comparison The table below describes what to do if the peaks in the curve window do not appear to be integrated properly for example if ghost peaks are labelled Action Click the Cancel button to quit the wizard Perform a peak integration see 12 1 2 How to perform a peak integ ration on page 330 and verify that the resulting curve is properly integrated Repeat the Multifile Peak Compare wizard
335. lock Peak_Fractionation Base SameAsMain Peak_Fractionation_900 0 700 ml 200 End Block Watch_Cone Greater_Than 100 0000 End_Peak_Frac End HEnd Block a A AS 1 p 1000 800 600 0 2 0 4 0 6 0 3 0 10 0 12 0 14 0 ep 233 10 How to view results 10 4 How to optimize the presentation of a chromatogram 10 4 How to optimize the presentation of a chromatogram Introduction This section describes some of the ways you can optimize the presentation of a chromatogram In this section This section contains these sub sections Topic See How to change the size of Fraction Injection and Logbook marks 10 4 7 03 0014 90 ep 234 10 4 1 Instruction How to view results 10 How to make changes in the Chromatogram Layout dialog box The Chromatogram Layout dialog box is used to make changes regarding chromatogram presentation The main features of the Chromatogram Layout dialog box regarding chromatograms are described in the subsequent sections in this chapter Features regarding peak tables are described in 12 1 2 How to perform a peak integration on page 330 The table below describes how to make changes in the Chromatogram Layout dialog box Action Open a result file e Right click the chromatogram window and select Properties or e Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed The view from which you activate the Properties command determines
336. locks C Method Notes C BufferPrep L Columns CJ Reference Curves O Evaluation Procedures E M Method Information CJ Signatures J Method Duration C Questions C Result Name CJ Instruction Set Cancel Help e Select the options you want to print e Click OK Note For comments on the different alternatives see The Print dialog box below pee Print dialog The table below describes some of the check box options in the Print dialog box ox Check box If you select this box Text Method all instructions will be printed including those in unused blocks 03 0014 90 ep172 How to edit methods 6 Check box If you select this box Text Method Current Ex the method will be printed according to the current pansion expansion in the Text pane Only available from the Text Instructions editor Exclude Unused Blocks only blocks that are used in the method will be prin ted Text Method Block List only the main method and a list of the blocks that are used in the method will be printed e p173 6 How to edit methods 6 10 How to export a method 6 10 Instruction 03 0014 90 ep 174 How to export a method You can easily export a method to another file and save it in another format for instance rtf This is useful when you want to enable others to read the methods without having access to UNICORN on their computers The table below describes how to expor
337. low describes curve name aP how to do this pearance Choose Edit Chromatogram Layout Result The Chromatogram Layout dialog box is displayed Click the Curve Names tab 03 0014 90 ep 236 How to view results 10 Step Action 4 e Select the appropriate boxes for Curve name appearance e Select the appropriate Curve legend position e Click OK Note It is usually sufficient to select the Curve Name option if only one chromatogram is being evaluated However confusion can arise when more than one chromatogram is shown so more complete names might be necessary e p 237 10 How to vi ew results 10 4 How to optimize the presentation of a chromatogram 10 4 3 The Curve Style and Color tab 10 4 3 Introduction Peak label settings Fraction text and Logbook t alignment ext settings How to change the color and style of a curve How to display and filter logbook information 03 0014 90 ep 238 The Curve Style and Color tab All curves within a chromatogram are represented by a default color and line style Curves imported into the chromatogram or newly created curves are automatically assigned a color and line style Peaks can be labeled on the Curve Style and Color tab of the Chromatogram Layout dialog box Use a combination of the following labels e Retention the default label e sequential Number e user defined Peak name Both Fraction text an
338. lt The procedures of the selected method will be displayed on the Select list 2 Select the desired procedure on the Select list Result The method name is displayed in the Import as field Note Click Procedure List to return to the list of UNICORN s global evaluation procedures If desired change the procedure name in the Import as field Note The imported evaluation procedure cannot have the same name as an existing evaluation procedure in the method If the default name is not allowed for this reason the Import button will be gray and disabled When you change the name in the Import as field the button will become available again Click the Import button Result The evaluation procedure is imported into the method Repeat steps 2 4 until you have imported all procedures Click the Close button How to delete The table describes how to delete evaluation procedures from the method evaluation proced ures Step Action Select the Evaluation Procedures tab Select the procedure s that you want to delete e p 137 6 How to edit methods 6 5 Run Setup 6 5 7 The Evaluation Procedures tab Step Action 3 Click the Delete button and confirm the deletion when prompted Result The deleted procedures are immediately removed from the method file How to rename The table describes how to rename evaluation procedures in a method evaluation proced ures Action Sele
339. ltiply function This function is similar to Normalise Size but each curve is repositioned with precise numbers instead of by eye and the instruction logged in the evaluation log The table below describes how to use the Multiply function Step Action e Make sure that a chromatogram with the relevant curves is open in the Evaluation module e Choose Operations Multiply Result The Multiply dialog box is displayed e Select the curve to be multiplied in the Source chromatogram list e Select a curve position in the Target chromatogram list e Type a new Curve name or accept the default e Select the axis axes along which the multiplication is to be made along the X axis Multiply retention along the Y axis Multiply amplitude e Type the multiply value s e Click OK ep315 11 How to edit results 11 8 How to import and compare different runs 11 8 5 How to produce a mirror image 11 8 5 How to produce a mirror image Instruction A very useful way to compare the features of two curves is to produce a mirror image of one curve The table below describes how to do this Step Action e Make sure that a chromatogram with the relevant curves is open in the Evaluation module e Choose Operations Multiply Result The Multiply dialog box is displayed e Select the curve to be multiplied in the Source chromatogram list e Select a curve position in the Target chromatogram list e Type a new Curve nam
340. lts 11 11 6 How to match protein activity to a curve Introduction You can compare data from the results of protein activity assays such as ELISA with the data contained in the UV curve The activity curve and the UV curve can be compared in a combined presentation The Activity Histo The illustration below shows the Activity Histogram dialog box gram dialog box Activity Histogram Ea Fraction marks Target chromatogram 1 Y 1 11 idi 48Quanttate001 1_ Fractions Y 16 17 id1 48Quartitate001 1_UV1_280nm 01 84SEM 18 Histogram name Fractions 11 HIST How to enter pro The table below describes how to enter the values from a protein activity assay in tein activity values comparison histogram for comparison Action Choose Operations Activity Histogram Result The Activity Histogram dialog box opens By default the fraction curve for the specific chromatogram is selec ted e If necessary change the source and target chromatograms All the component fractions of the fraction curve are listed in the Fraction Activity field e Type an activity value for each fraction in the Activity column e Click OK e p 285 11 How to edit results 11 7 How to rename chromatograms curves and peak tables 11 7 Instruction 03 0014 90 e p 286 How to rename chromatograms curves and peak tables The table below describes how to rename chromatograms curves or peak
341. ludes more than one peak The second droplist in the Peak identification field of the Identification Settings dialog box offers the following options e Highest peak maximum default e Closest to retention i e closest to the center of the window see the retention column in the peak table e Maximum peak area Examine the nature of the peaks enclosed by the window and select the option that differs between the wanted and the unwanted peaks Use Closest to retention if there are large peaks from components that are not going to be quantitated Note The selection applies to all peaks even the internal standard and reference if used When the Peak identification is set to Absolute retention the peak window width can be displayed as Absolute or Relative Select the appropriate button in the Identification Settings dialog box e Select Absolute to show the window width of each peak in minutes or the base volume unit e Select Relative to display the width of each component as a percentage of its retention If Peak identification is set to Relative retention Window is set automatically to Relative except for the reference peak e p417 13 The Analysis module 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Step 4 Howto When the component selection and identification settings are completed see Step create a calibra 3 the Quantitation table dialog box is opened tion curve and a
342. m ay Step Action e Right click in the chromatogram window and choose Properties on the shortcut menu Result The Chromatogram Layout dialog box opens e Choose the Curve tab e Select the Logbook curve 03 0014 90 ep 232 How to view results 10 Step Action e Choose the Curve Style and Color tab e Click the Filter button in the Logbook text alignment field Result The Filter Logbook dialog box opens e Select all the logbook items you want to display and click OK e Click OK in the Chromatogram Layout dialog box Result The selected logbook items are displayed in the chromato gram window How to view the complete logbook information At some breakpoints there can be more logbook information than what is possible to conveniently display in the chromatogram window The additional information that is not displayed is indicated by an arrow point symbol by the break point e Hold the mouse cursor over the break point to display the complete information in a flyover text box as shown in the illustration below End Block Block PeakFrac_Parameters_Level Base SameAsMain Peak_FracParametersUY Level 0 190 min 50 000 m4U 100 000 m4U mi End Block Block PeakFractionation Base SameAsMain Weatch_Cone Greater_Than 0 0000 Start_Peak_Frac End Block Block Linear_Gradient Base SameAsMain Gradient 100 00 B 20 000 CY Condition_Occured Conc Greater_Than 0 0000 Start_Peak_Frac a Base SameAsMain B
343. m Baseline example 1 ATIMFO06 1_UIV2_2S4ren Bateira example 1 ATJMFOOGI UVI 2r Step 2 How tose The table below describes how to select data to compare lect data to com pare Step Action e Use the drop down lists and Browse buttons in the Chromatogram selection field to specify the result files chromatograms and curves for comparison e Click the All button if you want to select all available results chromatograms or curves 2 e Click the Search button in the Found curves field Result A list of all curves that matched the search criteria is displayed in the Found curves field 03 0014 90 ep 290 How to edit results 11 Step Action e Select the check boxes or click the Select All button of the desired curves within the Found curves field e Click the Next button to proceed to the Peak Data Selection dialog box e If all the chosen curves have been integrated go to Step 3 How to select the peaks in this section e If any of the chosen curves have not been integrated the Curves not Integrated dialog box is first displayed Curves not Integrated x There are some curves that are not integrated You can select to integrate them with default parameters to skip the curves that are not integrated or to quit the wizard Peak must be one of fed largest Skip uit wizara Help e If desired change the default value for the peak number selection filter e C
344. mandatory Used to set averaging time for UV detector used to set peak frac tionation parameters Not mandatory Used to give a warn ing when saving or starting the method if the BufferPrep_pH value is higher than the set value D e p 531 D The Column list D 1 How to edit the Column List Parameter Unit Comment pH low value longterm e Not mandatory e Used to give a warn ing when saving or starting the method if the BufferPrep_pH value is lower than the set value pH high value shortterm e Not mandatory e Used to give a warn ing when saving or starting the method if the BufferPrep_pH value is higher than the set value pH low value shortterm Not mandatory Used to give a warn ing when saving or starting the method if the BufferPrep_pH value is lower than the set value Average particle diameter pm e Not mandatory e Information only Code no e Not mandatory e Information only Typical loading range e Not mandatory e Information only Mol weight range e Not mandatory e Information only Scan rate spectra sec e Not mandatory e Information only Note The values for the parameters Max pressure Default flowrate and Typical peak width at base used to set average time and peak fractionation parameter MinWidth are only copied into the method if the corresponding instructions are available as variables 03 0014 90 ep 532 The Column lis
345. manual chromatography software UNICORN 5 0 User Reference Manual Amersham e Biosciences Gr 03 0014 90 UNICORN 5 0 User Reference Manual 03 0014 90 91 Edition AB 2004 02 Office addresses Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences K K Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Amersham Biosciences China Limited 13 F Tower I Ever Gain Plaza 88 Container Port Road Kwai Chung New Territories Hong Kong www amershambiosciences com Trademarks UNICORN Drop Design FPLCdirector OligoPilot BioProcess Ettan KTA AKTAxpress AKTAbasic AKTAexplorer AKTAFPLC AKTApilot and AKTApurifier are trademarks of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham ple Adobe Acrobat and Distiller are trademarks of Adobe Systems Inc Microsoft and Windows are trademarks of the Microsoft Corporation in the United States and or other countries Terms and Condition of Sale Unless otherwise agreed in writing all goods and services are sold subject to the terms and conditions of the company within the Amersham Biosciences group which
346. methods They are available from the Instruction box in the Method Editor In this section This section contains these topics Topic See Base instruction 6 6 1 Instructions at the same breakpoint 6 6 2 Block and method length 6 6 3 Messages and Set_Marks 6 6 4 How to delay a method 6 6 5 Linear flow rates 6 6 6 Gradients and eluent concentrations 6 6 7 03 0014 90 p 154 6 6 1 Bases What base should I use Column paramet er named column Column paramet er Any How to select columns for a template or wiz ard How to edit methods 6 Base instruction Every method block must start with a Base instruction defining the base for calculating breakpoints Different blocks can use different bases The base can be one of the following e volume the unit depends on the scale defined in the system strategy e time minutes e column volume CV defined as a numerical value or taken from the column definition e SameAsMain all blocks apart from the main block which means that the block will inherit the base defined in the main block Method blocks that use a volume or column volume base Make sure that the flow rate is not zero Volume breakpoints are calculated from the flow rate of the pump and the method will not progress if the flow rate is zero Use the base that most closely suits the purpose of the block Column volume is recommended as the base for most steps in a run In s
347. mplete facilities for advanced editing of the methods Two modes The Method Editor interface operates in two modes e Run Setup e Text Instructions Run Setup Run Setup is a dialog box with a number of tabs that define the method properties See the illustration below Pe RRS Tea aa ae UN NUUN TURAL CD RE ON ENE R DEEN AG abc OM Mpa Oa Ue ee ay aia ead al a a Bajse o Bye Mes Rese cn o a eon ee SEAS NP am 03 0014 90 ep 28 UNICORN concepts 2 Text Instructions Text Instructions are used for advanced editing Up to five different display panes can be open at the same time e The Block pane e The Flow Scheme pane e The Gradient pane e The Text pane e The Instruction box pane See the illustration below paeeeaeeaeee EEEEEEEEEEEE ee ee eee el ee ee ae The Block pane The Block pane contains a graphical representation of the method organized in blocks See the illustration below Block m TCAE sasar W Stort instrushor Alann M A A in i 0 00 Cy 0 00 CV 0 00 CV a00 CV 0 00 CY 0 Watch oH Greates Than w e p29 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 2 The Method Editor module The Flow Scheme The Flow Scheme pane displays the configuration of the system components The pane pane is static and for information only See the illustration below The Gradient The Gradient pane provides a graphical overview of the block structure and eluent pang gradient in the curren
348. ms checkbox e Click OK ep 243 10 How to view results 10 4 How to optimize the presentation of a chromatogram 10 4 6 How to show part of a curve 10 4 6 Introduction How to use the zoom function 03 0014 90 e p 244 How to show part of a curve You can select a part of a curve in order to examine details more closely You can e use the zoom to magnify or e cut the axes It is also possible fix the axes see 10 4 4 How to change and fix the axes on page 240 In the active chromatogram window you can zoom in on a designated area of the chromatogram This is the easiest and quickest way to enlarge different parts of a curve The table below describes how to do this Action Open a result file e Place the mouse pointer in any corner of the area you want to magnify e Press and hold the left mouse button A magnifying glass icon will be added to the mouse pointer arrow on the screen e Drag a box to cover the area to be magnified and release the mouse button Result The selected region is now displayed in the entire chromato gram window together with appropriate scales for the Y and X axes Use the arrow keys on the keyboard to move around in the chroma togram at the current zoom scale Undo zoom Right click in the window and select Undo zoom to undo the last zoom step Reset zoom Right click in the window and select Reset zoom to reset all zoom steps at once
349. n Construction of the result file name How to edit methods 6 The Result Name tab The Result Name tab is used to specify e how the result files will be named for the results of a run e where the result file will be saved e the name of the special scouting folder where results from scouting runs will be stored The illustration below shows an example of the Result Name tab Frac 950 Variables Scouting Nees Gradient BufleiPrep Coumans RefetenceCuves Evaluation Procedures Method Information Stel Protocol Questions Resuk Name I Noresult I Changeable batch ID M Resuk directory Home Browse Scouting subdrecton M Resuk name T Add unique identifier to result name serial numbes is added to all result names C Method name C Date C Name Variable x The result file name is constructed by one of the base options listed below The serial number is changed automatically each time the method is run Base options of the result file name are e The Method name plus a 3 digit serial number e The Date of the run in an 8 digit format determined by the country setting in Windows 2000 or XP plus a 3 digit serial number e A freely specified Name within the file naming restrictions of the operating system plus a 3 digit serial number e A selected Variable from the droplist plus a 3 digit serial number Note If a result names includes decimal points e g numer
350. n The Instructions dialog box Curve settings Store and Time between samples System settings 14 Curves This section is a short description of the Curves system settings The illustration below shows the Instructions dialog box with the Curves instructions selected System Curves Instructions xi m Instructions UV1_215 Parameters C Alarms SES Store ON Time between samples 0 100 le OFF ON C Specials Cond Store ON Time between samples Ci Mordors Time between samples 0 100 jao A pH Store ON Time between samples 0 100 Store ON tore Curves Time helusen camnlac NINN lt FI Set Selected Parameter To Strategy Defaut Value a const e The curve settings determine which monitor signals that will be stored as curves in the result file Verify that Store ON is set in the Instructions dialog box for all signals that are to be stored Warning If a curve is set to Store OFF data from the specific monitor cannot be displayed in the curves window during a process run The data will not be recorded in any way The table below describes the function of the two curve settings Setting Function Store OFF ON This setting determines whether the curve data is stored or not Time between samples This setting determines with which frequency curve data is recorded It does not affect the reading frequency of the actual monitor Default value is the sho
351. n User info about The Administra tion and Technical Manual ep18 Question What do I need before I start What are the contents of the User Reference Manual How should I use the User Reference Manual Answer Knowledge of PC and Windows func tions and an understanding of the concepts and terminology of liquid chromatography Preferably previous experience with UNICORN e Detailed descriptions of UNICORN e Instructions on how to use the sys tem with suggested alternatives Most instructions are based on a model system Depending on your previous experi ence you can either read whole chapters from the beginning to the end or only selected sections for refer ence The questions and answers in the table below describes the features of the Administration and Technical Manual Question Who should read the Administration and Technical Manual What do I need before I start What are the contents of the Adminis tration and Technical Manual Answer System administrators e General knowledge of UNICORN e Knowledge of PC Windows and general network administration functions e An understanding of the concepts and terminology of liquid chroma tography Detailed instructions of e How to install and maintain UNICORN in a network environ ment e How to create and administrate user profiles Most instructions are based on a model system Introducing U
352. n the system MethodQueues are used to link several methods together on the same or on different systems Example A MethodQueue can be set up to conduct a CIP study of a number of columns through a controlled series of scouting runs ep2l 2 UNICORN concepts 2 1 Concept definitions Method Wizard Result files Scouting Strategy Template Variable 03 0014 90 ep 22 The Method Wizard is a user friendly tool to create new methods The Wizard takes the user step by step through the creation process Method Wizards are supplied with UNICORN installations for AKT Adesign systems UNICORN creates Result files when a method is run The Result files contain e Run data from the monitors in the chromatography system Example UV absorbance flow rate conductivity etc e Documentation from the run Example Logbook entries calibration settings scouting parameters text method etc e Saved results from evaluations of the run data Example Peak integrations simulated peak fractionations etc Scouting is used to repeat a series of Method runs automatically with predetermined changes in the values for one or more Variables A Scouting Scheme is defined as part of the method Scouting is used for optimizing chromatographic processes Part of the UNICORN software is specific for the system that it is set up to operate The system specific part is usually referred to as the Strategy The Strategy
353. n a value for explained variance if you have sufficient data points on the curve For instance if you only have two points for a Linear model or only three points for a Quadratic model the fitted curve will pass exactly through the points By definition this leads to an undefined value for explained variance In these cases the Statistics table will show a symbol instead of an explained variance value The Column list D D The Column list Introduction The Column List includes all available columns and their specific parameters This appendix describes how to edit the Column List In this appendix This appendix contains this section Topic See How to edit the Column List D 1 e p 527 D The Column list D 1 How to edit the Column List D 1 Introduction Available columns How to column 03 0014 90 rint the ist e p 528 How to edit the Column List This section describes how to edit the list of available columns When you create a new method and select a column certain column specific parameters are automatically copied into the method The list of available columns is found in the For column field of the New Method dialog box The Column List is not linked to a particular method although the columns are edited within the Method Editor Columns are either globally available to all users or only personally available It is best not to edit the globally available columns
354. n additional chromatogram la belled 12 that contains a small part of the curves collected during the execution of the evaluation procedure 03 0014 90 ep 446 How to change the scouting runs to be updated by average The Analysis module 13 The table below describes how to change the scouting runs so that the quantitation table is updated by average after each standard run Step 10 Action e Click the Evaluation Procedures tab in the Run Setup Editor e Click the Import button Result The Import dialog box opens e Select the current method from the Method file menu e Highlight Update_Quantitation in the Select field e Type anew name e g Update Average in the Import as text box e Click the Import button e Click the Close button Result The new procedure is added to the list of Evaluation Proced ures Select the new procedure and click the Edit button Result The Procedure Editor dialog box opens Highlight the existing Update instruction Use the scroll bar in the Parameter field to locate the Average or re place point droplist e Select the AVERAGE option e Click the Replace button Select File Close in the Procedure Editor dialog box to return to the Run Setup Select the Update_Average procedure and click the Quantitate button Result The Quantitation table dialog box opens Select the quantitation table and click OK Deselect all the procedures on the Evaluation Proc
355. n below is an example of the statistical information for an applied Quadratic curve model Statistics Ea Model peannt Model function values The table below describes the features of the Quadratic through origin curve fit model Feature Description Mathematical model The constants A and B are determined by linear least squares regression Minimum number of required points 2 at least 4 points recommended Measuring range for the calibration From the point with the highest value curve down to the origin e p 523 C Curve fit models and statistics C 1 Curve fit models The illustration below is an example of the statistical information for an applied Quadratic through origin curve model Statistics x Model TEE Model function values 1 E 001 un Dp 29 E 000 Jumber of point s 4 Help Bs Pii to point The table below describes the features of the Point to point curve model mode Feature Description Mathematical model As these curves are not based on a single equation no statistical data is available The statistics table only contains information on the number of points in the curve Minimum number of required points 2 Measuring range for the calibration Within the highest and lowest values curve for the points The illustration below is an example of the statistical information for an applied Point to point curve model Statistics Ea
356. nO1 quantitation02 an idi 48Quantitate001 1_UV1_ 280nm 01_P How to prepare the calibration curve for updating The table below describes how to open the function and prepare the calibration curve for updating Step 1 2 Action Perform a peak integration for the new run and save the result Select Quantitate Edit Quantitation Table Update Result The Update Quantitation Table dialog box opens e Select the Personal radio button if the table is located in your personal folder e Select the quantitation table that is to be updated in the Quantita tion table field Double click the result file in the Select peak table list to access the new data e Click the Current button if you want to use the result file that is open in the Evaluation module e Select the chromatogram on the Source chromatogram list e Select the peak table that contains the new data in the Peak table s list ep 423 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table Step Action 6 e Select the level you wish to update on the Level list e Ifthe selected quantitation table is based on concentration verify or edit the Inj Volume field e Click OK Result The Update Calibration Curve dialog box opens See How to update a calibration curve below The Update Calib Data on the selected components for the curve to be updated are sh
357. na 136 6 5 8 The Reference Curves Ta Diing iia dcaimticecnraaertg shire rneiaatutadeountidetevuanbeistvanoube ite dd 140 6 5 9 The Columns LAD i clare weacenscdeshutceuenedukdssenedqavatacect sheveradeeca tunes Aa ikt ETAS I aara 142 6 5 10 The BufferPrep TaD iy saissicuvnseanetiettresriceltaatedsl ai deked denaacedbcacesuciaselaesauea eons 143 6 5 11 The Method Information taba ws orewpeersrcurantnsute aie ten Mate edeereatniabe 146 6 5 12 The Result NAITO ADD acuta tac avmanriaawen atten tan cencutaduid ema lNemerdnindtivanante acaats 147 6 513 The Frac 950 al 55 0 cadectecetvved seats Seana ceed Aa EA Gepecahenolsmamobengeeucts 149 6 5 L4 Te Stark Protocol tabire a e a ETa TAE ERE 151 6 5 15 How to export the values in the Run Setup ssssssssrssessrsrrsrrsrrrrsrrrrrsrrrrn 153 6 6 How to use selected method instructiOnS cccccceessssseeseeeeeessseseeeeeeeeeesseeeeeeeeeees 154 6 6 L Base INStrUCGTION S ushia ea a ve rensee dues widths Eaa a Ras een tartan 155 6 6 2 Instructions at the same breakpointins ss icctiverscc vestige cineca vies 157 6 6 3 Block and method ANGE pss cove wrcienruemaverneintegehvinn vend anevada 158 03 0014 90 epii Table of Contents 6 6 4 Messages and Set MatkSicosiescdnucas eeremotedrsreathubnleenun unt he crammuinencatentiddyvan 160 6 6 5 How to delay a MetOd tens nwnsietna yeah Siete ve belts ccbat vats detent utsce vhs won deeye a 162 6 06 Linear ROW rates p a tied E a cence scree EEE
358. naxe Retention min Area wAU min Height mAV 2 Peak A 0 2 6 53 34 0082 95 761 3 Peak D 0 2 7 06 20 4377 125 560 _4 Peak C 0 2 7 76 36 9911 147 623 Note Peak tables can be copied from one chromatogram to another with the Edit Copy command However to display the table you must right click in the chromatogram choose Properties and then select the new peak table on the Peak Table tab of the Chromatogram Layout dialog box The peak retention times and several other peak characteristics are calculated automatically The table below describes how to display other peak characteristics Step Action e Right click in the active chromatogram e Select Properties from the shortcut menu Result The Chromatogram Layout dialog box opens Click the Peak Table tab e Select options from the Select peak table columns list e Click OK Result The selected items will be displayed in the peak table Evaluation 12 How to filter Peaks can be removed from display in a peak table The table below describes how peaks from view filter the peaks Step Action e Right click in the active chromatogram or peak table e Select Properties from the shortcut menu Result The Chromatogram Layout dialog box opens e Click the check boxes in the Filter Peaks field to select the filter criteria e Specify filter values e Click OK To filter peaks vs The table below describes
359. nction in the File Navigator is used to locate result files in the available folders The table below describes how to use the Find function to locate and open a result file Step Action 1 e Click the Find tab Result The Find list opens in the File Navigator x Recent Runs Files Find Result file name Value of variable Sample_ID Example Result001 Example Result002 u Example Result003 za 1 pu Example Result004 2 e Type a file name or part of a file name in the Result file name text box Standard wildcard characters can be used or e Type a Sample ID value in the Value of variable Sample_ID text box Note The defined variable name must begin with Sample_ ID 3 e Click the Quick Find button Result The located result files are listed in the File Navigator 4 e Double click the desired result file or chromatogram Result The file or chromatogram opens in the Evaluation module Note Click the Find button to open the Find Files dialog box where more search functions are available See 4 3 How to arrange and locate your files on page 74 for more information e p 223 10 How to view results 10 2 How to use the File Navigator How to opena The Recent Runs list shows all the available recorded recent runs based on the Recent Run selected user preferences The table below describes how to use the Recent Runs list to locate and open a result file Step Action e Click the Recent
360. ne between two points straight line Step Action Select the first of the two points in the point list Click the Draw straight to next point button Result The baseline is drawn through the points as a straight line 03 0014 90 ep 350 12 1 6 Introduction How to open the peak table for editing How to adjust the baseline How to Evaluation 12 edit the peaks Once a peak table has been generated based on an appropriate baseline it is possible to split or join peaks and to manually adjust the peak start and end points The peaks will then be renumbered and the peak values will all be recalculated The table below describes how open the peak table for editing The editing options are described below this table Step Action e Select Integrate Edit Peak Table Result If there are more than one peak table available the Select Peak Table to Edit dialog box opens The name of the baseline on which the peak table was based is displayed at the bottom of the panel e Select the peak table from the list and click OK e Select one or more Help Curves to be displayed for reference if necessary Result The Edit Peak Table dialog box opens Note The Edit Peak Table dialog box will be opened immediately if you select Save and Edit Peak Table as the last step of the peak integ ration Perform the changes described in the instructions below Click OK Result The Save Edited Peak Table
361. nformation overlaid on the Curves pane do the following e Click the Filter button Result The Filter Logbook dialog box is displayed e Select the appropriate check boxes and set the maximum block depth e Click OK to return to the Curve style and Color tab Click OK How to view the At some breakpoints there can be more logbook information than what is possible complete logbook o conveniently display in the Curves pane The additional information that is not information a f displayed is indicated by an arrow point symbol by the break point e Hold the mouse cursor over the break point to display the complete information in a flyover text box as shown in the illustration below 400 End Block Block Start_Conc_B Base SameAsMain Gradient 0 00 B8 0 000 CY End Block Block Column_Valve Base SameAsMain i ColumnPosition Position Bypass End Block Block et ial gt Compensation sun ZUU2 300 200 100 memar 0 0 0 2 0 4 0 6 1 0 1 4 For Help press F1 Run System_Volume_Compensation Astar lt S UNICORN Main lt 4qMethod Editor u 1 System Control 1 2 System Contr ep 203 9 How to perform method runs 9 2 How to monitor a method run 9 2 4 The Flow Scheme pane 9 2 4 Introduction Illustration How to stretch a flow scheme How to view and select flow scheme manual instruc tions 03 0014 90 ep 204
362. nformation summary of a selected systemthe systems Step Action 1 Choose Administration System Setup in the UNICORN Manager Result The System Setup dialog box is displayed System Setup k xj M Systems 3g amp amp Explorer Demo Mtp Test MultiStrat Oligo Demo B New Edit Delete Show Icons C List Strategy Summary Socket cise Help e p61 3 General system operations 3 5 How to connect to the chromatography system Step Action e Select the system you want a summary of e Click the Summary button Result The System Table Summary dialog box is displayed Demo system The system is a chromatography AKTA system Control unit 1 at computer ANIMECHVALLE controls the system Backup is taken every 5 minutes Strategy in use E100F400 The strategy is designed for the systems AKTAExplorer100 Air Strategy Version 4 00 Required Unicom version UNICORN v4 12 or later Required instrument versions AD 900 v1 00 or later Air Sensor Ai 900 v1 01 or later AutoSempler 4 900 v1 04 of later AutoSampler 4 905 v1 00 or later Fraction Collector Frac 950 1 00 or later pH C monitor pH C 00 v1 01 or later Sample Pump P 950 v1 12 or later UY Monitor UY 900 v1 04 or later Valves v1 04 or later Compiled withe z e Click the Print button to print the information e Click the Close button to exit the dialog box 03 0014 9
363. ng Variables dialog box e Select other scouting variables of interest e g Sample_ID Vial_Num ber etc e Click OK e Double click the Quantitation_Type variable table cell e Select the correct standard concentration level Note This corresponds to the text instruction QuantitationData You can also set this level after the run has been completed For more information about scouting see 7 1 How to set up a scouting scheme on page 176 ep 439 13 The Analysis module 13 5 Automated quantitation 13 5 1 How to set up for automated quantitation Step 5 Action Click the Evaluation Procedures tab e Select the Integrate_and_Print procedure Result This procedure will automatically use default baseline settings and integrate the first UV curve Save the method Perform all the standard runs In the Evaluation module select Quantitate Edit Quantitation Table New Create a quantitation table manually from the standard runs See 13 3 2 How to create a quantitation table on page 411 The Scouting tab The illustration below shows the Scouting tab in the Run Setup used to enter standard data before the standard concentration level is defined 03 0014 90 e p 440 Evaluation Procedures l Method Infomation l Stat Protocol Questions Result Name Fiac990 Block Aulosampier_ Injection Partial ample_ID uantitation Pe SSS aay E E Po E e E E Variables Scouting
364. ng part of the peak e Bisa partial peak width measured at a percentage of the peak height for the tailing part of the peak 10 peak height l AB How to change the Asymmetry Ratio The Asymmetry Ratio is selected in the Options dialog box in the UNICORN Manager The table below describes how to select a value Step Action e Choose the Administration Options menu item Result The Options dialog box opens e Type a ratio value in the Asymmetry Ratio at text box e Click OK Result The ratio value is changed and the dialog box closes Note You must repeat the peak integrations after the change to update the values based on the new asymmetry ratio The default ratio is 10 HETP formula Evaluation functions and instructions B The formula below is used to calculate the HETP value HETP L N N 5 54 x Vp wy assuming a Gaussian peak Where e N no of theoretical plates e L bed height in cm e Vp peak retention elution volume or time wy peak width at half height expressed in the same units as Vp e p 503 B Evaluation functions and instructions B 4 Procedure instructions B 4 Introduction Curve operation 03 0014 90 e p 504 Procedure instructions This section contains lists of procedure instructions with descriptions These instructions are used in the Procedure Editor Choose Procedures Edit New in the Evaluation module to view the Instruction
365. nge breakpoints Button Function Change This button shifts all subsequent instructions in the block according to the change in the breakpoint Change does not affect the relative order of instruc tions in the method You cannot change the break point of an instruction to earlier than the nearest previous breakpoint in a block The illustration shows an example where Fractionation is changed from breakpoint 0 to 5 Change Gradient 0 00 Base SameAsMain xe 5 00 Gradient 100 x8 20 00 base 20 00 Message End of gradient Screen No sound 25 00 Message End of gradient Screen 20 00 End_Block gt 25 00 End Block No sound ep1l15 6 How to edit methods 6 3 Method instructions 6 3 4 How to change or move method instructions Button Function This button moves the selected instruction but does not change the breakpoint of any other instruction Replace can change the relative order of instructions in the method The illustration shows an example where Fractionation is changed from breakpoint 0 to 5 Replace Gradient Gradient 0 00 Bare SameAeMain 0 00 Bare SomeArMain 0 00 Fractionabon 18mm 5 mi Fast ube Volume 0 00 Gradiemt 100 2B 20 00 base 0 00 Gradom 100 B 20 00 ibase 500 Fractionation Them 5 mil FirstT ube Volume 20 00 Message End of gradient Screen No sound 20 00 Message End af gradient Screen No k und 20 00
366. nimized A minimized System Control unit may control a process All modules will normally open when the program is started However a user profile may be set up so that not all modules are available Only the available modules will be displayed epll 1 Introducing UNICORN 1 1 About UNICORN Work flow The work flow in UNICORN can be divided into four distinct stages Each stage is described in separate chapters in this manual The flow chart below shows the work flow stages 1 Create a method Run the method 3 Evaluate the results 4 Compile a report Help functions An online help utility is included in the UNICORN software The table below describes how to access the help utility If you want to access Then the general help utility open the Help menu in any of the software modules context specific help e click the Help button in the dialog box topics or e press the F1 key on your keyboard the online manuals open the Help menu in any of the software modules and select Manuals Security The table below describes the main security functions in UNICORN Feature Function Access Security Only authorized users can access UNICORN Each user is assigned an access level which defines the func tions that the user is permitted to use 03 0014 90 p 12 Introducing UNICORN Feature Connection Security Data Security Electronic Signatures
367. ns E E E ET 477 A2 UNICORN ACCESS tics enuhiainrcntuarinddecastuccs iachdbie enea snneiahes raenqvedd r Naai 479 A 3 Methods and method HUNnSsactctihos te teen actsts ah oth vane cit Alena ed tes meets hgh eg 482 AAs Evaluati On a aa E A Clete E a ETA 487 A 5 AKTAdesign system Specific ProDIOMs ccccsesssceeeceecessecesssseaeeeneeeeeseseeneaes 488 B Evaluation functions and imStructiOns sssssssscceeesesessseeeeeeeeesseeeeeeeeeeesessneaeeeeeeneesees 489 B l Smoothing algon tA Sice card utasce eicnecs toca geeud e A EE aa T ADE ia E EGAT a ETRE 490 B 2 Baseline calculation THeOnys savas actin eencvass veaaavroasss tadvenh aaantdananeateartuen oataltenes 493 B 3 Peak table column COMPONENTS Ax vi entitets re cpaiatenene uadcei ts dace creer ee eeuealotaads 497 BA Procedure AMSMUCUIONS cher edrasmuratindaeiutiad enrannentnt ra eaaa E anO Eia OAT aa aTa 504 C Curve fit models and Statistics sssssscseeeesessssneeeeeeeeeesseeeeeeeeesssaneeeeeeeseeesssaeeeeeeneeeees 520 Cts CurvefitmodelS asee n n a satan ep astute E a asia nc AEA EEEN 521 G 2 Statisties esi a e a EY E A a Eea Ea EE 525 D The Coimisean beens aaa a va as anain b dedoniaen Kian i dania areca EEA 527 D 1 How to edit the Column LISE cases alctee Sierras eb ateeeda crake dete tecuncdeen gee teeweugectea se 528 E How to create and edit BufferPrep reCipes cccccssssssseeeeeeeeesseneeeeeseeeessseeeeeeeeneeeees 536 E 1 How to create a Buff
368. nstruction to another breakpoint 117 Instructions at the same breakpoints 157 Method runs Start from the UNICORN Manager 190 Start from System Control 190 How to define methods as menu commands 190 How to start an Instant Run 191 How to use the Start Protocol 191 How to start a method run when the system is busy 191 Run Data pane description 196 Curves pane in System Control module 199 Flow Scheme pane description 204 Logbook pane description 205 Scouting runs 216 How to perform a MethodQueue run 217 If network communication fails 219 Method templates How to create a method 85 Template information 86 Save a new method 86 How to create a template from a method 171 How to delete a template 171 Method Wizard How to create a method 81 How to save a new method 83 03 0014 90 epx Index MethodQueue How to create a new 183 How to use several systems in a queue 184 Relative timing of steps 185 Unattended execution 185 Temporary hold when system is busy 186 Folder handling 187 File handling 187 How to edit a MethodQueue 187 How to perform a MethodQueue run 217 Unattended operation 217 Start when the system is busy 217 How to display and edit pending and running MethodQueues 218 Methods How to create using a wizard 81 How to sign 91 Different method editing operations 96 Method variables general description 118 How to select a column 156 Hold instruction description 162 Pause
369. nstructions how to edit the instructions see table below 03 0014 90 e p444 The Analysis module 13 How to perform The table below describes how to edit the text instructions automated update in scouting runs step 2 Step Action Click the Text Instructions icon Select the last instruction in the method in the Text pane e Click the Other radio button in the Instructions field of the Instruc tion box e Select Evaluate on the Instructions list Select Update_Quantitation in the Procedure droplist of the Para meters field e Click the Var button in the Parameters field Result The Variable Name Definition dialog box opens Type a variable name for example Procedure and click OK Result The Evaluate instruction is inserted in the method By defining the evaluation procedure as a variable different procedures can be selected on the Scouting tab for different scouting runs e Proceed with the instructions on how to set up the scouting runs for the standards see table below How to perform The table below describes how to set up the scouting runs for the standards automated update in scouting runs step 3 Step Action Select View Run Setup and click the Scouting tab Click the Define button Result The Scouting Variables dialog box opens ep 445 13 The Analysis module 13 5 Automated quantitation 13 5 3 How to perform automated update Step Action Ed
370. nt instruction 164 Gradient breakpoints 164 Gradient instructions 165 Watch instructions 166 How to insert a Watch instruction 166 Watch parameter options 167 Text Instructions Editor When to use 88 How to edit instructions 88 Save a new method 89 How to select panes 95 Toolbar icons In the System Control module 33 Troubleshooting Logon problems 477 Strategy file error 478 Access problems 479 Connection problems 479 Method problems 482 Incorrect time and date 487 Evaluation procedure aborts 487 AKTAdesign system problems 488 U Unconditional call Description 100 Unconditional method instructions Base instruction 155 UNICORN Manager 03 0014 90 ep xx Limited access to 27 V Variables General description 118 Identification in text instructions 118 How to change method variable values 119 Breakpoints or gradient lengths 119 How to define new method variables 119 Variable names 120 How to rename a method variable 120 How to remove a method variable 121 W Warnings Description 461 Watch instructions Description 100 Standard Watch conditions 166 How to insert an instruction 166 Parameter options 167 Air sensors 167 Permanent settings 168 Temporary settings 168 Delta_Peak settings 168 Delta_Base settings 169 Watch Stable_baseline 170 Wizard How to create a method 81 Y Y axis How to choose the Y axis scale 231 Z Zero baseline Index e p xxi Index
371. ntitation table dialog box Step Action Click the IS and Table settings button Result The IS and Table Settings dialog box opens The illustration below shows the IS and Table Settings dialog box with an Internal standard selected IS and Table Settings Ea M General Multiply amount with fi 000 0 001 1000 Multiply conc with fi 000 0 001 1000 Amount unit label mg Injection volume Not used m Internal standard Internal standard peak intemal standard x S amount oso mg Quantitation peaks Area Height Cancel Help Type the amount and concentration multipliers in the General field Note These values are normally set to 1 See remarks below Select the internal standard component on the Internal standard peak droplist Note The default option is Not selected which is used for external standard quantitation and measurements of the recovery factor Type the injected internal standard amount for the standard and sample runs in the IS amount text box Select if the quantitation will be based on Area default or Height in the Quantitation peaks field Note Select Height if the peaks are not completely separated from those of other components Click OK Note The amount and concentration of the sample are multiplied by the multiplier values when the calibration curve is applied to a sample Change the default values if you want to determine the amount or c
372. nu or e Select Edit Fill Peak Result The Color and Pattern dialog box opens Color and Pattern 2 x Color E AMn Cancel e Select a color and a pattern e Click OK Result The peak is filled according to the selections Note The color and pattern selections will override the general Fill settings that can be selected for all peaks on the Peak Table tab in the Chromatogram Layout dialog box Evaluation 12 Peak start andend The beginning of each peak is marked with a drop line above the curve and the points How to split a peak end of each peak is marked with a drop line below the curve The illustration below shows an example of start and end point drop lines Peak start Peak end Where there are two peaks beside one another the end of the first peak will be at the same point as the beginning of the next peak Thus there will be a drop line below and above the curve at the same point See the illustration below It is possible to split the peak into two new peaks by inserting a drop line The table below describes how to split a peak in the Edit Peak Table dialog box Step Action e Click the Edit peaks icon M e Click the peak in the curve or in the peak table to select the peak e Right click and select Split Peak from the shortcut menu or e Select Edit Split Peaks Result A new drop line is inserted at the middle point between the two existing drop l
373. o attach a file Action The Attachments dialog box is displayed r Files Result Method System log Global loa Baseline example 1 res Baseline example 2 res r System information IV Computer amp operating system information ja ARTE Hara Were mionaton IV Integrity check e Depending on the character of the file to be attached select the appropriate tab Result Method System log or Global log e Attach the file Click the Add button Selecta file in the dialog box and click the Attach or OK button Result The selected file is added to the tab in the Attachments dialog box Note To remove a file select the check box and click the Delete button ep 471 15 System maintenance and error reporting 15 2 How to generate problem reports 15 2 1 How to generate a report from the UNICORN Manager Step 3 How to generate and save the report 03 0014 90 ep 472 Step Action To include more information in the report select the appropriate check boxes in the System information field By default all options are checked Computer amp operating system information A summary of the computer and operating system information for example type of processor processor speed RAM hard disk capacity and printer AKTA hardware information A summary of the specific AKTAdesign hardware for example the instrument and PROM version for every instrument that is connected
374. o enhance data file security The table below describes how to sign a result file electronically in the Evaluation module Step Action Choose File Sign Result Result The Sign the Result dialog box opens Signing View Signatures m Sign as user User dtr xf Pasw Full name pen S Poston a Signature Meaning E integration performed e The Sign as user field shows the properties for the current user You can also choose another user from the droplist If you choose a new user the corresponding password must be typed in the Password text box e Type a short text description for the signed operation in the Meaning field e g Peak integration performed e The Lock check box is selected as default to lock the result file from further changes e Type your signature password in the Password field and click OK Note You should only lock the result when you are sure that the result file will not be modified anymore Signatures associ The View Signatures tab of the Sign the Result dialog box provides a list of all ae with the res signatures associated with the current result The information on this tab is for viewing purposes only and cannot be changed e p 325 11 How to edit results 11 11 How to save results and exit the Evaluation module 11 11 Introduction How to delete un wanted curves How to save the results How to exit the Evaluation mod ule 03 0
375. ocol is not displayed at the beginning of each run 03 0014 90 ep 216 9 5 Instruction Unattended Meth odQueue opera tion MethodQueues when the system is busy How to perform method runs 9 How to perform a MethodQueue run The table below describes how to run a MethodQueue Step Action e Make sure that all systems used in the MethodQueue are connected with control mode connections 2 Select a MethodQueue in the Methods pane in the UNICORN Manager and e choose File Run or e right click the MethodQueue icon in the Methods pane and select Run from the shortcut menu or e double click the MethodQueue icon in the Methods pane and click the Run button in the MethodQueue Editor dialog box Result The MethodQueue will start in accordance with the conditions defined in the MethodQueue setup See 8 1 How to create a new MethodQueue on page 183 for information about how to create a MethodQueue The Start Protocol for the first and each subsequent method step in the MethodQueue is displayed when the corresponding method is run If you require unattended MethodQueue operation after the start of the first method step make sure that subsequent method steps do not include a Start Protocol Note If the Start Protocol for a method in the queue is cancelled the MethodQueue is paused Select MethodQueue Display Running in the UNICORN Manager and Restart or End the run in the displayed dialog box
376. od notes 135 Evaluation Procedures tab 136 How to import evaluation procedures in the method 137 How to edit method procedures 138 Reference curves 140 How to add reference curves 140 Columns tab 142 BufferPrep description 143 How to create a BufferPrep method 144 BufferPrep correction factors 145 Method Information tab description 146 Result Name tab description 147 Result file serial numbers 148 Result file unique identifier 148 Batch ID 148 Start Protocol contents 151 How to export values 153 Scouting tab buttons 176 S Scouting Specify folder for storing results 148 e p xvii Index Start Protocol settings 152 Usage 176 Changing variables 176 Change variable values 176 How to set up a Scouting Scheme 177 How to edit a Scouting Scheme 177 How to rename variables 179 How to delete a variable 179 How to change a variable into a detail variable 179 How to copy variable content 180 How to define columns 181 How to perform a Scouting run 216 Result files 216 Scouting runs Change of variables during a run 216 Searches General functions 37 Security Backup 78 Set_Mark Usage 160 How to issue 161 Slope values Usage 369 How to measure 370 SMART Manager How to import data 319 Smoothing algorithms Moving average 490 Autoregressive 490 Median 491 Savitzky Golay 492 Snapshots How to view 41 How to add a text instruction 90 Standard addition quantitation How to per
377. oduce a peak table Use the peak table from the standard to produce a molecular size table Each peak is represented by a retention value Select the relevant peaks and input data for the corresponding mo lecular sizes Result The software plots these values as a molecular size curve This curve has an inverse relationship between the logarithm of the molecular size and retention Retention 125 log Mol size ep 449 13 The Analysis module 13 6 How to measure molecular size 13 6 1 Overview of molecular size determination How to calculate The table below is a brief description of how to use the molecular size table to the molecular size calculate the molecular size of the components in the sample in the sample Step Action Use the sample peak table to obtain retention values for each of the components of interest mau 13 5 Use the molecular size curve to obtain the molecular sizes of the components in the sample The molecular sizes are presented in the peak table Retention 30 025 05 O75 1 125 E Molecular size of sample component log Mol size 03 0014 90 ep 450 13 6 2 Introduction Before you start The Molecular size table dialog box The Analysis module 13 How to determine the molecular size This section describes the technique for measuring molecular size in detail Before you create the molecular size curve you need to do the following e P
378. odule 13 3 How to prepare for quantitation 13 3 2 How to create a quantitation table Identification set tings How to adjust the identification set tings 03 0014 90 ep 416 The criteria by which peaks are identified are set in the Identification Settings dialog box The criteria are valid for all the selected peaks in the Define Component s dialog box These settings also affect the information provided in the peak table in the dialog box Description By default peaks are identified by their absolute retention values and the highest peak maximum within the window In most cases it is not necessary to change these default settings Peak identification by absolute retention works well when there has been little or no drift in retention between successive runs of the standard Quantitate will find corresponding peaks in these successive runs providing any drift in retention does not move a peak outside the peak window Instruction If you have drifting retention that makes peak identification difficult you can choose to identify peaks according to their position relative to a reference peak The table below describes how to adjust the identification settings in the Define Component s dialog box Step Action Identify a component peak that can be used as the reference Note Choose a peak that is well separated from any other peaks This enables the window to be set relatively wide and the system can accommodate a l
379. og box The table below describes how to save the wizard settings Action e Click the Save Wizard Settings button Result The Save Wizard Settings dialog box opens Type a name in the Wizard settings name field e If the settings are to be used by all users on the system select the Global wizard settings check box e Click OK e Click Cancel to close the wizard Note The Global wizard settings check box can also be used to toggle between lists of stored global and stored user settings How to open the The table below describes how to open the saved wizard settings saved wizard set tings Step Action e Choose the File Multifile Peak Compare Start Wizard With Settings menu item Result The Select Wizard Settings dialog box opens e Select the desired saved settings from the list e Click OK Result The Multifile Peak Compare wizard opens with the saved settings Note The Global wizard settings check box is used to toggle between lists of stored global and stored user settings ep 301 11 How to edit results 11 8 How to import and compare different runs 11 8 2 How to import and compare chromatograms 11 8 2 Introduction Commands to use How to import chromatograms with the command File Open to com pare 03 0014 90 ep 302 How to import and compare chromatograms This section describes e how to import chromatograms from other result files e how to compare wi
380. ok all items are selected by default e Click the OK button Result Only the selected items will be displayed in the logbook The Logbook title in the upper right corner will show the text Filter on to indicate that not all items are visible All items will still be logged in the result file e p 205 9 How to perform method runs 9 2 How to monitor a method run 9 2 5 The Logbook pane How to find log The logbook can be searched for specific text entries The table below describes book text entries ihe function Step Action Right click in the Logbook pane and choose Find Result The Find dialog box opens e Type the text you want to locate e Select search criteria if necessary e Click OK Result The located logbook entry is highlighted 03 0014 90 ep 206 How to perform method runs 9 9 3 Manual system control Introduction This section describes how to control the system with manual commands and instructions In this section This section contains these topics Topic See The toolbar and status bar 9 3 1 Manual instructions 9 3 2 Alarms and warnings 9 3 3 e p 207 9 How to perform method runs 9 3 Manual system control 9 3 1 The toolbar and status bar 9 3 1 Toolbar buttons Manual Direct Commands Direct command button functions 03 0014 90 e p 208 The toolbar and status bar The toolbar at the top of the System Control module contains
381. om the list by clicking the Remove button The Remove all button clears the whole list Note Remove only clears the list the files are not deleted 03 0014 90 ep 224 How to view results 10 How to set prefer The File Navigator will display Recent Runs based on the individual user preference Runs for Recent settings The table below describes how to adjust the preference settings Step Action 1 e Click the Preferences button Result The Preferences dialog box opens x Maximum number of files to keep fi 0 Maximum no of days to keep files fi 0 Files created by the current user only C Files created by the specified users Specify All accessible files IV Remove files when saved OK Cancel Help i 2 e Type the maximum number of files to keep on the list e Type the maximum number of days to keep the files on the list 3 Select which files to display on the list e Only files created by the current user e All files created by specified users Note Click the Specify button to open a dialog box and select from a list with all accessible users e All accessible files regardless of the creator 4 e Choose Remove files when saved if the files are to be removed from the list when they have been saved e Click the OK button Result All new results will be displayed on the Recent Runs list based on the changed preferences ae As close the To close the File Navigator ile Navigator Click the
382. omatically from a run controlled from a remote station in a network installation the results will be printed on the printer currently set up on the local station not on the remote station If you execute the procedure interactively from the Evaluation module on the remote station the results will be printed on the printer set up on the remote station where you are working Evaluation procedures are defined in the Evaluation module Procedures imported to a method can also be viewed and edited in the Method Editor To do this select the required procedure on the list and click the Edit button To select procedures to run select the procedure s that are to be executed at the end of the run The procedures will be executed in the order they appear on the list How to edit methods 6 How to import The table describes how to import global evaluation procedures evaluation proced ures Note Procedures saved with one method file can be imported to another Step Action Select the Evaluation Procedures tab and click the Import button Result The Import dialog box is displayed Choose either option 1 or 2 below Option 1 Select a global UNICORN procedure 1 Select a procedure on the Select list Result The evaluation procedure name is displayed in the Import as field Option 2 Select a procedure from another method 1 Select a method that contains a procedure in the left part of the dialog box Resu
383. omatograms 10 5 How to create and print reports 10 6 Run documentation 10 7 03 0014 90 p 220 10 1 Introduction How to opena result from the UNICORN Man ager How to opena result in the Evalu ation module How to view results 10 How to open a result file All contents of the result files are opened in the Evaluation module By default the chromatograms in a run are shown as opened windows The chromatogram window on top is the active window There is also a minimized Temporary chromatogram window See 10 3 Basic presentation of chromatograms on page 226 for further information about chromatograms Note It is not possible to open the same result file from two different locations simultaneously To open a result file from the UNICORN Manager do one of the following e Double click a result file in the Results window of the UNICORN Manager or e Select a result file icon in the Results window of the UNICORN Manager and select File Open or e Click the Evaluation icon in the UNICORN Manager open the Evaluation module and select a result file from the Open Result dialog box lt a To open a result file in the Evaluation module e Do the following Select File Open Select a result file from the Open Result dialog box or e Do the following Select View File Navigator Locate and select a result file from the File Navigator Note See 10 2 How to use the File Navigator on pa
384. ome situations however it may be more suitable to use a time or volume base for individual blocks To change the base for an existing method Be careful when changing the base for an existing method Changing between time and volume bases can affect the relative duration of steps in the method if different steps use different flow rates If anamed column is selected for the Column parameter in the Other Base instruction the volume specified in the selected column definition will automatically be used for column volume in the method block The column volume for base CV cannot then be changed in the instruction or defined as a variable However the Column parameter should be defined as a variable Choosing a column definition also enables linear flow rate and column performance calculations If the Column parameter in the Other Base instruction is set to Any and the Base parameter is set to CV the column volume is set numerically by the Volume parameter The column volume may be defined as a variable allowing the scale of the run to be decided when the method is actually run In cases where a template or wizard generated method and column are chosen it is easy to select other columns for that method on the Variables tab in Run Setup Note This might not be possible for methods that you have created yourself ep 155 6 How to edit methods 6 6 How to use selected method instructions 6 6 1 Base instruction How to select
385. on 03 0014 90 e p 378 Automated evaluation procedures An evaluation procedure is a recorded sequence of interactive operations in the Evaluation module which can be executed for automated data evaluation and report generation The concept is similar to the macro facilities in other programs This section describes how to work with automated evaluation procedures This section contains these sub sections Topic See How to create a new procedure 12 3 1 How to edit a procedure 12 3 2 How to run a procedure 12 3 3 How to rename and remove procedures 12 3 4 12 3 1 Introduction The Procedure Editor dialog box How to record a procedure How to create a new procedure Evaluation 12 You can use the Procedure Editor to record or create a new procedure The Procedure Editor can also be used to view and edit the instructions within a procedure This section describes how to use the Procedure Editor to record new procedures The illustration below shows the Procedure Editor in Record mode Q Procedure Editor Untitled Fie Control Help a e Instruction p Parameter Sora e 5 AMP_MUL Integration C z AMP_SHIFT File operation CLEAR C Export COPY co CUT Cc hom DERIVATE Other Div C Test UICTACDAL z The table below describes how to record a new procedure Step Action Open the result file in the Evaluation module Choose Procedures Record
386. on How to edit methods 6 Step Action The Answer type determines what is displayed in the question defin ition field to the right of the Answer type field For each answer type do as follows Input field Enter a default answer if required Multiple choice e Click in the text field under Alternatives e Enter the answer e Click the Add Delete button Result The new alternative is added at the end of the list e Repeat this procedure to add new alternatives To remove an al ternative mark the alternative in the scroll list and click the Add Delete button No answer No action taken Value Enter maximum and minimum limits Select the Integer box if the question is to accept integers only as answers Click the Insert button Result The new question is added to the list The table below describes how to preview the questions as they will appear in the Start Protocol Step Action e Select a question e Click the Preview button Result The question is displayed Click the Edit button to return to the question editing mode The table below describes how to edit a question Step Action 1 Select the question you want to edit ep 131 6 How to edit methods 6 5 Run Setup 6 5 4 The Questions tab Click the Replace button How to delete a Do one of the following to delete a question question e Select a question and click the Delete button to remov
387. on 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm When to change the Max baseline level How to set the Max baseline level 03 0014 90 e p 346 In rare cases the top of a broad flat peak can be incorporated as a baseline segment This is one of the very few situations where it is useful to change the Max baseline level The illustration below is an example The table below describes how to set the Max baseline level Step Action Right click in the chromatogram and select Marker Result A vertical line is set in the chromatogram A text box in the top left corner of the chromatogram displays the X axis and Y axis values of the curve at the point where the vertical Marker line crosses the curve e Move the Marker with your mouse e Measure the height of the peak you want to exclude from the baseline Choose Integrate Calculate baseline e Select the Classic checkbox as the Chosen algorithm e Type anew value for Max baseline level Set the level slightly lower than the value that you measured in step 2 e Click OK Evaluation 12 Example of a cor The illustration below is an example of a correct baseline after the Max baseline rect baseline level has been changed e p 347 12 Evaluation 12 1 Peak integration 12 1 5 How to edit the baseline manually 12 1 5 The Edit Baseline dialog box How to use the zoom function 03 0014 90 ep 34
388. on Type the text string you want to find Match whole word only Select the check box if you only want complete string matches not partial matches Select the check box if you only want matches which correspond according to upper case and lower case letters Search from top of docu Select the check box to start the search from the top ment of the document otherwise the search will start from the cursor position Direction Choose whether to search upwards or downwards in the document Commands Use the commands below to find more occurrences of a text string after you have found the first one Press F3 to search for the next occurrence of the string or right click and choose Find next Right click and choose Find previous to search for a previous occurrence The default setting is to search in all result files or chromatograms User entered search filters to a maximum of 10 will be saved in the drop down menus for both Result and Chromatogram selections More than one string can be used as a search delimiter insert between strings and search filters are automatically saved and stored within user profiles Click All to return to the default setting to search in all result files or chromatograms UNICORN concepts 2 2 2 6 Help functions and manuals Introduction There are different ways to get help and instructions in the UNICORN application e From the Help menu in each module e From the con
389. on of the reference point e the minimum maximum and average values for the curve interval between the reference point and the new position How to change The Curves pane displays graphs for the selected curves in different colors with ser a any reference curves included with the method as dashed lines The table below describes how to change the curve colors and styles Step Action Select View Properties Result The Properties dialog box is displayed Select the Curve Style and Color tab e Select a curve from the Curve list e Select an appropriate color and style How to change In most cases the Y axis is automatically scaled for each of the curves Values on ey i of the Y the Y axis apply to the curve with the same color as the axis markings To get the correct Y axis click the legend The table below describes how to fix the scale of individual curves Step Action 1 e Select View Properties Result The Properties dialog box is displayed e Select the Y axis tab 03 0014 90 ep 200 How to perform method runs 9 e Select the appropriate curve e Select Fixed and type a minimum and maximum range in the fields within the specified limits Repeat step 2 for other curves if needed Click OK How to change The table below describes how to change the scale of the X axis the scale of the X axis Step Action e Select View Properties Result T
390. oncentration in the starting volume of the sample instead of in the injected volume of the sample The Analysis module 13 Quantitation stat The Statistics field in the Quantitation table dialog box displays the Correlation and istics Explained variance values when available Click the More button to open the Statistics dialog box for a complete display of available data Statistics x Model TET Statistical reference values e The correlation only available for linear models should be as close as possible to 1 00 e The explained variance value should be as close as possible to 100 Note that the value is usually rather high even for poor models A value of 90 indicates a very poor model The explained variance is not shown for curve models that are drawn through the origin Note If the point to point curve model is selected no statistics are available ep 421 13 The Analysis module 13 3 How to prepare for quantitation 13 3 3 How to edit and update a quantitation table 13 3 3 How to open an existing table The update func tion 03 0014 90 e p 422 How to edit and update a quantitation table The table below describes how to open an existing quantitation table for editing in the Evaluation module Step Action Select Quantitate Edit Quantitation Table Open Result The Open quantitation table dialog box opens Select a quantitation table from the Quantitation table
391. onding recipe in the method will not be updated automatically When you open the method you will be asked if you want to update the parameters in the method recipe The recommendation is that you answer Yes Note The question will not appear if you only change the Correction factors The Correction factors in the method recipe will not be updated How to determine Correction factors can be set to fine tune a recipe around a specific pH to obtain Neer oe high pH accuracy The table below describes how to run the BufferPrep manually changed at 0 and 100 in the System Control module to determine if the Correction factors need to be changed Step Action 1 Choose Manual Other Result The System Other instructions dialog box opens 03 0014 90 ep 542 How to create and edit BufferPrep recipes E Step Action e Select the recipe from the Recipe Name droplist and click the Ex ecute button Result The recipe instruction is added e Click the Pump radio button and select BufferPrep_pH e Set the pH value in the pH parameter box and click the Execute button Result The BufferPrep pH value is added and the run starts e Select Flow e Set the flow rate in the FlowRate parameter box and click Execute Result The new flow rate is added Check the pH reading when it is stable in the BuffPre_pH meter in the Run Data pane Note At least 30 ml of eluent must pass through before the reading stabilizes To displ
392. onnections in accordance with the method notes description or e edit the method instructions in accordance with your system setup How to ei the A new method created from a method template is untitled and must be saved new metho under a method name before it can be used The table below describes how to save a new method Action Click the Save Method toolbar icon or choose File Save e If required save the method in a folder other than the default home folder e Enter a Method name for the method The total path can be up to 256 characters long The method name must be unique for the chosen system within the folder e If you have more than one system connected to the computer choose the System for which the method is intended The method can be run on any system that uses the same strategy Remember that different systems may have different configurations and control capabilities e Choose the Technique for which the method was written e Click OK Result The method is saved but remains open in the Method Editor so that you can continue editing if you wish 03 0014 90 ep 86 How to create amethod 5 Note You might want to sign your method If you do so you can choose to lock the method so that nobody else will be able to change the method See 5 4 How to sign the method on page 91 for further instructions ep 87 5 How to create a method 5 3 How to use Text instructions 5 3 How to use Text instru
393. ope limit will cause the up slopes of the peaks to be recognized as baseline segments The example above was improved by the shorter baseline segments but the high slope of the short segments in the region between the second and the fourth peak still makes the baseline unacceptable In the example below the Slope limit is increased by a factor of 2 5 which produces a correct baseline 03 0014 90 ep 342 Evaluation 12 Too high slope A too high Slope limit value can cause peak limits too high up on the peaks This limit can be the case when the chromatogram includes a very large flow through or solvent peak The large peak affects the calculation of the default parameters and leads to too high values for the Slope limit Note A too high value for the Noise window can have the same effect and be caused by the same situation often also in combination with a high Slope limit Peak limits are defined on peaks in the example below due to the high Slope limit The example below has a much lower Slope limit and a lower Noise window ep 343 12 Evaluation 12 1 Peak integration 12 1 4 How to optimize the baseline with a classic algorithm Noise window 03 0014 90 ep 344 Sometimes you get too many peaks after the peak integration usually because noise on the baseline is erroneously detected as peaks The solution to this is to increase the Noise window parameter However this can result in peak limits too high
394. opens e Click the Browse button to locate the desired picture file Select the picture file and click the Open button Note The file formats bmp emf jpg and tif can be used Result A preview of the selected picture is displayed e Select the desired Settings and click OK Result The picture is inserted onto the page e p 255 10 How to view results 10 6 How to create and print reports 10 6 1 How to create and print a customized report How to adda The table below describes how to add a chromatogram to the report The layout chromatogram or can also be defined to include a peak or pool table if desired peak table Step Action e Click the Chromatogram icon e Press and hold the left mouse button on the report page and drag out a box to the size of the chromatogram Release the mouse button Result The Setup Chromatogram dialog box opens Setup Chromatogram x Selected chromatogram s m Settings r Fonts Tl Thick lines a Chromatogram I Start on new page Peak table T Full page ene Header text I Original file name Eat eA T Current _ Deep Cancel Help Select which chromatogram s to insert from the Selected chromato gram s droplist e Active chromatogram inserts the chromatogram that currently is active in the Evaluation module e All chromatograms inserts all chromatograms that are open in the Evaluation module
395. or of the Pause button is strategy dependent The Pause button suspends execution of a method and stops all pumps so that the system comes to a standstill For KTAdesign systems valves remain in the posi tion they were in before the pause Accumulated time and volume is not increased during a Pause Method instructions are not executed until Continue is pressed Resumes execution of a paused or held method e Terminates method execution e Puts the system into an End state Note You can choose to save the partial result or discard it Note The commands can also be found on the Manual menu Windows buttons The table below describes the functions of the Windows buttons Button Function Opens a dialog box where you can choose which window panes to display This button is equivalent to the menu command View Panes Opens the documentation pages Run notes can be entered on the Notes tab and settings can be changed Other tabs are displayed for information only This button is equivalent to the menu command View Documentation e p 209 9 How to perform method runs 9 3 Manual system control 9 3 1 The toolbar and status bar System Access buttons Status bar connec tion status 03 0014 90 ep 210 Button Function i Opens the properties pages This button is equivalent ii to the menu command View Properties There are two functions of the System Access buttons
396. ost as a result of computer failure or other incidents 03 0014 90 ep 78 How to create amethod 5 5 How to create a method Introduction Chromatography runs are programmed as Methods in UNICORN Before you can proceed with a chromatography run you need either to use an existing method or create a new method This chapter describes how to create new methods It also contains instructions for signing a method In this chapter This chapter contains these sections Topic See How to use the Method Wizard How to use the Method templates How to use Text instructions How to sign the method 5 4 e p79 5 How to create a method 5 1 How to use the Method Wizard 5 1 Introduction Are wizards al ways available 03 0014 90 e p 80 How to use the Method Wizard This section describes how to use a Method Wizard to create a new method For most purposes customized methods can be created simply by setting appropriate values for the method variables Note Each method is written for a specific strategy The function of the method cannot be guaranteed on systems having other strategies Method Wizards are available for some KTAdesign systems delivered with standard strategies Method Wizards are not available for process systems How to create amethod 5 How to createa The table below describes how to create a method with the Method Wizard new method Step Action Click the Me
397. ot be used user profile Note Contact the system administrat or if you are not authorized to change your user profile The table below describes some connection problems and their solutions Problem description Solution The connections are not available The connections are not available even though e the connection between PC and chromatography system appears to e be correct e the power is turned on s Check the connection between the PC and the chromatography sys tem Check that the power to the chro matography system is turned on Quit UNICORN Shut down and switch off the computer Switch off the chromatography system Restart the entire system e p479 A Troubleshooting A 2 UNICOR N access The Connection field in System Control displays a NO X 03 0014 90 e p 480 Problem description A system is not available when you attempt to establish a connection You receive the error message Can not connect to system in a network installation You can establish a connection but cannot control the system that is the Manual menu commands in the System Solution Check that you have access rights to the system Access rights are not automatically assigned for a newly defined system e Check that the local computer to which the system is connected is turned on and logged on to the network Check that the computer whe
398. otein concentrations Calculation formula 282 Q Quality control How to create a control procedure 557 How to add a control procedure to a method 559 Quantitation General description 394 Process steps 394 Procedure instructions 396 How to use an external standard 397 External standard reliability 399 Internal standard description 400 How to use an internal standard 400 Standard addition description 404 How to use standard addition 404 Standard addition reliability 405 Recovery calculation description 406 How to use recovery calculation 406 Recovery calculation reliability 407 General reliability factors 408 Preparations before 410 Standard concentration levels 410 Reject peaks 410 How to create a quantitation table 411 How to select table components 414 Peak identification 415 Relative retention 416 How to adjust peak identification settings 416 Peak identification criteria 417 How to create a calibration curve 418 How to enter standard data 418 How to create a calibration curve 418 How to enter standard data 418 How to select an Internal Standard 420 Statistics 421 How to open a table for editing 422 03 0014 90 epxiv How to update a calibration curve 423 How to rename the table 427 How to delete a quantitation table 427 How to prepare for 429 How to calculate the amount and concentration 430 How to view the results 431 Calculation error signs 431 Standard addition stages 4
399. othing parameter value Note If you increase the parameter value the smoothing effect is also increased Note The filter algorithm only accepts integer parameter values between 1 and 25 Median The table below describes the process when the Median smoothing algorithm is used Stage Description For each data point in the source curve the processed curve is calcu lated as the median of the data points within a window centered on the source data point e The width of the window is determined by the parameter value expressed as number of data points 2 When the source point is less than half the window size from the beginning of the end of the curve the median is calculated symmet rically round the source point over as many data points as possible e If you increase the window width the smoothing effect is also increased e To completely remove a noise spike the window width should in effect be slightly more than twice the width of the spike Note The filter algorithm only accepts odd integer parameter values between 1 and 151 If an even number has been given it is incremented by one e p 491 B Evaluation functions and instructions B 1 Smoothing algorithms Savitzky Golay The table below describes the process when the Savitzky Golay smoothing algorithm is used Stage Description The algorithm is based on performing a least squares linear regression fit of a polynominal of degree k o
400. ou have any unsaved data in the Method Editor or Evaluation module e Click Yes to continue to close the program Your unsaved data will be lost when the program is closed e Click No to return to the program and save your data The Leave Control of system dialog box opens Select the locked or unlocked option as in the logoff procedure Note This step only happens when a system is connected Click OK Note Do not shut down Windows 2000 XP or turn off the computer if you quit UNICORN with a separation run in progress If you are performing a Scouting run or a MethodQueue run you cannot quit the program at all In case your access to the UNICORN Manager is restricted you will still be able to quit the program e p49 3 General system operations 3 2 How to create a new user 3 2 Introduction Default user 03 0014 90 ep 50 How to create a new user This section describes how to create a new user and assign a home folder for the user s methods and results A default user is created when the system is installed The default user has unrestricted access to all UNICORN functions You log on with this profile when you access a newly installed system for the first time The table below describes how to log on as the default user Step Action Select user default from the user drop list Type password default if necessary Note The default user is the only user that is allowed to use the user
401. ound_Sample Block VolumeFractionation Block Lincar_Gradient Block Gradient_Delay Block Fractionation Stop Block Clean_after_Elution If the method is a scouting run click Run X to move between runs ep 159 6 How to edit methods 6 6 How to use selected method instructions 6 6 4 Messages and Set_Marks 6 6 4 When to use a message How to adda Message instruc tion When to use a Set_Mark 03 0014 90 ep 160 Messages and Set_Marks Messages are used to inform the operator of the progress of the run It is a good idea to issue messages at critical points in the method for example when Watch instructions are used for conditional events The Message instruction can be used to set up a message that will be displayed for the user during the execution of the method run The message can be for information in a screen only or it can require a signature before the user can control the system The messages are all added to the logbook text See F 6 Appendix Messages on page 555 for examples The table below describes how to add a Message instruction to the method Step Action e Select Other in the Instructions field of the Instructions box e Select Message in the instructions list Type a message in the Message text box in the Parameters field Select one of the display options on the Mode menu e Screen i e only a text message is displayed e Noscreen i e the message will not be displ
402. ow to edit the Column List Step Action Click the Export button Result The Export Column dialog box opens e Click the checkbox for each column you want to export e Click Export Result The Export Column to file dialog box opens e Select the desired folder in the navigation window e Type a new file name if neccessary e Choose the type of file to export column file or Excel file e Click the Save button Result The column file is saved and the dialog box closes Note If a column is selected in the Column List when the Export Column dialog box is opened this column will automatically be selected in the Export Column dialog box earth to importa The table below describes how to import a column column Step Action Choose Edit Column List Result The Column List dialog box opens Click the Import button Result The Import Column dialog box opens e Click the Browse button to locate the column file Result The Import Column from file dialog box opens e Select a column file e Click Open Result The Import Columns dialog box opens e Select the columns to import from the list e Select Import as global to add the columns to the global column list if desired e Click Import Result The selected columns are imported and available in the column list 03 0014 90 ep 534 The Column list D Note Select Import as global to import the columns to the global column list e p
403. ow to print descriptions of the instructions in your particular strategy Step Action Select File Print in the Method Editor e Select the Instruction set option to print the full set of instructions e Click OK The Snapshot instruction can be used to record the curve values at a specific point in the method run For example a snapshot can be inserted to record the curve values immediately before an injection The values are recorded in the result file and can be viewed in the Snapshots tab of the Documentation dialog box See 10 7 Run documentation on page 270 Up to 500 snapshots can be recorded in each result file The table below describes how to add a snapshot instruction to a method e In the Text pane select the instruction immediately before the position where you want to insert the Snapshot instruction e Select Other in the Instructions field of the Instructions box e Select Snapshot in the instructions list 3 Type a name in the Name text box in the Parameters field e Click the Insert button Note Snapshots can also be taken in the System Control and Evaluation modules However these snapshots will only record the data for a specific moment For more information about the Snapshot function see 2 2 7 Snapshots on page 41 How to create a method 5 5 4 How to sign the method Instruction If you sign the method you can choose to lock it so that nobody will be able to change it The table b
404. ow to sign results electronically 11 10 How to save results and exit the Evaluation module 11 11 e p 273 11 How to edit results 11 1 How to reduce noise and remove ghost peaks 11 1 Introduction How to reduce noise and remove ghost peaks Sometimes the chromatograms contain curves with a noisy baseline The noise can be caused by several factors for example a dirty flow cell air bubbles electrical noise dirty buffers etc The amount of noise can usually be reduced by taking proper precautions for example filtration of buffers and instrument maintenance You can also use the smoothing function to reduce or remove background noise from a selected curve Smoothing is always a compromise between noise removal and preservation of peak shape How to smootha The table below describes how to select a smoothing function and smooth a curve curve 03 0014 90 e p 274 Step Action Select Operations Smooth Result The Smooth dialog box is displayed Select the curve to be smoothed and its target destination Select the Filter type to be applied The options are e Moving average Use this if you have noise along most of the curve It affects peak height but not retention There is little effect on the peak area e Autoregressive Use this if you have periodic noise along the whole curve It affects peak height and retention although this has little effect on the peak area e Median Use this if
405. own in the ration Curve dia log box 03 0014 90 ep 424 Component name table When a component is highlighted its calibration curve is displayed above in the Calibration curve before update field The calibration curve to be updated is shown without taking the new point into consideration A new point is shown either in green or red If it is green the area falls within the set Limit value and this point will be used for calculation of the new calibration curve instead of the old point If it is red it falls outside this range Update Calibration Curve x m Level no 2 Calbration curve before update m Update by fuea Average Replace 40 8519 0 0614 5 1077 112 5148 28 4709 14 0643 How to update a calibration curve Update by Aver age The Analysis module 13 Peak size deviation The Deviation column of the Update Calibration Curve dialog box shows how much the peak size for the proposed new point differs from the existing size The Limit column displays the set limit for the deviation The default value is 12 5 of the existing peak size You can edit the Limit value Use the Deviation and limit as radio buttons to specify if both of these columns are expressed in Absolute or Relative units Instruction The table below describes how to use the Update Calibration Curve dialog box for calibration curve updates Step Action Choose to update by Averag
406. peak at the point where the skim line and the curve slope are equal The illustration below is an example of how a drop line A and a skimmed peak B affects the area under the main peak and the peak shoulder The peak shoulder area is marked in gray Drop line ay to skim The table below describes how to select a ratio to skim peaks in the Integrate dialog pee box Determine the ratio when peak skimming should be applied based on the relationship in the illustration below Note The default ratio value is 10 03 0014 90 p 360 How to integrate part of a curve Evaluation 12 Step Action 3 Type the ratio value in the text box Part of a curve can be selected in the Edit Peak Table dialog box and integrated with settings that differ from the rest of the curve The table below describes how to do this Step Action e Choose Integrate Edit Peak Table Result The Select Peak Table to Edit dialog box opens e Select the peak table to edit and click OK Result The Edit Peak Table dialog box opens e Click the Peak Window icon Result Two vertical cursor lines are displayed e Drag the cursor lines to the beginning and the end of the selected part of the curve No Penk name Retention min Penk start min Peak end min Area mAU ain 2 Internal stt 5 83 5 53 6 28 61 1651 2 Peak A 0 5 Ta Peak 6 0 8 4 Peak 0 5 4l Note All operations descri
407. peak area total area 0 813459 03 0014 90 ep 434 The Analysis module 13 4 3 How to calculate the recovery factor 13 How to prepare The table below briefly describes how to prepare for the quantitation for the quantita tion Step Action Prepare a quantitation table for the components of interest Note An external standard quantitation must be used Internal standard quantitation tables cannot be used Perform a sample run with the unspiked sample and a run with the spiked sample Peak integrate the sample curves to produce the peak tables for the unspiked and the spiked samples Note The sample curves must use the same X axis base unit as the standards during the integration Time is the recommended unit for highest reliability e Check that the integration is correct e Optimize the integration if necessary e Open one of the sample result files e Use File Open Peak Tables to copy the other peak table to that result file Select File Save to save the result How to calculate The table below describes how to calculate the recovery factor the recovery Step Action Select Quantitate Calculate Recovery Result The Calculate Recovery Factor dialog box opens Calculate Recovery Factor f Quantitation table C Geiba G Personal Addon component Added amount Ew_Std_Rito Yj Rbonuclease A z 0 3 mg r Select peak table Source chiomstogram Addition chromatogram fi
408. peak table for both the spiked and the unspiked sample Result The difference in peak area between the spiked and the un spiked sample represents the peak area from the added amount 4 With the assumption of a linear proportionality between the peak area and amount and with the added amount known the software calculates the amount of the component of interest in the sample Peak area from unspiked sample Peak area spiked sample Peak area unspiked sample Unspiked sample amount Amount added x Reliability Standard addition is the least precise of the quantitation techniques since it is restricted to a single concentration level and the amount in the sample is calculated by extrapolation Below are factors that determine if standard addition can be used with reliable results e The component of interest must be completely resolved from all other components in the chromatogram Overlapping peaks will produce unreliable results e The peak integration parameters baseline settings must be correctly selected The default settings will be satisfactory in many cases but the integration results have to be checked for all chromatograms e The standard addition technique assumes a linear through the origin relationship between the amount of component and peak size This is a good approximation for small quantities under normal conditions e Standard addition has no way of compensating for changes that are made between th
409. pecified block or an instruction when a particular monitor signal meets a given condition As long as the condition is not met the block is not activated Note Watch instructions are shown in the Instruction box of the Text Instructions editor indicated in the Block pane by a green line that shows the start and duration of the watch The breakpoint when the Watch instruction is issued determines when the watch begins not when the block is activated A watch is active from the point at which it is issued until e the Watch condition is met or e anew watch is set for the same monitor or e a Watch_Off instruction is issued for the monitor Watch instructions are inserted in the Instruction box of the Text Instructions Editor The table below describes how to do this Action In the Breakpoint field select the appropriate breakpoint This decides when the watch begins e Select Watch in the Instructions field e Select a Watch instruction from the list e Select appropriate values under Test Value and Action in the Parameters field Click the Insert button Result The new Watch instruction is inserted on the list of actions in the Text window How to edit methods 6 Test optionsinthe The table below describes the Watch options that are available on the Test Parameters field drop down list of the Parameters field Option Explanation Greater_Than The signal exceeds a certain value Less Than The s
410. ple run Retention scale Evaluation 12 How to find slope values With AKTAdesign systems it is possible to only collect peaks during fractionation The way to find suitable slope values for a particular run is described in this section The slope values can be used in the Method Editor e as StartSlope and EndSlope values in the Peak_FracParameters instruction e as parameters for the Watch instruction Using slope values for Watch instructions Conditional Watch instructions can be set up to let the progress of a run be determined by the events during the run e g start to collect fractions when the first peak emerges The slope of the curve can be set as a condition to satisfy a Watch condition in the method during the run It is important to use accurate slope values for the specific Watch instruction parameter You must first make a separation run with the sample you intend to purify The result from this separation run is then used to find the slope values Time should be used as the X axis scale for retention Step Action Click the Chromatogram Layout icon e Click the X axis tab e Select Time e Click OK e p 369 12 Evaluation 12 2 Other evaluations 12 2 2 How to find slope values Hosto differenti The slope values are measured on a differentiated curve The table below describes ate the curve How to measure the slope values how to create a differentiated curve S
411. pline mode draws the curve as a smooth line near but not through every point e Click the Spline through checkbox to draw the curve through all of the curve points Move a point e Select the point and drag it to the new position Result The curve is redrawn 03 0014 90 ep 374 Evaluation 12 Step Action Delete a curve point e Double click the curve point or e Select the point and click the Delete button or e Select the point right click and choose Delete Point from the shortcut menu e Click the Zoom icon to focus on details in the curve Note Right click and select Reset zoom to return to the full view e Right click in the chromatogram window and select Marker e Position the Marker bar over peaks in the help curve to measure the coordinates Result The coordinates are displayed in the Marker text box in the top left corner of the chromatogram Note Click the Marker text box to display the coordinates for the created curve Click again to return to the help curve coordinates Click OK Result The Save Curve dialog box opens Type a new name if desired and click OK ep 375 12 Evaluation 12 2 Other evaluations 12 2 4 How to create curves Curve example The illustration below is an example of a curve created by using the Draw Spline command in the Create Curve chromatogram window Create Curve 15 x 2 ga idi QuantitateOO1 1_UV1_220nm id 148 Qwandt
412. point where the vertical Marker line crosses the curve See the illustration below Note The color of the Marker is the same as the selected curve Move the Marker with your mouse to display the peak data Click the curve name legend above the chromatogram to change to another curve Result The Y axis is changed to the one corresponding to the new curve Right click and select Marker again to de select the function e p 363 12 Evaluation 12 1 Peak integration 12 1 8 Measurements How to set arefer The table describes how to set a reference point ence point Right click in the chromatogram and select Set Marker Ref Point to define a reference point for the marker position When the marker is moved from the reference point the X axis and Y axis values for the new position are displayed together with e the new position in relation to the position of the reference point e the minimum maximum and average values for the curve interval between the reference point and the new position a ic record a The table below describes how to record a Snapshot of the current curve values napshot Step Action e Right click in the chromatogram and select Snapshot from the shortcut menu Result The Snapshot dialog box opens 2 The dialog box displays all the curve data that was current at the moment the snapshot was taken e Click the Save to file button to save the snapshot as an Excel
413. pools e Click the numbered marker to select the pool and click the Delete button Click the Delete All button to clear all pools To restore the pools created by UNICORN e Click the Default Pool button Other curves can be selected for the operation e Select another source curve from the Source curve droplist and click the Default Pool button or e Select another baseline curve from the Baseline droplist and click the Default Pool button or e Select another fraction curve from the Fraction curve droplist and click the Default Pool button Result The pooled fractions in the list are replaced by the pooled fractions for the selected curve How to createa The pooled fractions can be stored as a new curve pool fraction j curve Note You must store the pooled fractions as a new curve in order to be able to proceed with other operations using the pooled fractions Step Action 1 e Choose Operations Create Pool Fraction curve Result The Create Pool Fraction Curve dialog box opens 03 0014 90 ep 280 How to edit results 11 Step Action 2 e Select a position where the curve will be stored from the Save curve in list e If needed type a new name in the Curve name text box Note The suggested curve name will have the default suffix POOL e Click the OK button Result The Pool Fraction curve is displayed in the chromatogram How to show only The active chromatogram will no
414. pter contains these sections Topic See Log on and log off routines How to assign user properties How to change your passwords and user attributes How to connect to the chromatography system How to back up and restore system data How to set up a printer 3 7 e p45 3 General system operations 3 1 Log on routines and log off routines 3 1 Introduction Username and password How to start the program How to log on 03 0014 90 ep 46 Log on routines and log off routines This section describes how to start and quit the UNICORN program and how to log on and log off Normally the system administrator defines the users and creates your first password The program can also be set up so you can log on without a password Note The first time after UNICORN has been installed you may need to log on as a default user and create a user profile This process is described in 3 2 How to create a new user on page 50 Note if UNICORN is already started by a previous user proceed to How to log on There are two ways to start the program If you start with Then a UNICORN icon on double click the icon your desktop Ab Ove if TORN the Windows Start locate the program under Programs Unicorn and click menu in Windows the UNICORN logo 2000 the Windows Start locate the program under All programs Unicorn and menu in Windows XP click the UNICORN logo The table below describ
415. r 15 2 1 How to generate a report from the UNICORN Manager Introduction The Generate Report Wizard is used to generate problem reports This section describes how to generate a problem report from the UNICORN Manager Step 1 How to The table below describes how to create a report with the Generate Report Wizard create the report Step Action 1 Select Administration Create System Report in the UNICORN Manager module 2 The first step is a Welcome screen e Click the Next button Result The Systems dialog box opens with a list of the available systems for the logged on user e Select a system for which the report is to be generated and click the Next button Result The Description dialog box opens 3 Add the following information in the dialog box e a short description of the problem e the circumstances under which the problem occurs e the consequences of the problem Click the Next button Result The Reproducibility dialog box opens 4 Specify whether the problem is reproducible or not Select one of these alternatives e Yes Provide a short description in the text box of how the problem can be reproduced e No e Unknown Click the Next button to proceed to attach example files see table below 03 0014 90 ep 470 System maintenance and error reporting 15 el a toat You can attach result files method files and or log files to the problem report t m The table below describes how t
416. r all chromatograms All integrations must be performed using the same X axis base unit For highest reliability time is the recommended unit The concentration levels of the standard have to be accurately prepared Errors in the amount or concentration values will lead to unpredictable results Self imposed limitations such as the use of a small number of concentration levels of the standard also limits precision Precision is improved by the appropriate choice of the concentration range of the standard The range should extend across the presumed amount in the sample Use of the most appropriate curve model will maximize precision Accuracy is improved if several runs are performed at each level All the runs should be performed consecutively to reduce systematic errors and thereby maximize precision Refer to statistical reference books for more detailed information about quantitative analysis An example is Statistics for Analytical Chemistry 3rd Edition 1993 J C Miller and J N MIller Ellis Horwood PTR Prentice Hall The Analysis module 13 13 3 How to prepare for quantitation Introduction This section describes how to use peak data from standards to prepare quantitation tables and calibration curves for use with External standard Internal standard and Recovery quantitation In this section This section contains these sub sections Topic See Preparations before quantitation 13 3 1 How to create a q
417. rPrep Reference Curves Columns used in method Column Data HiLoad_16 10_Q_Sepharose_HP HLoad 16 10 Q Sepharose HP Normal Parameters Advanced Parametess Method_Wwizard_Mex_PlowAlete_Column 7 Parameter j Value Height mand 10 0 cm Diameter mand 1 6 cm Column volume 20 106 mi Column volume unit mand ml Defaut tlownale mend 30 mW min Max thunale 5 0 mimin Typ peak width at base 15 0 mi pH High value longteim 13 pH Low value longterm 4 pH High value shortterm 14 pH Low value shoilterm 3 Average paiticle dam pm Code no 17 1064 01 Typical loading range 200 1200 mg Mol weigh range kDa Scantale Spectra sec 03 0014 90 ep 142 6 5 10 BufferPrep usage Illustration Stock solutions How to edit methods 6 The BufferPrep tab BufferPrep allows a buffer of different pH and salt concentrations to be prepared on line from four stock solutions This removes the need to manually prepare new buffers every time the pH needs to be changed Linear and step salt gradients can be run and pH can be used as a variable scouting parameter BufferPrep is optimized for cation and anion exchange chromatography For a complete description of BufferPrep see the user manual for AKT Adesign systems Note BufferPrep is only available for some AKT Adesign systems The illustration below shows an example of the BufferPrep tab Evaluation Procedures Method Infomation S
418. ractionation ends 0 00 End Block How to issue a Set_Marks are issued from the Instructions box of the Text Instructions editor The Set_Mark table below describes how to do this Step Action Select Other Set_Mark in the Instructions box Type the message in the Mark text field Click the Insert button Result A new line with the Set_Mark is added to the text instruction ep 161 6 How to edit methods 6 6 How to use selected method instructions 6 6 5 How to delay a method 6 6 5 Introduction Hold Pause Hold_Until 03 0014 90 ep 162 How to delay a method A method can be programmed to be delayed at critical points There are three instructions for this purpose Hold Pause and Hold_Until These instructions are described below The Hold instruction suspends the execution of the method but continues to pump eluent at the current flow rate and concentration settings For example this instruction is useful for giving the operator time to load a sample loop Resume the method The method may be resumed if you click Continue on the System Control toolbar The Pause instruction suspends execution of the method and stops the pumps so that the system comes to a standstill In AKT Adesign systems valves remain in the position they were in before the pause The pause may be defined as indefinite or for a given number of minutes This instruction is most useful for stopping the
419. radient tab 6 5 5 The Notes tab 6 5 6 The Evaluation Procedures tab 6 5 7 The Reference Curves tab 6 5 8 The Columns tab 6 5 9 The BufferPrep tab 6 5 10 The Method Information tab 6 5 11 The Result Name tab 6 5 12 The Frac 950 tab 6 5 13 The Start Protocol tab 6 5 14 How to export the values in the Run Setup 6 5 15 03 0014 90 ep 122 How to edit methods 6 6 5 1 Overview of Run Setup Introduction To access Run Setup either e Click the Run Setup icon on the Method Editor toolbar B or e Select View Run Setup Illustration of Run The illustration below shows an example of the Run Setup with the Variables tab Setup selected Evaluation Procedures Method Information StetProtocd Questions Resuk Name Frac 950 Variables Scouting Netes Gradient BwlePiep Columns Reference Curves Man Column mi 0 100 0 100 99999 000 flow Role Plow Rale imin aoo fooo 1o0o0 Colman Presswie Limit Colma Presswelimk MPat Jaoo fooo 1oo Stat_Insiuctions Wavelength tin o ow y poo O Oo a o ooe pooo O a 190 700 BulferValveAltnit BulterValve At Ir et ANT EluentAtniet a a CCisdC fEluent BIdet Puro B inet o o O oo Cohena valve Cohen Posion fFlowlhvough Fiactionation Fiowthrough_FiacSize mb h00 59953000 Fractionation Ele Frac Sze im 0 000 3989 000 O o Psem o o 0 000 9989 00 Linear Giadert Tamet Cons iB o flen Gradient CV E f ee SSS
420. ration x m Quantitation table TTT Global C Personal m Select peak table Source chromatogram 1 v Peak table s Injection volume id148Quantitate001 1_ UV1_ 280nm 01PEAK fo OK Cancel Help Select a quantitation table on the Quantitation table droplist Select the chromatogram that contains the sample curve on the Source chromatogram droplist Select the sample peak table from the Peak table s list Check the Injection volume value and type a new value if necessary Note For internal standard quantitation the injection volume must be the same as used for the standard runs e Click the OK button Result The peak table is updated 03 0014 90 e p 430 The Analysis module 13 How to view the The results of the quantitation are shown in the Concentration and Amount peak aaa res table columns of the Evaluation module The Peak Names are shown in the table and the type of quantitation is also listed See illustration below A ID 265002 10_UV2_215ine 02 PEAK1 No Peak name Retention min rea mAU min Height mAv i Cone mg ml Amount mg 1 Component 1 8 49 106 7160 584 031 2 Component 2 9 24 75 4914 466 665 7 957 0 596 3 Component 3 9 77 98 1309 593 738 7 939 0 397 4 Component 10 45 175 9518 694 142 7 900 0 395 5 6 Tocal nurber of detected peaks 60 7 Total area wAU min 886 0878 8 Acea in evaluated peaks mAU min 456 2401 To Ratio peak area
421. re logged for every run with date time and current user name where appropriate The logbook thus provides a complete history of any given run The log is saved in the result file The illustration below shows an example of the Logbook pane 0 00 min Method Run 3 26 2002 8 47 16 AM Method sdtest Result v _ Niktas ches001 res 0 00 min Batch ID 4E9820F 4 3804 11D6 AC46 00008728BCCO 0 00 min Base CV 0 10 mil 0 05 min Block Flow_Rote 0 05 min Base SemeAsMain 0 05 min Flow 1 00 ml min 0 05 min End Block 0 05 min Block Column_Pressure_Lima 0 05 min Base SarneAsMain 0 05 min Alarn_Pressure Enabled 400MPe 0 00MPa 0 05 min End Block 0 05 min Block Stert_Instructions 0 05 min Base SameAsMain 0 05 min Wovelength 260 nm OFF OFF 0 05 min AveregingTimeUV 2 56 sec 0 05 min End Block 0 05 min Block Butte Valve_A1_Inlet z Note The second logbook line is the BatchID that is automatically generated The Logbook pane can autoscroll to display the latest entries Right click in the pane and select Autoscroll You can also select the Autoscroll option in the Properties dialog box View Properties and select the Logbook tab You can choose to display only selected items in the logbook The table below describes how to activate the filter Action e Right click in the Logbook pane and choose Properties Result The Properties dialog box opens e Choose the Logbook tab e Select the items you want to display in the logbo
422. re you try to establish a connection is logged on to the network e Check that the limit of 8 connec tions to the system has not been exceeded e Check that no other user has a control mode connection Control are grey e Check that you have sufficient ac cess rights to control the system manually Note The Method Wizard can be used on a local system even if the network connection is not established The table below describes some connection problems and their solutions Problem Description The Connection field in the Run data pane in System Control says NO 1 or NO 2 Solution Check that the UNICORN PC Control board is configured according to the settings made during the installation of the program The same Control unit number Address and IRQ must be set at the Control board see the Administration and technic al manual Hardware installation The communication may also fail if there is a conflict between the UNICORN PC Control board configurations and other boards in the PC If so select a free Address and a free IRQ during UNICORN installation and at the Control Board see the Administration and technical manual Hardware installation Troubleshooting A Problem Description Solution The Connection field in Choose Administration System Setup in the UNICORN the Run data pane in Manager System Control Bays Select the system with problems in
423. reate and print a customized report Step Action 3 e Select the desired options and click OK Result The object is inserted onto the page Note If you want to edit an object later double click the object box How to add free The table below describes how to add free text to the report text Step Action e Click the Free Text icon it e Press and hold the left mouse button on the report page and drag out a box to the size of the text Release the button Result The Setup Free Text dialog box opens e Type text in the edit field e Select if the text is to start on a new page e Select if the text box should be automatically sized e Select if the text should appear in the same position on all pages for example as header and footer text e Click the Font button to change the default font Result The Font dialog box opens e Make the necessary changes and click OK to return e Click OK Result The text object is inserted onto the page 03 0014 90 ep 254 How to view results 10 How to adda pic The Picture dialog box is useful to insert logos pictures or other figures in the turg report The table below describes how to add a picture object to the report Step Action e Press and hold the left mouse button on the report page and drag out a box to the size of the picture item Release the mouse button e Click the Picture icon ia Result The Picture dialog box
424. ree levels 15 0 19 5 46 0 6 5 17 0 min Area Standard Area Standard Area Interna Standard Amount tndard Amount intemal amp ndard 13 2 3 Internal standard quantitation Step 5 03 0014 90 ep 402 The Analysis module 13 Step Action 6 e Prepare data from the sample in the same way as the data from the standard runs to produce peak sizes relative to the internal standard peak size e The resulting relative value is applied to the calibration curve to determine the amount and concentration of the component of interest See illustration below Area component of interest Area Interna Standard Amount component of interest Amount ternal Standard Reliability Internal standard quantitation is potentially the most reliable of the quantitation techniques However if the internal standard component is not selected carefully the reliability will probably be worse than with the external standard technique There are some specific factors that can affect the reliability e There is an increased risk of overlap when the extra component the internal standard is added if the sample contains many peaks e The addition of the internal standard must be accurate in both the standards and samples otherwise the precision of the quantitation will be reduced dramatically ep 403 13 The Analysis module 13 2 Quantitation overview 13 2 4 Standard addition quantitation 13 2 4 General
425. res e Click the Close button e Click the check box to de select the quality control procedure Note If the quality control procedure is selected it will initiate a new manual run at the end of the method run e p 559 F Method examples F 7 Quality control procedure 03 0014 90 e p 560 Step Action e Click the Text Instructions icon z e Select the last instruction in the method e Select Other Evaluate in the Instructions field e Select the quality control procedure in the Procedure list e Click the Insert button e Choose File Save or e Click the Save icon Result When the method run is performed the quality control pro cedure will create a second chromatogram If the controlled value is outside the acceptable range the system will be paused A Alarms Description 461 Alarms and warnings Description 215 Effects on the system 215 Analysis External standard quantitation 397 Recovery calculation description 406 Quantitation reliability factors 408 How to create a quantitation table 411 Molecular size determination overview 449 How to create a molecular size table 452 How to calculate molecular size 455 Analysis module How to install 391 Automated quantitation Basic conditions 439 How to prepare the quantitation table 439 How to set up the sample runs 441 Automatic update with Replace 442 Automatic update with Average 443 How to perform updates in scout
426. result from the test run is unacceptable the system can be paused so that the error is not repeated in subsequent runs How to create the The easiest way to create the quality control procedure is to edit an existing To a procedure that includes a peak integration The table below describes how to do this Step Action e Choose Procedures Edit Open in the Evaluation module Result The Open Procedure dialog box opens e Select the procedure Global Integrate_and_Print e Click the OK button Result The Procedure Editor opens with the procedure displayed e Select the REPORT instruction in the procedure e Choose Other and QC_TEST in the Instruction field e Type appropriate values in the Parameter field See QC_TEST Parameter descriptions below e Click the Replace button Result The REPORT instruction is replaced by the QC_TEST instruc tion e Choose File Save As Result The Save As dialog box opens e Type a name for the procedure for example QC_test e Select the Global procedure check box if the procedure is to be available to all users Note If you select File Save to save the procedure it will replace the Global Integrate_and_Print procedure ep 557 F Method examples F 7 Quality control procedure Illustration The The illustration below shows the Procedure Editor with the QC_TEST instruction Procedure Editor displayed amp Procedure Editor Integrate_and_Print mp
427. rget peak table 2 pet 01 125200101 1_UV1_280nm eaktableL 02 125200101 1_U 2_250nm accor 03 125200101 1_UV3_Onm Pe blef 04 125200101 1_Cond a ri 05 125200101 1_Cond Pecktableth 06 125200101 1_Cone 07 125200101 1_pH 08 125200101 1_ Pressure Peak table name 09 125200101 1_Flovw 10 125200101 1_Temp JUv1_280nm 01 PEAK 13 125200101 1_SamplePres 14 125200101 1_SampleFlow Peak window I Accept negative peaks Baseline Calculate baseline 7 Reject peaks I Peak skim jio ratio Baseline settings Column height Jao 00 cm Cokumn 320 ml SE Cancel Help e p 331 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration Peak integration results How to display peak characterist ics 03 0014 90 e p 332 The peak table is displayed underneath the active chromatogram The start point and end point of each peak are marked by vertical marks drop lines in the chromatogram The peaks are automatically labelled according to what is selected in the Curve Style and Color tab of the Chromatogram Layout dialog box This is an illustration of the results after a peak integration Q Uvabsstion Lcample Result002 1 V UMCORN Loca iii Niklas resut Example ResuRO02Z Biample ResokooZres lik Fie Edt View Integrate Operations Procedures Window Help al jx o 2 e p wo 120 140 Oo wo wo mn A Example Ren a0021_UV1_21 SeenG01 PEAKI No Peak
428. rite of baselines and peak tables e Prompt for column before manual runs e Select a size definition and type a value for the Fraction mark height e Select a size definition and type a value for the Injection mark height e Select a size definition and type a value for the Logbook mark height Click OK to finalize or select another definition to edit The Advanced The Advanced window pane is used to define password policies for the user dialog page Normally this is only used by the system administrator i i User properties x E Eric Eric Lemming M Advanced User A Attributes mb essHord aoei Advanced ans Oly rere IV Expires in 30 days BAA Instructions M Password uniqueness V Remember Bo passwords Account lockout IV After Bo bad logons m Automatic workstation IV Lock after minutes I Logoff after J m Signature Password one 03 0014 90 ep 54 How to define ac cess to folders and systems How to define available manual instructions Access groups General system operations 3 Note This dialog page is only available if a required password was selected when the software was installed The Access dialog page is used to define the folders and systems that the user has access to Click the check box for each selected folder and system Up to 20 folders can be set up to be shared Th
429. rm Quit or Lo You might be running a Scouting goff from UNICORN for a connection method or a MethodQueue These functions require a control mode con nection in order to start subsequent cycles correctly Action Stop the Scouting method or MethodQueue before you quit or log off Monitor signals do not appear in the Curves panel in System Control Error message Couldn t create result file Destin ation path could not be found The Method Sys tem Connection dialog box keeps appearing Troubleshooting A The table below describes a problem and its solution Problem description Monitor signals do not appear in the Curves pane in System Control Solution e Choose System Settings in System Control Result The System Instructions dialog box opens e Choose the Curves group in the In structions field e Set the Store option to ON UV1 Parameters Store C OFF R ON i Signals for which Store is set to ON can be selected from the View Proper ties Curves dialog box in System Con trol The table below describes a problem and its solution Problem description If you receive the error message Couldn t create result file Destina tion path could not be found at the end of a method the local computer was unable to access the folder spe cified in the result file path Solution This may happen if the specified folder is on the networ
430. rst curve in the desired sequence in the left field e Select the second curve in the sequence in the middle field e Click the OK button to add the two curves together in a new result curve e Open the Add dialog box again e Select the result curve ADD from the previous addition in the left field e Select the next curve in the sequence in the middle field e Click OK to add the two curves together in a new result curve Repeat steps 3 and 4 until all curves have been added together The final curve should be the cumulative curve for the whole run Note All curves created using the Add operation receive the ADD suffix by default The default curve name can be changed as needed The original curves are distinguished in the chromatogram by underlined curve names e p 277 11 How to edit results 11 4 How to enter and edit text in the chromatogram 11 4 How to enter and edit text in the chromatogram How to enter text Text can be added to the chromatogram The table below describes how to do this Step Action e Right click the curves view of the chromatogram window and select Add text from the menu or e Choose Edit Text Add e Click where you want to insert text in the chromatogram Result A text box opens e Type the text e Click outside the text box to set the text How to edit the The table below describes how to edit inserted text text Step Action Choose Edit Text Edit
431. rtest possible time between samples e p 463 15 System maintenance and error reporting 15 Introduction In this chapter 03 0014 90 ep 464 System maintenance and error reporting This chapter describes the system maintenance and error reporting functions This chapter contains these sections Topic See System maintenance functions 15 1 How to generate problem reports 15 2 System maintenance and error reporting 15 15 1 System maintenance functions Introduction Some strategies support the capacity to view system information for the components in a chromatography unit The system information can be used to issue maintenance warnings for the components This is featured in the strategies for the lab scale AKTAdesign family This section describes the system maintenance functions How to open the The system maintenance functions are controlled in the Maintenance manager dialog Maintenance box in the System Control module manager e Select System Maintenance Result The Maintenance manager dialog box opens with the Info tab selected The connected chromatography system is scanned for its components After a while the components are displayed The illustration below shows the Maintenance manager dialog box with the Info tab selected and general information about the pump displayed Maintenance manager E x Information Warning Info Warming S
432. rves can also be moved see 11 8 4 How to stack and stretch curves on page 312 How to edit results 11 How to use The table below describes how to import individual curves into an active File Open Curves chromatogram with the File Open Curves command Step 1 Action Make sure that the destination chromatogram for the imported curve s is active on the screen e Select File Open Curves in the Evaluation module Result The Open Curves dialog box is displayed Select curves in the Open curves dialog box e Select the folder and the result file in the upper part of the dialog box e Select a chromatogram on the Chromatogram drop down list Usually there is just one chromatogram Result The available curves are listed on the Available list e Click the check boxes on the Available list for the curves that you want to import and click the Select button Result The selected curve s is displayed in the Selected curves list To remove a curve from the Selected curves list click the check box and then click the Remove button e Repeat step 2 if you want to import curves from other chromato grams e Click OK when you have selected the curves you want Change some comparison settings e Choose Edit Chromatogram Layout to open the Chromatogram Layout dialog box e Select or de select the check boxes on the Curve tab to compare a different set of curves e On the Y Axis tab the curves can be scaled
433. s rights to be able to delete global tables The table below describes how the molecular size curve is used to calculate the molecular sizes of the components in the sample Step Action Perform a sample run and peak integrate the curve to produce a peak table Note The sample curve must use the same X axis base unit as the standards Use volume for molecular size calculations ep 455 13 The Analysis module 13 6 How to measure molecular size 13 6 2 How to determine the molecular size Step Action Select Mol Size Calculate Mol Size in the Evaluation module Result The Molecular Size dialog box opens m Molecular size table MolSize_3C 7 Global Personal Select peak table Source chromatogram a Peak table s id 19301 1_UV1_280nm 47 PEAK Cancel Help e Select Global or Personal according to the location of the molecular size table e Select the molecular size table on the Molecular size table droplist e Select a chromatogram on the Source chromatogram droplist e Select a peak table on the Peak table s list and click OK Result The results of the molecular size calculation are shown in the Mol size peak table column See illustration below The Mol size The illustration below shows the Mol size peak table column peak table column No Retention min ol size kDa a 6 42 gt 30 38 22 42 31 15 59 19 57 43 14 05 271 S
434. s 1 3 for other desired peaks in the current curve Use the Next curve and Previous curve buttons to navigate forward and backward among your selected curves and manually check the selections made by the software if necessary Other possible actions you can perform e Ifthe current curve does not prove useful for your comparison click the Delete curve button to delete it from the comparison e Click the Back button to navigate back to the Data Selection dialog box and add new curves to your comparison See also How to change the peak identification below ep 293 11 How to edit results 11 8 How to import and compare different runs 11 8 1 How to use the Multifile Peak Compare wizard How to change the peak identifica tion 03 0014 90 ep 294 Step Action 7 When all peak selections and identification settings are complete click the Next button to proceed to the Peak Data Selection dialog box Note Click and drag in the curve window to zoom into selected peaks to simplify accurate peak identification Right click and click the Reset Zoom button to reset the zoom to the full view In the Peak identification settings table each column identifies a peak parameter to be compared among all peaks If UNICORN has identified other peaks than the intended ones you can change the peak identification manually The table below describes how to change the identification If you want to then remove a pe
435. s 267 10 7 R n COC UAV FILL OM arieni eae ea a iaa A wan E TAR SEE ATOE SENETA 270 11 How t edit 1eSUNtS i emcee nia eal a 273 11 1 How to reduce noise and remove ghost Pp akS cccceccseceseeeeeseeeeeeeeeeneeeneers 274 11 2 How to subtract a blank run CURVE cicnis ccsduessanisite a eneeueiaprova aa mwvanieats 275 TL3 Howto add CUNE Smeti netee eena are A ee Ee Aaea 277 11 4 How to enter and edit text in the chromatogram ss ssssssssrrsrerrrrrerrerrerrerrere 278 TDs OW TO p ol fractions ee e aa a a a E E A 279 11 6 How to match protein activity to a CUTV sssessssssrssrersrrrrrsrrrrrrrrererrerrerrent 285 11 7 How to rename chromatograms curves and peak tables cccccseeeeeeeneee eee 286 11 8 How to import and compare different runs ccccccssssseccseeeseesssseeeeeeeenesseeeeeeeeeees 287 11 8 1 How to use the Multifile Peak Compare WiZard cccccseceseeeeeneeeeeenaeenes 288 11 8 2 How to import and compare Chromatograms ccccccsceseeeeseneeeneeeaeenseenes 302 11 8 3 How to import and compare CUIVES s ctsccrrecercetontuadetaseornsdevacnentytetemcnumenaeted 305 11 8 4 How to stack and stretch CUIVES ccccccseccsecceeeseeeneceseceneeesaseneneneenenenes 312 11 8 5 How to produce a mirror MMAR sein caoteacanie washes cabs tanta Cradesidia eatinacshceenenes 316 11 9 How to import and export reSults ccccceessssseeeeeeeessssseeeeeeeeeesssnaeeeeeeesesneeeeeeeenees 3
436. s and method runs After Windows logout and login you cannot get a system connection Print screen does not send a copy of the screen to the printer 03 0014 90 ep 486 The table below describes a system connection problem This applies only to local systems not remote systems Problem description You have logged out of Windows 2000 and then logged in again but you cannot get a system connection in UNICORN Reason If you shut down Windows 2000 with the command Start Shut down Close all programs and log in as a different user you will not be able to obtain a System Control connection in UNICORN the next time you or another user logs on This is because the described shutdown procedure automatically shuts down a number of processes including those needed for system connection The services are only started when the computer is booted up Solution Restart the computer in order to ob tain a system connection in UNICORN The table below describes how to solve a printing problem Problem description The Print screen command only makes a copy of the screen to the clipboard and not to the default printer Solution If you want to print the view on the screen press the lt Print Scrn gt key and paste the image from the clipboard into an appropriate program such as Microsoft Paint and then print out the image A 4 In this section Incorrect date and time in the result
437. s been selected The appearance of the box to the right of the Answer type field depends on the answer type option selected Answer type _ pValue Input field fires Min C Multiple choice C No Answer Value Ruci e p 129 6 How to edit methods 6 5 Run Setup 6 5 4 The Questions tab The table below describes the different answer types Answer type This option accepts any alphanumerical input as the answer Input field questions may have a default answer Multiple choice allows the user to choose one of a defined set of an swers To allow a blank answer enter a space in one of the predefined answers is used to e display important information or e to split a question over more than one line by set ting all but the last line in a question to No answer Normally each question consists of one line only It is impossible to give an answer to questions with this option selected Value accepts only numerical answers Value questions must have specified maximum and minimum limits and may be defined to accept only integer values How to inserta The table below describes how to insert a question question Step Action If there are questions on the list select the question that should be followed by the new question Enter the question text status answer type and answer option as required 03 0014 90 ep 130 How to preview questions How to edit a questi
438. s including e AKTA design systems e BioProcess systems Note All examples in this guide are based on an AKTAexplorer 100 system that operates with the E100F400 strategy If you use another system you may find that the descriptions and instructions do not match your system on every point In that case you also need to refer to the user documentation for your specific chromatography system Introducing UNICORN 1 System networks UNICORN can be installed on a stand alone computer to control only a single locally attached system However a stand alone computer can control up to four separate systems In a network installation each computer workstation can operate many systems regardless if they are locally connected or not Each system can only be operated by one workstation at a time but several may view the output data T me Software modules The UNICORN control software consists of four integrated modules Module Function UNICORN Manager File handling and administration e g definition of systems and user profile etc Method Editor To create and edit methods for pre programmed control of chromato graphy systems System Control To control and monitor the separation processes online through method based or manual control Evaluation To evaluate and present stored results from separation processes Note All modules are active when the program is operating and are not closed when they are mi
439. screen i e the message will not be displayed but only inserted into the logbook e Authorize i e the message will require a signature from the user before the user can interact with the system again e Select a sound on the Sound menu if desired e Click the Insert button Note If the Message instruction is inserted in a conditional block it will only be displayed if the conditions of the block for example a Watch is fulfilled All messages are erased when the system reaches the End status This also includes Authorize messages A message can be set up in the beginning of a method to protect the method run from unauthorized interference Once the message is issued the system is locked from interaction by any user unless the user provides an authorization signature The only command that is available without authorization is Pause The illustration below shows the text instruction for the message described above E Main 0 00 Base C 0 965 ml RESOURCE_Q_1_ml Column Message Protected Run Authorize No sound E 0 00 Block Flow_Rate Block Column_Pressure_Limit H 0 00 Block Start_Instructions Samco E 2 e p 555 F Method examples F 6 Messages Pausing a method A message can be set up to pause the method until a sample has been injected run for a manual sample injection 03 0014 90 e p 556 manually If a message requiring an authorization is followed by a Pause instruction th
440. search path must be entered in answer to the question If all parameters to this function are OFF then no documenta tion is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file 03 0014 90 ep514 Evaluation functions and instructions B Instruction EXPORT_DOC_XLS EXPORT_DOC_ASCII Description Exports the documentation in the current result file in XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered fol lowed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters to this function are OFF then no documenta tion is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file Exports the documentation in the current result file in ASCII format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters to this function are OFF then no documenta tion is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in
441. sed from the Variables tab 03 0014 90 p 128 6 5 4 Introduction Question status Answer type How to edit methods 6 The Questions tab The Questions tab of Run Setup is used for viewing and adding questions that the system asks a user at the start of a run These questions provide a means for entering structured run specific information Method wizards and templates supplied with UNICORN are defined with a set of questions for sample column and eluent identification Note For questions to be shown in the start protocol the Questions option must be checked on the Start Protocol tab of Run Setup Different types of questions have different status The illustration below shows the Question field an example of a question and the status alternatives that can be used gt Question Sample Volume and Type I Mandatory T Authorized M Chromatogram The table below explains the different alternatives Question status Explanation Mandatory These questions must be answered before a method is started Authorized These questions must be signed with the users signa ture password to unlock and continue the method Chromatogram These questions will be printed with the answers on the same page as the chromatogram if a question is chosen in an evaluation report A question has to be defined to accept one of four types of answers The illustration below shows an example where the Value option ha
442. should only be performed on chromatograms that have been integrated and saved Time is the recommended base unit for quantitation and it must be used for all integrations The table below describes the general steps in quantitation The steps are described in detail in the sections about the different quantitation techniques Step Action Run the different concentration levels of the standard e Integrate the curves to produce peak tables e Check the integration Identify the components for which calibration curves will be pro duced Enter the known concentrations for the different standards to pro duce a calibration curve for each selected component Run the sample and integrate the curve Let the program calculate the concentration and amount of the components of interest in the sample Note The steps above do not apply to Standard addition See Standard addition quantitation below Illustration of the work flow The four quantita tion techniques External standard quantitation Internal standard quantitation Standard addition quantitation The Analysis module 13 The quantitation work flow is illustrated below Area Comporert 3 Area Comporert 2 Area Component 1 Quartitated san ple Am ount Calikration curves Standards at different concentration levels The Analysis module provides four different quantitation techniques External standard quan
443. shows the currently selected last tube The table below describes how to re define the last tube In the Frac 950 dialog box select the Define box in the Last tube field to select the last tube position Place the cursor over the appropriate tube circle within the tube matrix and click again Note When using different sized tubes in the same rack the last tube can be set for both tube sizes Use the Tube type drop down list to choose the desired tube size and then follow the procedures outlined above to select the last tube If you want to return to the default last tube position click the Reset to default button of the Frac 950 dialog box in the Start Protocol when you start a method run How to edit methods 6 6 5 14 The Start Protocol tab Introduction The Start Protocol tab determines which items of the Run Setup are displayed at the start of a method run Click the Start Protocol tab and select the items that you want to be displayed Checkboxes The table below describes the check boxes of the Start Protocol tab Checkbox Displays the Frac 950 setup parameters which can be changed Variables values for method variables that can be changed at the start of the run These values will override the default values for the particular run and be saved in the result file The de fault values stored in the method are not affected the scouting scheme which can be changed at the start of the run
444. ssword rules and the folders and chromatography systems that the user can access This section describes how to assign properties The user properties are defined in the User Setup dialog box in the UNICORN Manager module The table below describes how to open User Setup Step Action Select Administration User Setup Select a user in the Users list Click the Edit button Result The User properties dialog box opens The User properties dialog box is used to edit the user definition and assign properties for passwords folder and system access and available manual instructions The table below describes how to edit the user definition in the User properties dialog box Action Select the User item Select an access group from the Group drop down box Note A pre defined access group is assigned a certain level of access to UNICORN Select a folder from the Home folder drop down box Click the check boxes to select Administrator Attributes Click OK to finalize or select another definition to edit The table below describes how to edit the attributes in the User Attributes window pane Step Action 1 Select the Attributes item ep53 3 General system operations 3 3 How to assign user properties Step Action Select applicable attribute items in the User Attributes pane e Use large toolbar icons e Show unused variables e Show variable details e Default overw
445. standard quantitation 13 2 3 General informa tion General assump tion Advantages What is a suitable internal standard How to perform Internal standard quantitation 03 0014 90 e p 400 Internal standard quantitation Internal standard quantitation uses peak tables prepared from the standard similar to the External standard quantitation However a fixed quantity of an additional component is added to every separation run including the sample The peak sizes of the standards and the sample are then related to the peak size of the internal standards to compensate for any changes that may have occurred between the runs The internal standard technique relies on the assumption that any changes in the injected amount of the component s of interest e g due to sample preparation losses correspond to equal changes in the injected amount of the internal standard component Internal standard quantitation reduces errors that are caused by changes in the system between successive runs with the sample and the standard concentration levels For example there may be unpredictable losses during the sample preparation procedure or unintentional changes in the amounts that are injected A suitable internal standard must meet the following conditions e It must be well separated from the components in the sample not just from the components of interest e It must not be present naturally in the sample s
446. structions in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If 2 is entered fol lowed by text e g Enter a file name as File name a full search path must be entered in answer to the question Exports multiple curves previously defined with EXPORT_SEL_ CURVES instructions in XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If 2 is entered fol lowed by text e g Enter a file name as File name a full search path must be entered in answer to the question Normalizes retention when exporting multiple curves Exports the peak table in Peak table source to the file defined in Export to file in ASCII format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question Exports the peak table in Peak table source to the file defined in Export to file in WKS format If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question 03 0014 90 ep512 Evaluation functions and instructions B Instruction Description EXPORT_PEAKTABLE_XLS Exports the peak table in Peak table source to the f
447. supplies them A copy of these terms and conditions is available on request Any use of this software is subject to Amersham Biosciences Standard Software End User License Agreement Copyright Amersham Biosciences AB 2004 All rights reserved Table of Contents Table of Contents 1 Introducing UNICORN ocak scaccsccns viccecctwtcviesncs aicenscannbeannc eos bas ntudannemiceasealacenlasvonsinveieanentde 9 Delp DOUL NPC OR Nils cnr eshte E E E E ET 10 eZ POOLE thiis manual seenen a a ie e i 14 1 3 About the UNICORN user GOCUMENTATION cccecceeceeceeeeeeeceeeseeeesereeeneeeneenaees 17 2 UNICORN CONCEP S aa araara aa aana aaa a a A Ka rE CE aAa D Ea oand EaU bent seat 20 2 1 Concept SGeLIMITIOMS tcrus het iiancdseesaiietedenededdades shi rttr ttr rttr ert AE SEEE SEEEEEEE EEEE EEEE EEEn 21 2 2 The UNICORN user inteHaGe i sic ind ict ahaa 24 2 2 1 UNICORN Wana ela caviar arate trey eii e E AEAT EE 25 2 2 2 The Method Editor module sssssssssesrsrrerssrrsrrrrrsrsrrerrertererrtrrerrererrrererrnne 28 2 2 3 The System Control module sssssssssesrsrrrrsrrsrrrrrsrsrrsrrsrtsrrrtrrnrtarrrrrnrerrnn 32 2 2 4 The Evaluation MOG Cai ais terrace aaaeatvevemeanierea saa variandk auautiaratowauunda dynes tarda 35 22o SLACH PERCHIONS avarice irs oueat scatters A aa delete S re 37 2 2 6 Help functions and MaMa lSiscinsortataredenwnteiaiwasnentadetacmimee dermbanbetedienente hts densh 39 Le Erde SWAPS AOL Se ar dacctatec erates
448. system in the event of an unexpected condition Resume the method The method may be resumed if you click Continue on the System Control toolbar The Hold_Until instruction is a special kind of Watch instruction The method is put on hold until a specific condition is met signal test or value or the time out is reached Thereafter the remaining instructions in the method are executed Instructions that share the same breakpoint as the Hold_Until instruction but are placed after it in the method will be executed after the Hold_Until conditions have been met How to edit methods 6 6 6 6 Linear flow rates Introduction Linear flow rates cm h can be specified for Flow instructions The volume flow rate is calculated from a specified linear flow rate and the column diameter as given in the column definition How to use linear The table describes how to use linear flow rates flow rates Step Action Select a specific column on the Variables tab of the Run Setup or Insert a column for the Base instruction of the block in the Text In structions Editor In the Instruction box of the Text Instructions editor select Flow and select the Linear Flow option as shown in the illustration below Instruchons Pump Pasometers Flonase 0 00 623 00 Gide va po H cn pean C Flowpelh SampkeFlow DiectLoad AlemstMon PumpwiashE xplorer c Pump WashPurtier Frac Pump WashBasic SyslemWach C Wa
449. t 267 How to edit a customized report 268 Result file name Name options 147 Serial numbers 148 Unique identifier 148 Result files How to open in the UNICORN Manager 69 How to open in the File Navigator 69 Automated printing of 136 Specify folder for storing 148 How to open in UNICORN Manager 221 How to open in UNICORN Manager 221 Electronic signature 325 How to save 326 Run Data pane Description 196 How to change the appearance 196 How to change text color or background 197 How to set pressure units 197 How to view and edit manual instructions 197 Run Setup Description 28 Run Setup from the Method Wizard 82 Tabs description 123 Variable tab view options 125 How to change variable values 125 Blue variable values 125 How to delete variables 126 03 0014 90 epxvi Index How to rename variables 126 How to change a variable into a detail variable 126 How to change a detail variable into a regular variable 127 Scouting tab 128 Questions tab description 129 Question status alternatives 129 Mandatory questions 129 Authorized questions 129 Chromatogram questions 129 Questions answer types 129 How to insert a question 130 How to edit questions 131 Gradient tab description 133 Gradient zoom function 133 Gradient marker line 134 Gradient change X axis base 134 Gradient hatch marks 134 Notes description 135 How to write method notes 135 How to search for text in the meth
450. t Column Valve jEluent_A_Inlet JEluent_B_Inlet JEnd_Eluate_Frac OFlow_Rate Flowthrough_Fractionation Fractionation_Stop Gradient Delay zi Select the blocks you want to delete and click OK Click Yes to confirm The table below describes how to delete an unused method block Step Action Highlight the method block e Press the lt delete gt key or e Right click and choose Delete on the shortcut menu Result The Delete Block dialog box opens Note that the Move button is not available Click the Delete button Result The unused block is deleted and cannot be called upon again in the method ep 105 6 How to edit methods 6 2 Method blocks 6 2 5 How to rename method blocks 6 2 5 Instruction 03 0014 90 ep 106 How to rename method blocks The table below describes how to rename blocks Action Right click the block you want to rename in the Text pane and select Rename Result The Rename Block dialog box is displayed Select the block to rename Buffervalve_A1_Inlet Column_E quilibration Column_Pressure_Limit Column_Valve Eluent_A_Inlet Eluent_B_Inlet End_Eluate_Frac Flow_Rate Flowthrough_Fractionation Gradient_Delay Linear_Gradient Sample_Injection Sample_Injection_ Start_Conc_B Start Eluate Frac Start_Instructions Start with Pumpwaat Explorer System Volume Compensation x New name REE Hename Note By def
451. t D How to edit The table below describes how to edit column parameters in the Method Editor column paramet ers Action Choose Edit Column List Result The Column List dialog box opens Select a column and click the Edit button Result The Edit Column dialog box opens Select the desired parameters and change the value settings e Click the Save button or e Click the Save as button to save the column under a new name Note If a column has been selected and saved in a method and the parameters for the column are changed later the column in the method will not be updated automatically When you open the method you will be asked if you want to update the parameters The recommendation is that you answer Yes How to delete a The table below describes how to delete a column column Action Choose Edit Column List Result The Column List dialog box opens Select a column and click the Delete button Result The Delete Column dialog box opens e Click the checkbox for each column you want to delete e Click OK Result The selected columns are deleted How to exporta The column information for a system can be transferred to another by using the column export and import functions in the column list The table below describes how to export a column Step Action 1 Choose Edit Column List Result The Column List dialog box opens e p 533 D The Column list D 1 H
452. t File Close from the menu in the Procedure Editor dialog box Result The Procedure Editor dialog box is closed and the procedure is saved automatically ep 139 6 How to edit methods 6 5 Run Setup 6 5 8 The Reference Curves tab 6 5 8 The Reference Curves tab Introduction Reference curves are curves from existing result files that you can display in the Curves pane of System Control during a run How to choose You can include up to five reference curves in a method You choose which curves and display refer to display during the run with the View Properties Curves command in System ence curves Control see 9 2 3 The Curves pane on page 199 Reference curves are only displayed during the run Reference curves are not saved in the result file How to add refer The table below describes how to add a reference curve from a result file ence curves Step Action Select the Reference Curves tab and click the Import button Result The Import Reference Curve dialog is displayed e In the left field select the result file containing the curve to be added Result The Select list displays the available curves for the result file e Select the curve you want to add from the Select list e If desired change the curve name in the Import as field Note The curve name has to be changed if a reference curve with that name already exists e Click Import Repeat steps 2 and 3 if you want to add more curves
453. t UV curve with default baseline settings and updates the selected quantitation table with the new standard An update report is then printed Click the Quantitate button Result The Quantitation table dialog box opens Select the quantitation table from the Global or Personal folder Time must be selected as the X axis base unit Result The quantitation table is copied into the Update_Quantitation procedure Note You can only perform one run at each level since the old points in the quantitation table will be replaced after each run Save the method with a new name Perform the run s Result The quantitation table will be updated automatically after each run Note The quantitation table will not be updated if the peak area or peak height of the new and the previous results differ more than the Limit value The Limit value is defined either for peak area or height 03 0014 90 ep 442 The Analysis module 13 How to perform The table below describes how to automatically update a quantitation table with automated update the A verage with the Average y Be Open option Action Open a method in the Method Editor e Click the Scouting tab in the Run Setup dialog box e Click the Clear All button to clear the scouting scheme e Double click each Quantitation_Type table cell and select the correct concentration level for the standards e Click the Evaluation Procedures tab e Select the Update_Quant
454. t a method and save it to another file Step Action In the Text Instructions editor or the Run Setup select File Export Meth od Result The Export Method a box is displayed Export Method 0 Export Method 0 Method C Block list there T Main only I Current expansion M Exclude unused blocks Export Cancel Help Do the following e Select whether the current method should be exported as a Method or as a Block list e Select the appropriate boxes in the Options field to define the level of detail in the information e Click the Export button Result The Export Method to file dialog box is displayed e Enter a file name and select the target drive and folder e Click the Save button Scouting 7 7 Scouting Introduction Scouting is used to repeat a series of Method runs automatically with predetermined changes in the values for one or more Variables A Scouting Scheme is defined as part of the method This chapter describes how to set up a Scouting Scheme and define columns The chapter also provides some usage examples In this chapter This chapter contains these topics Topic See How to set up a Scouting Scheme How to define different columns for scouting 72 e p 175 7 Scouting 7 1 How to set up a Scouting Scheme 7 1 Introduction When to use scouting Variable values Scouting tab but tons 03 0014 90 ep176 How
455. t a specific column is selected Choose the File New Method menu command click the New Method icon the shortcut menu Select the system for which you want to create the method in the For system drop down list Select Template in the Use field Select a chromatographic technique from the Technique drop down list Select a method template from the Template list Select a column from the For column list and click OK ep 8d 5 How to create a method 5 2 How to use the Method templates Note Only columns for the selected technique are displayed If Any is selected as technique all columns are displayed Right click in the textbox to open a list of the column categories to limit the number of displayed columns If you type the beginning of a column name in the textbox UNICORN will automatically complete the column name If you do not find your specific column it can be added to the list The column value recommended flow rate pressure limit and averaging time for the selected column will be automatically copied into the method thus reducing the need to edit the method Method notes Click the Notes and then the Method Notes tabs in the Run Setup The notes describe important information about the template and how the system should be connected so that the method will work correctly Note If your system does not correspond to the description on the Method Notes tab either e rearrange the valves and tubing c
456. t and compare different runs 11 8 3 How to import and compare curves 03 0014 90 e p 308 Step Action If you selected the Stack option in step 3 the Shift Curves by Offset dialog box is displayed Shift Curves by Offset x Offset Unit fe fmu r Selected curves will be shifted M01 Superdex 75 NCC test 3001 1_UV Select All V02 Superdex 75test 4001 1_UV M03 Superdex 75test5001 1_UV Clear M04 Superdex 75test5001 1_U 01 BASEM Cancel Help e You can set the Offset value to increase or decrease the offset distance between the curves e Click OK Result Depending on your previous choices the imported curves are now displayed in the source chromatogram or in a newly created chromatogram Note If curves with several different units have been selected the curves with each different unit will be grouped together with separate offset from the other groups Change some comparison settings e Choose Edit Chromatogram Layout to open the Chromatogram Layout dialog box e Select or de select the check boxes on the Curve tab to compare a different set of curves e On the Y Axis tab the curves can be scaled individually all with the same scale click the All with this unit button e Click OK to display the curves If you stacked the curves and want to change the stack offset e choose Operations Shift offset e type a new Offset value and click OK Note The individual cu
457. t curves from result files into one chromatogram e File Open to compare This is the preferred option if you want to automatically search result files that are stored in the same folder to locate all curves of a specified type for example all UV curves This is especially useful for comparison of curves from scouting runs Moreover the imported curves can be automatically overlaid stacked or presented as mirror images See How to use File Open to compare below e File Open Curves This is the preferred option to import individual curves See How to use File Open Curves below Note Original curves are underlined in the chromatogram imported and created curves are not underlined e p 305 11 How to edit results 11 8 How to import and compare different runs 11 8 3 How to import and compare curves How to use The table below describes how to import curves to a chromatogram with the oe to com command File Open to compare Step Action In the Evaluation module e choose File Open to compare Curves or e click the Open curves to compare toolbar button Result The Open Curves to Compare dialog box opens m Chromatogram selection Folder fe Defeuts Browse Resa Peakskmereroe z bowe a Chromatogram Poo Browse All Curve name uv Browse All m Found curves Clear Select All Peak skim example HIPREPO2 1_UV1_280nm Peak skim example HIPREPO2 1_UV2_406nm TTS
458. t method See the illustration below The Text pane The Text pane displays the method as a list of text instructions The instructions can be organized in blocks denoted by blue square symbols The blocks can be expanded to show the instructions within the block See the illustration below E Main 0 00 Base CY 1 000 Column mi Any H 0 00 Block Flow Rate 18 0 00 Block Column _Pressure_Limit 0 00 Block Start_Instructions 0 00 Block Alarm_Sample_Pressuretimit Alarm_Sampte_PressureLimit 0 00 Base SameAsMoin 0 00 Alarm_SamplePressure Enabled 1 00 4 Sample_PressureLimit MPa 0 1 0 00 End_Block gt 0 00 Block Eluent_A_Inlet Eluent_A_Inlet 0 00 Base SameAsMain 03 0014 90 ep 30 UNICORN concepts 2 The Instruction The Instruction box pane is used to enter edit or delete instructions See the illustration below box pane Toolbar icons in The table below describes the toolbar icons in the module the Method Editor Icon Function The New icon opens the New Method dialog box The dialog box is used to create a new method The New Block icon opens the New Block dialog box which is used to add blocks to a method The Open icon displays all available method files and method folders in the Open dialog box The Save Method icon saves the edited method The Print icon opens the Print dialog box Select the method elements that you want to print The Customise
459. t the method because the system is occupied You have three options Wait until the system is free and start the method again Add the Method to a Method Queue and open the Method Queue editor NOTE This method contains a start protocol that will be executed when the method starts Hep e Select the Add the method to a MethodQueue that will execute as soon as the system is free option e Click OK Result A MethodQueue will automatically be created in the default queue folder The name of the MethodQueue will be the same as the method name followed by a five digit sequence number The method will be executed as soon as the system is free Note A warning note is displayed in the System Busy dialog box if the method includes a Start Protocol The Start Protocol must be completed at the start of the method run before it can be executed 03 0014 90 ep 186 8 2 Method Queues are saved in a sep arate folder How to edit a MethodQueue file Instruction MethodQueues 8 How to edit a MethodQueue MethodQueues are saved in a separate folder within the folder that you specified when you saved the MethodQueue The MethodQueue folder is represented by a special icon in the Methods window of the UNICORN Manager a A MethodQueue folder contains the MethodQueue definition and copies of all included methods The MethodQueue files are copies of the original method files If changes are made
460. tables in the Evaluation module Step Action Choose Edit Rename and the relevant option Chromatogram Curve or Peak Table Result The Rename dialog box opens e Select the appropriate object e Type a new name in the Name field e Click OK Note The original raw data curves cannot be renamed They will not be listed as options in the dialog box 11 8 Introduction In this section How to edit results 11 How to import and compare different runs This section describes how to make comparisons between curves or chromatograms from different runs how to present curves or chromatograms from different runs how to compare curve parameters among curves from different runs how to view several chromatograms at the same time how to overlay curves from different runs in one chromatogram how to stack curves from different runs in one chromatogram how to stretch curves to make comparisons easier how to create mirror images This section contains these sub sections Topic See How to produce a mirror image 11 8 5 e p 287 11 How to edit results 11 8 How to import and compare different runs 11 8 1 How to use the Multifile Peak Compare wizard 11 8 1 How to use the Multifile Peak Compare wizard Introduction This section describes how to use the Multifile Peak Compare wizard to make comparisons between different results for example by comparing area retention etc The differenc
461. tart up phase of the method run You can start a method from the UNICORN Manager in two ways e Select a method in the Methods window and select File Run e Select a method right click and select Run from the displayed menu The table below describes how to start a method run from System Control Step Action Select File Run or click the Run button Result The Run dialog box is displayed Note The Run button will open the method that was used for the previous run if a run has been performed since you logged on Select a method and double click the method icon Result The method run starts If the method includes a Start Protocol this must be completed before the actual method run starts Se further instructions below How to add meth For methods that are used frequently for example column cleaning methods or ods to the File menu 03 0014 90 ep 190 routine separations it may be convenient to define the methods as commands in the File menu The table below describes how to define a method as a command 2 Click the Add button and click OK Result The method name will appear as a command in the File menu If you choose the command the method will start How to start an instant run How to use the Start Protocol How to start a method when the system is busy How to perform method runs 9 You can start a method template or wizard directly if your system has defined
462. ted objects to that of the highlighted object Align bottom Matches the bottom alignment of all selected objects to that of the highlighted object 03 0014 90 ep 260 How to view results 10 Function Adjust to margins Stretches the selected object s to the left and right margins Adjust to left margin Adjusts the selected object s to the left margin Adjust to right margin Adjusts the selected object s to the right margin Adjust to centre Adjusts the selected object s to the center of the page Make same size Adjusts the selected objects to the same size as the highlighted refer ence object Eee Make same width Adjusts the selected objects to the same width as the highlighted reference object Make same height Adjusts the selected objects to the same height as the highlighted reference object Note The Make same size and Make same width functions can only be used to resize the width of chromatograms free text and picture objects How to print the The table below describes how to print the report report Step Action 1 e Choose File Print or e Click the Print icon amp Result The Print dialog box opens Note Printers are set up in the File menu of the UNICORN Manager e p 261 10 How to view results 10 6 How to create and print reports 10 6 1 How to create and print a customized report Step Action 2 e Select the printing ran
463. tep Action Select Operations Differentiate Result The Differentiate dialog box opens e Select the UV curve you want in the Source chromatogram list e Click the First order radio button e Click OK Result The differentiated curve opens in the chromatogram Sometimes the differentiated curve must be filtered to reduce noise and ghost peaks before the measurements See section 12 2 1 Peak purity and peak identification on page 366 The table below describes how to measure the slope values on the differentiated Step Action 1 Click the name of the differentiated curve above the chromatogram window to select the curve Use the zoom function to magnify the curve over an appropriate area Right click and select Marker from the short cut menu Result A vertical cursor bar opens in the chromatogram 4 Place the Marker at the beginning of a peak where you want the Watch conditions to be fulfilled i e where the slope becomes higher Read the actual slope value in the active Marker text box in the top left corner of the chromatogram window g lt Q Note The unit for the differentiated curve is mAU min or AU min Any Y axis value for the differentiated curve is the UV curve slope at the selected retention point Peak fractionation If your system is an KTAdesign system measure the slope at the beginning and for AKTAdesign 03 0014 90 e p 370 the end of the smallest flattest peak of all the pea
464. tes Result The Change user attributes dialog box opens Change User Attributes xj IV Use large toolbar icons J Show unused variables J Show variable details I Default overwrite of baselines and peak tables Quick view curve fi Fraction mark height 2 Character heights bd Injection mark height Bo Percent of window height Y Logbook mark height 3 Character heights Cancel Help Dialog check box options The dialog check box options are described below e Use large toolbar icons Display large toolbar icons in all modules e Show unused variables Show variables that are not used in the method on the Variable page of the Start Protocol e Show variable details Show detailed method variables on the Variable page of the Start Protocol e Default overwrite of baselines and peak tables When new baselines and peak tables are created the old ones are overwritten Mark heights Select a size definition and type the height for the following marks e Fraction mark e Injection mark e Logbook mark Click OK e p 57 3 General system operations 3 5 How to connect to the chromatography system 3 9 Introduction How to establish a connection Remote connec tions 03 0014 90 ep 58 How to connect to the chromatography system A computer can have up to four chromatography systems connected at a time This section describes how to connect to the systems and different connect
465. tetProtocd Questions ResukName Frac 350 Variables Scouting Notes Gradient BufleiPiep Coimns Reference Curves r Status G ON C OFF r Recipe FM Stock solutions Piperazine pH 6 0 6 7 Global gt Inlet 417411418 Bulfer 100 8 1M 0 1 M Piperazine pH Range 6 67 Notes BUFFER SOLUTION 2000mt Piperazine x 6 H20 38 84g Mw 194 2 ID SOLUTION 1000ent Inlet A2 Acid Base Use ampoule 0 1 HCI OIM HO SALT SOLUTION 2000r Natl 233 89 Mw 58 44 Inlet B1 Water Defauk conection factors 0 2 at 048 and 0 4 at 100 B Inlet B2 Sak 2M Nai The solutions and the inlets to which they should be connected are displayed to the right of the dialog box Accuracy of preparation is essential The four stock solutions consist of e a mix of buffering components there can be up to five different buffering components enabling a broad pH range to be covered e an acid HCI or base NaOH for pH on line titration e distilled water e an inert salt for example NaCl for salt gradient formation ep 143 6 How to edit methods 6 5 Run Setup 6 5 10 The BufferPrep tab How to createa Jfa suitable template or wizard is not available you can create a BufferPrep method BuieEce meth yourself The instruction BufferPrep_pH must be available at breakpoint zero at the beginning of the method The method must not contain the instructions PumpAlnlet or PumpBlnlet The table
466. text sensitive help in each dialog box e By selecting the Online Manual from the Help menu e By pressing the lt F1 gt key e By right clicking an instruction in the Method Editor and selecting the What s This menu item The Help menu e From the Help menu in each module you can access the Help file e From the Help menu of the UNICORN Manager module you can also access the installed manuals The illustration below shows the Help menu of the UNICORN Manager module Help Help for UNICORN Manager Index About The Help file The table below describes how to open and use the Help file Step Action Choose Help Index Result The Help file is displayed e Type a word you want help on in the text box in the left pane Result The closest matches are displayed in the list e Select a match and click the Display button Result The associated help text is displayed in the right pane e You can also click the Contents tab to view the contents of the Help file divided into sections e Click the plus signs to expand the tree structure e Click a topic to read the associated help text ep39 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 6 Help functions and manuals Manuals Context sensitive help 03 0014 90 e p40 When UNICORN was installed the administrator selected which manuals to install Therefore the available manuals may be different on your system than in t
467. th chromatograms in an already opened result file Two commands in the Evaluation module can be used to import chromatograms from result files into an already opened result file e File Open to compare This is the preferred option when you search for many chromatograms in a specific folder based on defined selection criteria See How to import chromatograms with the command File Open to compare below e File Open This is the preferred option to import any individual chromatograms from result files in different folders See How to import chromatograms with the command File Open below The table below describes how to import chromatograms with the File Open to compare command The search is performed at specific locations or with specific search criteria This method is useful if you for example want to import chromatograms from all files of a scouting folder Action Step 1 Choose File Open to compare Chromatograms in the Evaluation module Result The Open Chromatogram to Compare dialog box is displayed Chromatogram selection fe Default M Browse f z Browse All Chromatogram i v Browse All Found chromatograms Baseline example 1 1 Baseline example 2 1 1 1 4 S Clear Baseline example 3 did Select All i Peak skim example 1 OU L How to edit results 11 Step Action 2 e Click the Search button in the
468. the Block pane of the Method Editor Description Each block is represented by a gray bar with the block name and the length of the block The line is shifted down to indicate calls to other blocks Click on the line that represents a block in the Block window to expand the block in the Text pane and select the first instruction in the block Figure The figure below is an example with a Watch instruction to start the fraction collector which is active throughout the gradient elution block Loop to repeat a group of instructions and Hold_until instructions are also indicated in the Block pane ooo lsm Cone B Woke Foal iuioZetn UV Ehua Fi aci Waih O olumeF action Gn deri Gindert Dola Fiactenation Si Clean ow cy ooo Soo cv Owy CV Enele Iniectio owt AON B00 mt at 5 Miach Coos Gister Than 0 38 Stat Eliae Fran loog Hok Urti Cond Stable Barsine 5 Mirne 5 00 base Blocks are represented in the Gradient pane of the Method Editor by marks on the X axis The marks show the length of each block The name of the block in which the cursor line is currently placed is shown at the top of the pane The figure below describes the Gradient pane Gradort Block name 85 00 0 00 Sample _kryection e p99 6 How to edit methods 6 2 Method blocks 6 2 2 How to call method blocks 6 2 2 General descrip tion Types of calls Watch instruc tions 03 0014 90 ep 100 How to call method blocks
469. the Evaluation module e Right click and select Marker in the menu Result A vertical line indicating a certain point is displayed Click the marker line and drag it to the desired point where you want to take a Snapshot Right click and select Snapshot in the menu Result The Snapshot is displayed in the Snap Shot dialog box 01 125200101 1_UV1_280nm 11 69 mAU 02 125200101 1_UV2_250nm 237 85 MAU 03 125200101 1_UV3_0nm 0 00 mAU 04 125200101 1_Cond 0 47 mSicm 05 125200101 1_Cond 0 50 06 125200101 1_Conc 100 00 8 5 89 1 00 mimin 26 60 gC 0 00 mimin 07 125200101 1_pH 09 125200101 1_Flow 10 125200101 1_Temp 14 125200101 1_SampleFlow e Click the Save to File button if you want to save the information as an Excel file xlS or a tabbed text file txt e You can also copy the information to the clipboard Click and drag the mouse in the table to select the information you want to copy Press CTRL C The information can now be pasted in a text editor e Click the Print button if you want to print the information e Click the Close button 03 0014 90 ep 42 UNICORN concepts Step Action 2 Repeat steps 2 to 4 if you want to view more Snapshots How to view Snapshots during a method run The table below describes how to view Snapshots in the System Control module during a method run Step Action A method is running and the System Control is displayed
470. the block selected for point of insertion Instruction The table below describes how to import method blocks Action Choose Block Import Block As in the Method Editor Result The Import Block dialog box is displayed ix Aut_PressuieFlove Regualion Aut_PressureFlow_Reset oZero_ Explorer Demo S03KB Method File BufferValve_At_Inlet Explorer Demo 303KB Method File Clean alter Elution Explorer Demo 204KB Method File Explorer Demo 304KB Method Fi Couma Pressure_Limk Column Valve DelayOp02min 4 Block name Column _Equ bration Cal Fron Ab pa cv oe e Select the method from which you want to import a block e Select the block Result The name of the selected block is displayed in the Block name field ep 109 6 How to edit methods 6 2 Method blocks 6 2 7 How to import method blocks Step Action In the Call field do the following e On the From drop down list select a block into which the block will be imported e In the At field select the breakpoint value for the block to be im ported Click the Import button Note The imported block cannot have the same name as an existing block in the method If the default name is not allowed for this reason the Import button will be gray and locked If this occurs change the name of the imported block so that the Import button becomes available e Repeat steps 2 and 3 if needed e Click the
471. the dialog NO 3 box and click the Edit button Check that the strategy computer name and the control number are correct according to the installation at the local station which is physic ally connected to the system See the Adminis tration and technical manual System defini tions e If you connect remotely to a system check that the local station which is physically connected to the system is turned on check that the network is functioning at both the remote and the local station e Check that the limit of eight connections to the system has not been exceeded ep 481 A Troubleshooting A 3 Methods and method runs A 3 In this section Cannot perform Quit or Logoff 03 0014 90 e p 482 Methods and method runs This section describes how to solve the following method and method run problems Cannot perform Quit or Logoff Monitor signals do not appear in the Curves pane in System Control Error message Couldn t create result file Destination path could not be found The Method System Connection dialog box keeps appearing The Method Editor window does not fit on the screen There are red instructions in a method After Windows logout and login you cannot get a system connection The Print screen command does not send a copy of the screen to the printer The table below describes a problems and its solutions Problem description Solution You are unable to perfo
472. the major differences in the effect of filtering peaks to reject peaks compared to excluding the peaks by rejection Filter peaks Reject peaks excludes the peaks from display permanently excludes peaks from the integration does not exclude the peaks from the excludes the peaks from the calcula calculation of the total peak area tion of the total peak area can be reversed cannot be reversed e p 333 12 Evaluation 12 1 Peak integration 12 1 2 How to perform a peak integration Peak labels Peaks can be labelled with their retention sequentially numbered or be marked with specific identification names See table below for an instruction on how to display peak labels The label type can be selected on the Curve Style and Colour tab in the Chromatogram Layout dialog box De select all label options to hide the labels e g for presentations The illustration below shows the Chromatogram Layout dialog box with the Curve Style and Colour tab opened Chromatogram Layout 1 x Header CurveNames YAss XAxis Curve PeakTable Curve Style and Colour Edit Texts Layout Library Select curve to modify colour and linestyle for Colour Line style 01 id148Quantitate001 1 UV1_ 280m O E 02 id148Quantitate001 1_U 2_Onm 03 id148Quantitate001 1_UV 3_Onm HH 04 id148Quantitate001 1_Cond a SS 05 id148Quantitate001 1_Cond mn EEL DE id 48Quantitate001 1_Cone 07 0
473. the tab that is displayed in the Chromatogram Layout dialog box Carry out the changes on the different tabs to get the desired layout for header curves and peak table Select Apply to all chromatograms if you want to apply changes made in the Chromatogram Layout dialog box to all open chromatograms Click OK e p 235 10 How to view results 10 4 How to optimize the presentation of a chromatogram 10 4 2 The Curve tab and Curve Names tab 10 4 2 The Curve tab and Curve Names tab The Curve tab The Curve tab of the Chromatogram Layout dialog box contains a list of all the curves included in the chromatogram Select the curves you want to display in the chromatogram and click OK Curve name ap You select options for the curve name appearance on the Curve Names tab This is pearance an example of a default curve name Result 11_UV1_280 The table below describes the three components that make up the default curve name Component Description Example Result name Name of the result Result Chromatogram name Number given automat 11 ically during a run or a name defined by the New_Chromatogram in struction Curve name Curve type for example UV1_280 detection of an eluted component In this example the sys tem uses a variable wavelength detector so the wavelength 280 for the UV curve is also giv en How to choose You can choose to view only part of the curve name The table be
474. thod Wizard icon on the Method Editor module or choose File Method Wizard 1 ay A o Result The Method Wizard dialog box appears Method Wizard for System Explorer Demo i x Main Selection Column and BufferPrep Main Selection Anion_Exchange Column Ary Column Position l Posilionl Bypass z J Flexible Flow Rates NOTE Different Flow Rates are Set on Variable Page I Flow Regulation of the System Pump I BulferPrep X lt Back Next gt Frnsh Cancel Help Set Defaut Note If several systems are available you must first select which system you want to use Select the appropriate parameter values and click the Next button Note Click Set Default on the first wizard page to restore all settings to the default values In each new dialog box select the appropriate parameter values and click the Next button to continue Note Select a column even if you want to perform a test run without a column Use a small column Replace the column with a piece of tubing when you run the method e p81 5 How to create a method 5 1 How to use the Method Wizard Step Action 4 Click the Finish button in the last dialog box Result The Run Setup opens The Run Setup The Run Setup consists of a number of tabs Click on the appropriate tab at the top to select it Reference Curves Evahiation Procedures Method Information Stat Protocol Result Name Frac
475. ti verses atecuventiotatene triacs cc T E A AEE 196 9 23 NUEVOS a1 steer Vase a a Pataca ee a areal E ngs eS A AE 199 9 2 4 The Flow Scheme PANO i tcso sa tene tenn Nachle darteieorts dandedited ara aari ee ate toriacuuanuee 204 9 2 5 The Logbook Panlesg ear tates concn cs con sans Avene ianiaayaeactdetesdetersencudsestigekeaeseer crates 205 9 3 Man al system C NtrO laer ra arrana aar ea aaar ae a secce seceeeantddeuedecssvireret icra recast 207 9 3 1 The toolbar and status Dal con siavintea atacand eee ios 208 9 3 2 Man adl ISEMUCHONS 2 5 Mcsncnetes vaetuttetarraanent 1s ceacnuitldetesemuandedadontetetcasententetes 212 9 3 3 Alarms and Wan Scne thn cy hasta na yentcx vous ty voces inne ch uae uereguctearuaneemeaGe EEEa EEEn 215 9 4 How to perform a scouting FUME igeaneriantresricetaatedsly ai duted cena ateddcasesuciaselersaumteceante 216 9 5 How to perform a MethodQueue run yi snaveesvernciveverivenSats wily van Reataadverieud aay 217 9 6 If the network connection TANS iiccdassncvadlaaaieatemcanwmanae nua iNauraxantaddiwtianteasnts 219 10 HOW tO VIGW results niiina eaaa eaaa aada aaa adaa ene A 220 10 1 How to open a result TIE isichivaicte tinier hauleeG rarlesectndetencbated eGesticenadelahna sewn vee destecsanen 221 10 2 How to use the File Navigator cnt tcccraiciminen ae aniiaketsiaenanaee ans 222 10 3 Basic presentation of Chromatograms cssssesceeeessssseceeeeeeeeeessseeeeeeeseseeeeeeeeeeees 226 10 3 1 Introduction
476. tion module e Choose Integrate Peak Integrate or e Click the Peak Integrate toolbar icon ful e a Result The Integrate dialog box opens e Select a source curve e Select a baseline or a calculation method from the Baseline list e Click OK to integrate with the default selections or e Proceed with steps 4 to 6 to change the default selections Note See also 12 1 3 How to optimize the baseline with a morpho logical algorithm on page 336 and 12 1 4 How to optimize the baseline with a classic algorithm on page 340 e Click the Baseline settings button to change the calculation al gorithm in the Settings dialog box The default algorithm is Mor phological e Change the selections or values e Click OK e Click the Peak window button to edit the peak window limits if necessary e Click the Reject peaks button to set the parameters for peak rejec tion if necessary e Edit the Column height or Column V values if necessary Evaluation 12 Step Action 6 e Click OK to integrate and close the dialog box or e Click Save and Edit Peak Table to save the integration and open the integrated curve for editing See 12 1 5 How to edit the baseline manually on page 348 See 12 1 6 How to edit the peaks on page 351 See 12 1 7 How to integrate part of a curve and how to exclude or skim peaks on page 358 Illustration This is an illustration of the Integrate dialog box Chromatogram Ta
477. tional parameters are required Click the Insert button Result The instruction will be inserted in the block e at the position of the breakpoint of the new instruction if there are no other instructions at that breakpoint e immediately after the currently highlighted instruction if the highlight is at the same breakpoint as the new instruction e as the last instruction at the breakpoint if there are several in structions at the same breakpoint and none of these is highlighted Note Instructions that are placed at the same breakpoint are ex ecuted simultaneously with the exception of Block instructions which are executed in the sequence in which they are written If you use AKTA systems the Pause Hold and Hold_until instructions will stop execution at this breakpoint that is instructions following after Pause Hold and Hold_until at the same breakpoint will not be executed until a Continue instruction is issued e p113 6 How to edit 6 3 Method instructions methods 6 3 3 How to delete method instructions 6 3 3 Instruction How to suspend execution t arily 03 0014 90 empor e p114 How to delete method instructions The table below describes how to delete method instructions in the Text Instructions Editor Step Action Select the instruction in the Text pane 2 Use one of the following alternatives e Right click the instruction and choose Delete in the displaye
478. tions B 4 Procedure instructions Instruction EXPORT_DOC_400_WKS EXPORT_DOC_400_XLS EXPORT_DOC_WKS Description Exports the documentation in the current result file in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters to this function are OFF then no documenta tion is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file Exports the documentation in the current result file in MS Excel XLS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full search path must be entered in answer to the question If all parameters to this function are OFF then no documenta tion is exported If at least one of them is ON then the documentation will be exported and the corresponding parts will be included in the exported file Exports the documentation in the current result file in WKS format to the file defined in Export to file If is entered as File name the current Result file will be used If is entered followed by text e g Enter a file name as File name a full
479. tips when you move the cursor to ded variable cells fields that can have several functions Note The options to show detail and unused variables can be set up as default options in the Administration Change User Attributes settings in the UNICORN Manager Enter new values in the appropriate fields to change the default variable values For some variables pre set values are available on drop down menus Save the method when you have made your changes Note The Variables box must be selected on the Start Protocol tab if you want to be able to change variable values at the start of a method For variables with values shown in blue the value input can be toggled between OFF INFINITE or other single position values and a variable range To change the value right click the value cell Variables can be changed in the Text Instructions Editor as well as on the Variables tab of the Method Editor Changed values will be displayed for the corresponding instructions in both windows ep 125 6 How to edit methods 6 5 Run Setup 6 5 2 The Variables tab How to delete or The table below describes how to delete or rename a variable in the Run Setup rename variables Step Action e Click the Edit Variable button on the Run Setup Variables tab or e Choose the Edit Variable Method Editor menu option Result The Edit Variables dialog box opens The variables are listed alphabetically Select the variable to ed
480. titation e Internal standard quantitation e Standard addition quantitation e Recovery calculation Each technique is described below One or several component s of interest are run to produce a calibration curve The amount and concentration of the component in the sample is then determined from the calibration curve This technique is fairly simple and usually produces accurate results Peak areas of the components of interest are related to the peak area of an internal standard added in a fixed amount to each concentration level of the standard and to the sample This technique reduces errors that are caused by changes occurring between the separation runs and is therefore the technique that can produce the highest precision if a suitable internal standard can be selected The sample is spiked with a known amount of the component of interest The areas of the spiked and unspiked sample are then compared and the amount in the unspiked sample is determined No calibration curves from standards are used Only one component can be quantitated Compared to other techniques results can be obtained more quickly when you are performing a small number of sample runs with standard addition However the precision is limited e p 395 13 The Analysis module 13 2 Quantitation overview 13 2 1 General information about quantitation Recovery calcula Recovery is used to determine the losses that can occur during the sample non prepar
481. titation vcsniscterdecuiversis vend daviaveneeveiavent 439 epv Table of Contents 13 5 2 How to perform automated QUaNETALON sai ccsecevealaccaiecethswwbersbobe te sowberthaas eas 441 13 5 3 How to perform automated Update cccceeccseseeeseeeeeeeeeeeeeeeeeeeeeneeeneees 442 13 6 How to measure molecular SIZC cceceeeeeseeeeeeeeeesseneeeeeeeeeeeseeaeeeeeeeenseeeeeeeeeeees 448 13 6 1 Overview of molecular size deterMination cccceccseceeeeeeeseeeeeeaeeeneeeneees 449 13 6 2 How to determine the molecular SIZC ccccccecseeeeceeceeceeceeeeeeeeeeeeeeeaeeas 451 14 System Seting Saraan ante aana en nee aE 458 14 1 General information about system settings sssssrssreersrrrrrsrrrrrrrrererrrrrerrere 459 TA 2A Alat MS a a a a a lana cra E E AEE inet cd 461 IA Se AE AS EEE EEE E O EAEE A E A EEEE 463 15 System maintenance and error repOrting cssseseeeeeeeessseeeeeeeseeessseeeeeeeeesesseneeeeeeeees 464 15 1 System maintenance TUNCTIONS weiss wise sein sccantieels codetediviadet evar acidvesekeeaeseodtees 465 15 2 How to generate problem reports ccscssessesessssessesscessssssseeseeeeeeeeeeeeeeeeenseesennneees 469 15 2 1 How to generate a report from the UNICORN Manager 470 15 2 2 How to generate a report from the System Control ccccceseeeceeeeeaeenaeeees 473 Bs TEQUDIGSMOOUING sesso E E bee thas sacssed ost E E E NA 476 Bizet IGOR EE eu sescatean as Suntan Sen sire
482. to customize System Control panes 9 2 1 Introduction Illustration How to select what panes to dis play 03 0014 90 e p 194 How to customize System Control panes The System Control module displays the status of the current system On the Windows taskbar there may be up to four System Control modules available that can be connected to different systems Separate systems may be controlled and displayed independently of each other The illustration shows the System Control module with the Run Data Curves Flow scheme and Logbook panes displayed lt gt DEMO Systom Cortiol 1 Exptorert 00 Method c Defouk Quoucs _Example700002 Cxampte7 mt Resuk c Deloutt Examele7001 r05 Fale EF Fie View Hawd System Help Run Hold Continue End W f m es I DOE O oe ie ie Gem s gt PAU oo Sangeres SanglePow Aunint A00 ATeno 0 00 min Rian started 1 58 min Pouse 1 68 min Corfinue For Help geess FI run Bleek j j No watch Corio By dloa Each System Control module displays up to four panes for monitoring different aspects of the run To select what panes to display either e click the Customize Panes icon i or e choose View Panes How to perform method runs 9 How to customize Change the size System Control aes Select a split bar and drag up and down to change the size of a specific pane Maximize restore or hide Right click a pan
483. to exclude or skim peaks on page 358 for more information e p 357 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks 12 1 7 How to integrate part of a curve and how to exclude or skim peaks Introduction There are several possibilities to improve the results if the peak integration is unsatisfactory This section describes e How to select only part of a curve for integration e How to exclude peaks e How to skim peaks These operations can be performed both in the Integrate dialog box in preparation for the peak integration or in the Edit Peak Table dialog box to adjust an unsatisfactory peak integration This section describes both alternatives How to select part The table below describes how to select only a part of a curve for peak integration of a curve in the Integrate dialog box Step Action e Choose Integrate Peak Integrate Result The Integrate dialog box opens e Click the Peak Window button Result The Peak window dialog box opens Pook window S25 KI gJ wo G Wo w wo Leni PS Baie paw o i e Type new X axis values for the Left limit and the Right limit or e Drag the vertical cursor lines to define the limits 03 0014 90 ep 358 How to exclude peaks How to include negative peaks Evaluation 12 Step Action 3 Click OK Result The baseline will be calculated from the whole c
484. to rename and remove procedures The procedures that you have created can be renamed or removed from the list of available procedures This section describes how this is done How to rename a The table below describes how to rename a procedure procedure How to delete a procedure Action Choose Procedures Edit Rename Result The Rename Procedure dialog box opens Select a procedure Result The procedure name is displayed in the New name text box Type the new name Click OK Result The procedure name is changed The table below describes how to delete a procedure Action Choose Procedures Edit Delete Result The Delete Procedure s dialog box opens Select a procedure e Click OK e Click the Yes button to confirm Result The procedure is deleted Global procedures It is not advisable to edit existing global procedures Open the global procedure instead and save a copy under a new name Use this copy for editing purposes e p 387 13 The Analysis module 13 The Analysis module Introduction This chapter describes how to use the Analysis module This module is an optional feature that must be ordered separately and installed after the regular UNICORN installation The Analysis module is accessed in the Evaluation module The Analysis module uses functions in the Evaluation module that are presented in the previous chapters It is recommended that you are famili
485. to runa single procedure Batch runs How to perform a batch run 03 0014 90 e p 384 How to run a procedure You can run the saved procedures either for a specific chromatogram or as batch runs The table below describes how to run a procedure for a specific chromatogram Step Action Open a result file Select Procedures Run Result The Run Procedure dialog box opens Select the procedure from the list and click OK Result The procedure is executed Note You can also open the procedure in the Procedure Editor dialog box and choose Control Run or click the Play button It is possible to apply an evaluation procedure to a designated batch of result files if they are not open in the Evaluation module An open file will not run and an error message will be displayed The batch run is performed in the background of the Evaluation module and the results of the run are not seen with the exception of prints and documentation that are defined as steps in the procedure For example batch runs are useful to perform integration with the same parameter settings on many results e to print a number of results with the same settings The table below describes how to perform a batch run Step Action Choose Procedures Batch run Result The Open Procedure dialog box opens Select the procedure from the list and click OK Result The Batch Run dialog box opens Evaluation 12
486. ton in the System Control window if they are not to run to completion If you click the End button for a pending MethodQueue it is deleted from the pending list e The End All button terminates the running MethodQueue and de letes all MethodQueues from the pending list e The Close button closes the dialog box 03 0014 90 ep 218 How to perform method runs 9 9 6 If the network connection fails Results will be If the results of a method run are stored on a server or other location and there is ote the Failed network communication failure during a method run that has been started from a remote station the method run will continue and the results will be saved in the Failed folder on the local station A control mode connection can be established on the local station to control the running system See the Administration and Technical Manual for more details e p219 10 How to view results 10 How to view results Introduction A result file is automatically generated at the end of a method run and contains a complete record of the method run including method system settings curve data and method run log The Evaluation module offers extensive facilities for presentation and evaluation of curve data This chapter describes how to present the chromatograms and curves of your result file and how to create and print reports In this chapter This chapter contains these sections See How to print active chr
487. turn to the default value if necessary Result The default setting that was defined in the system strategy is restored Only the selected parameters will be restored e e e Click OK Limits for monitor If the system strategy allows limits for certain monitor signals can be set in the signals in methods yy ethod These limits will only work locally in the method and override the global settings as long as the method is in operation This feature can be used to set the pH warning threshold to one value during the process operation and another during the system cleaning 03 0014 90 p 460 14 2 Introduction Alarms and Warn ings Alarms in a net work System settings 14 Alarms This section is a description of the Alarms system settings The Alarms settings define the upper and lower Alarm and Warning limits for process monitor signals The table below describes the difference between Alarms and Warnings If the signal exceeds then the Alarm limits e an alarm sounds e an alarm message is displayed e the process is paused i e the method execution is suspended and all pumps are stopped e the alarm is noted in the logbook The situation must be acknowledged and corrected before the process can be continued the Warning limits e a warning message is displayed e the process continues e the warning is noted in the logbook Note The message text in an Alarm
488. uantitation The method that is used for the sample runs must be the same as for the standard runs If the method is created from a wizard or a template for AKT Adesign systems select Sample in the variable Quantitation_Type on the Variables tab in the Run Setup The table below describes briefly how to prepare for the quantitation Step Action Prepare a quantitation table for the components of interest See 13 3 2 How to create a quantitation table on page 411 for further information Perform a sample run Note If internal standard quantitation is used the internal standard must have been added to the sample prior to the sample preparation procedure The injected amount must be the same as on the standard levels Open the sample result file and peak integrate the sample curve to produce a peak table Note The sample curve must use the same X axis base unit as the standards during the integration Time is the recommended unit for highest reliability Select File Save to save the peak table ep 429 13 The Analysis module 13 4 How to quantitate the sample 13 4 1 External and internal standard quantitation How to calculate The table below describes how to calculate the amount and concentration in the the amount and l sample concentration Step Action Select Quantitate Calculate amount and conc Result The Calculate Amount and Concentration dialog box opens Calculate Amount and Concent
489. uantitation table 13 3 2 How to edit and update a quantitation table 13 3 3 e p 409 13 The Analysis module 13 3 How to prepare for quantitation 13 3 1 Preparations before quantitation 13 3 1 Description Concentration levels Reject irrelevant peaks 03 0014 90 ep 410 Preparations before quantitation The table below describes the preparations before the quantitation Step Action Create a method to be used for all the standard runs The method and the injection volume must be the same for all the runs Perform at least one run for each concentration level of the standard Peak integrate the curves to produce a peak table for each of the standard curves Note When integrated all standards must use the same X axis base unit Time is the recommended unit for the highest reliability Check that each integration is correct and consistent Select File Save to save all the peak tables The standard series should include standard concentrations that extend beyond the lower and upper limits of the sample amount If an internal standard is used the internal standard must be added in the same concentration in all standards Methods created from a wizard If the method is created from a wizard for AKTAdesign systems you may select the correct standard concentration level in the variable Quantitation_Type You can also set the level after the run has been performed Each level is an al
490. ude subfolders e Select date limits in the Date drop down boxes e Type text strings to match Question or Answer texts e Type a variable name and if desired a value e Type a Batch ID Note You can search for a sample ID provided the sample ID is defined as a variable Click Find Result The search results are listed in the Found folders and files field The search is limites to either methods or results and to the folder including its subfolders that is currently displayed Double click a file in this list Result The dialog box is closed and the selected file is highlighted in the UNICORN Manager window Note If you click Close you will return to the UNICORN Manager window with no file highlighted regardless if you have selected one in the dialog box or not 4 4 Introduction How to copy or move files and folders The function Copy to External Files and folders in UNICORN 4 How to copy delete rename and backup files and folders UNICORN has some file and folder handling functions that are slightly different from the general Windows functions This section focuses on the differences Note You need explicit authorization in your user profile to copy move and delete files There are some restrictions to how you can copy or move files and folders e Files and folders can only be copied or moved to folders that are specific to your user name e You can also copy files to and from
491. ump Methodbase instruction is included in the method the default setting SystemPump will be used How to edit methods 6 6 6 2 Instructions at the same breakpoint Description Instructions placed at the same breakpoint in a block are executed simultaneously Exceptions Exceptions are successive Block instructions which are executed in the sequence in which they are written This can have important consequences in some situations The instruction sequence below shows an example of instructions with the same breakpoint where the AutoZero_UV will start after the Wash block is completed Breakpoint Instruction Block WASH AutoZero UV Block ELUATE e p157 6 How to edit methods 6 6 How to use selected method instructions 6 6 3 Block and method length 6 6 3 General descrip tion Block length 03 0014 90 ep 158 Block and method length The time or volume of a method run is determined by the sum of the block lengths In turn the length of a block is determined by the breakpoint of the last instruction in the block Note Depending on how conditional calls are used see 6 7 Standard Watch conditions on page 166 the overall method time or volume may vary according to watch events during the run A block in which all breakpoints are set to 0 will take no time or volume during a method run The illustration below shows an example of this W 0 00 Block Initial_Eluent_Conditions_BP Initial Eluent
492. urve but the calculation of the peak areas is only performed on the selected section You can define criteria to exclude peaks from integration The table below describes how to define peaks to be excluded in the Integrate dialog box Action Click the Reject peaks button Result The Reject Peaks dialog box opens e Select the appropriate checkboxes and type values for height width and area Define how many of the largest peaks you want to include e Click OK Select the Accept negative peaks checkbox of the Integrate dialog box to include negative peaks in the integration Result The negative peaks will be reported as negative areas in the peak table By default negative peaks are not included in the integration e p 359 12 Evaluation 12 1 Peak integration 12 1 7 How to integrate part of a curve and how to exclude or skim peaks Peak skimming vs The area under a peak can be calculated either using separating drop lines or peak drop lines skimming e Drop lines are vertical marks that split two peaks at the valley Drop lines are used mostly for peaks of relatively similar size When a peak has a shoulder splitting with drop lines will cause the first peak to lose too much of its area to the peak that forms its shoulder e The Peak skim option can be used to skim off the smaller peak with a straight line that starts in the valley between the peaks and ends at the other side of the smaller
493. urves e Click OK Note You can also subtract corresponding blank runs if there are blank runs available Repeat steps 2 and 3 for the second UV curve Select Operations Divide Result The Divide dialog box opens e Select the first result curve from the subtractions in the first list of curves e Select the second result curve from the subtractions in the second list of curves Click the checkbox for Threshold and type values for each curve This results in the following e The quotient is set to 1 0 if either of the sample values is closer to zero than the threshold value Very high quotient values are prevented if division is performed with values close to zero Very low quotient values are also prevented Note Default Threshold values are entered by UNICORN The values can be changed Click OK The resulting curve can be filtered to reduce noise and to remove ghost peaks The table below describes how to filter the curve Step Action Select Operations Smooth Result The Smooth dialog box opens e p 367 12 Evaluation 12 2 Other evaluations 12 2 1 Peak purity and peak identification Step Action 2 e Select the Source Chromatogram e Select a Filter Type Note The Median filter is recommended to remove noise that appears as spikes or occurs in a small area of the curve e Click OK 03 0014 90 p 368 12 2 2 Introduction Where to use slope values A sam
494. utton e executes all instructions in the list at the same time or e executes the currently marked instruction if the list is empty Note Although all instructions are executed simultan eously some for example gradient and fraction in structions may take some time to complete in the li quid handling module If you click the Close button without first clicking the Execute button commands in the list e will not be executed e will be deleted from the command list e p213 9 How to perform method runs 9 3 Manual system control 9 3 2 Manual instructions How to save manual results 03 0014 90 e p214 When you choose to run the system manually as opposed to a Method run the results are automatically stored in a folder called Manual Runs The Manual Runs folder stores the ten most recent results from your manual runs To save a result file from the Manual Runs folder more permanently you need to move or copy it to another location An alternative way to save the results from a manual run is to record the results manually in a result file The table below shows how to do this e Choose Manual Other e Select the instruction Record On at the beginning of the run e Click the Execute button Result UNICORN will prompt for a result file name 9 3 3 Introduction Limits for monitor signals Effects of alarms and warnings In a network sys tem How to perform method runs 9
495. utton in the Integrate Peak integrate dialog box The Morphological algorithm searches for all parts of the source curve where The curve parts come into contact at both ends of a horizontal line of the length defined in the Structure width parameter The default value of this parameter is based on the widest detected peak in the curve The horizontal line is moved along the curve up the peak until it reaches the contact points The curve parts below the horizontal line and the line will now forma curve with a plateau The center point in the plateau formed by the horizontal line will be the data point for the baseline The data points fulfil the Minimum distance between data points This parameter reduces the total number of data points that are created from a curve The Classic algorithm searches for all parts of the source curve where The curve parts are longer than the Shortest baseline segment This parameter determines the minimum length for a part of the source curve to be considered a possible baseline segment The curve has no point outside the Noise window The noise window is defined as a rectangular corridor parallel to the slope of the curve and centered on the first and last points within the currently inspected segment The slope is less than the Slope limit This limits the maximum slope of the baseline to differentiate baseline segments from peaks The curve parts are lower than the Max baseline level This paramet
496. ver at least k 1 data points around each point in the curve to smoothen the data The derivate is the derivate of the fitted polynominal at each point The calculation uses a convolution formalism to calculate 1st through 9th derivatives The calculation is performed with the data in low X to high X order If the input trace goes from low to high it is reversed for the calcu lation and is re reversed afterwards Note See Gorry Peter A General Least Squares Smoothing and Differentation by the Convolution Savitsky Golay Method Analytical Chemistry 1990 Volume 62 570 573 for more information on the Savitzky Golay algorithm 03 0014 90 ep 492 B 2 Overall process Baseline segment definition Morphological al gorithm Classic algorithm Evaluation functions and instructions B Baseline calculation theory The table below describes the overall process of a baseline calculation Description The baseline segments are defined The baseline points are selected The baseline is drawn Baseline parameters are used to find the baseline segments The default values for the parameters are determined from the source curve The baseline segments are found by different parameters that are based on the type of algorithm that is selected Note The parameters can be displayed in the Evaluation module if you choose Integrate Calculate baseline function You can also click the Baseline settings b
497. vse E ancient vans cudseaie A E 41 2 9 UNG Start GUIE eri n a eaa E EE teen E E E E E AE 44 3 General system operationS s sevssssesucdansthwcees sewasucuatoedsavssatusaud scctuandasueumaenittwdau dewiditanestaond 45 3 1 Log on routines and log off TOUTING rcs cc cused sanweinwsseutuiatne bias th Risvexanwadsevaveeanideccads 46 3 2 How to create a NeW USE is icccsic fo ecsuncedes suede des anes Sukae pbwe lace Samp andue enone edocs eee aeeoass 50 3 3 How to assign user PrOPeRL CSovie stir iiteutbi ren sanwiun Seed Muy etier aneelahvertetmatieeudineeiatants 53 3 4 How to change your passwords and user attributesS sssssesrsrresrerrerrsrrrrerrerne 56 3 5 How to connect to the chromatography SySteM cccceeeceeeeeeeeeeeeeeeeeeeeeeeaees 58 3 6 How to back up and restore system data cccceccseccseceeecseeeeeeeeeeneeeeeeneeeneeeaeenaees 63 SJ How toset upa printerne a E a a E a Ea EIEE EEE 66 4 Files and folders in UNICORN snnssnnnnnnnnnnnnnennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmnnn nnna 67 4 1 How to create folderS enenennnnsrsrrsrerrrrrrnrsrnnrsrrrrrrrrnrsnnnrsrsrrrrrrrrennnrsrsrrrreenee 68 4 2 How to open and preview THES seco tate reseed sens kepsnedavecgecevadscevaansededecadatiadedenceatects 69 4 3 How to arrange and locate your fileS ssssssssssrrsrssrerrsrrrrrsrrerrrrrrrsrrtrrnrrsrerrr rne 72 4 4 How to copy delete rename and backup files and folderS ccccccec
498. w show both the original and the pooled fraction pi pooled frac curves The table below describes how to show only the pooled fractions Step Action e Choose Edit Chromatogram Layout or e Right click in the chromatogram and choose Properties from the shortcut menu Result The Chromatogram Layout dialog box opens e Select the Curve tab e De select the check box for the original fraction curve remove the check mark Result The original fraction curve is de selected and is not displayed ep 281 11 How to edit results 11 5 How to pool fractions How to calculate concentration and amount in the pools How to determine a pool target volume How to use the Pooling Protocol 03 0014 90 e p 282 Protein concentrations The protein concentration in the fractions are calculated using the following formula Concentration mg ml A d 1000 Ext Coeff A Average fraction absorbance Area Volume mAu d UV cell path length cm Ext Coeff Protein coefficient at used wavelength 1 g cmt Protein amounts The total amount of protein found in the pool fraction is calculated using the following formula Amount mg Concentration mg ml pooled fraction volume ml How to calculate the concentrations and amounts e Type the UV path length expressed in centimeters in the Path Length text box e Type the extiction coefficient in the Ext Coeff table cell for each pool
499. w to edit results 11 8 How to import and compare different runs 11 8 2 How to import and compare chromatograms Step Action 4 e Repeat steps 2 3 if you want to import chromatograms from other result files e Click OK Note If the names of the imported chromatograms already are used they will be sequentially numbered for identification purposes Up to 10 chromatograms can be made available at the same time in the Evaluation workspace How to display The table below describes how to simultaneously display and compare the imported and compare the chromatograms imported chroma tograms Step Action In the Evaluation module select e Window Tile to display the chromatograms side by side or e Window Cascade to display the chromatograms in layers Note Chromatogram windows can be individually sized and the presentation of the curves changed Display all chromatograms on the same scale e Open the Chromatogram Layout dialog box for any chromatogram e Make the changes to the chromatogram axes e Select the Apply to all chromatograms option Note Imported chromatograms cannot be shown with column volume as the X axis base 03 0014 90 ep 304 11 8 3 Introduction Commands to use How to edit results 11 How to import and compare curves This section describes how to import or copy curves from different runs into one chromatogram for comparison Two commands can be used to impor
500. w to export 323 How to select contents 364 Peaks How to filter from view 333 Labels 334 How to display peak labels 334 How to open the peak table 351 How to delete a peak 353 03 0014 90 epxii How to add a fill color and pattern 354 Drop lines description 355 How to split a peak 355 How to join peaks 356 How to add peak names 356 How to exclude before integration 359 Include negative peaks in integration 359 How to select a skim ratio 360 Edit integration for part of a curve 361 Peak purity 366 Peak identification through the absorbance ratio 366 Peak parameters 497 How to change the peak resolution algorithm 500 How to change the Assymetry Ratio value 502 Pool Fraction print list How to use 282 PrimeView How to import data 320 Problem reports How to use the Generate Report Wizard from the UNICORN Manager 470 How to use the Generate Report Wizard from the System Control 473 Procedure instructions Curve operations 504 Integration 507 File operations 508 Export functions 509 Chromatogram functions 515 Miscellaneous 516 Test instructions 518 Procedures How to record 379 Global procedures 380 How to build a procedure with instructions 380 How to edit 382 How to add instructions 383 How to run a single procedure 384 How to batch run 384 How to rename 387 How to delete 387 Protein activity Match to UV curve 285 Index e p xiii Index Protein amounts Calculation formula 282 Pr
501. want to define new scouting variables The Scouting Vari ables dialog box is displayed and you can select variables to be used in the scouting series Note The variables that have been selected for scouting cannot be changed on the Variables tab Clear All to clear all runs This converts the scouting run to a non scouting run so that it contains only the original method and val ues Scouting 7 Click the button if you want Delete to remove a run from the Scouting tab Click on any variable in the run you want to remove and then click the Delete button Edit Variable to rename or delete a variable or change a variable into a detail variable to insert a new scouting run before an existing run Click on a run column and then click the Insert but ton The new run will inherit the variable values from e the preceding run or e from the default values in the method if the run is inserted at the beginning of the scouting series e to add a scouting run if there are no runs previ ously in the scheme Default values will be used for the first run or e to add a scouting run after all other runs in the series The new run inherits the values from the run that precedes the new run to set up a series of runs with differing inputs How to set up or The table below describes how to set up or edit a scouting scheme edit a Scouting Scheme Action Create a method If you
502. x Fie Control Help Sinica BASE TIME REJECT_PEAKS OFF OFF OFF OFF 20 NEGATIVE_PEAKS OFF CALCULATE_BASELINE_MORPH 01 17 DEFAULT DEFAULT DEFAULT SUB 01 17 47 PEAK _INTEGRATE 47 A OC_TEST A 0100 RETENTION 1 RETENTION 10 11 PAUSE Retention out of range Instruction p Parameter al C Curve operation al QC Action jarca Integration RECEN MOLSIZE PAUSE C Benice C Espat QUANTITATE 7 Chom REPORT Message text Dne Other RUN_PROGRAM Retention oul of range x UPDATE Test QC_TEST para The table below describes the parameters for the QC_TEST instruction meter descriptions The example values are used in the illustration above Parameter Description Peak table source The peak table indicated in the PEAK_INTEGRATE in struction Example A Left limit The retention value where the control instruction will begin Example 0 Right limit The retention value where the control instruction will end Example 100 Note The control instruction will be applied to the run up to the sequence in the method where the con trol instruction is inserted e The controlled part of the run will end at the Right limit if this retention value is reached before the control instruction is reached in the method e If not the controlled part of the run will end when the control instruction is reached in the method Peak sele
503. x opens e p51 3 General system operations 3 2 How to create a new user Home folders 03 0014 90 ep 52 o N n A eS Ng ge Action Enter a user name in the User name text box Enter the full name of the user in the Full name text box Enter the position of the user in the Position text box Select or create a Home folder e Select a Drive and a folder from the Name drop list and proceed to step 9 or e If you need to create a new home folder proceed with step 6 Click New Result the Create New Folder dialog box opens Select a Drive and type a folder name Click OK to create the folder and return to the Create New User dialog box Click OK Result The new user is created and added to the User Setup list e Click Close or e Click the New button and repeat steps 1 8 to create more users Each user must be assigned to a home folder The Default folder can be used if you do not want to assign an individual home folder Note If you create a home folder on the C local drive it will not be accessible from other computers If you select a network make sure that is addressed by the same drive letter from all computers in the network 3 3 Introduction How to open User properties How to edit the user definition How to edit the user attributes General system operations 3 How to assign user properties A user is assigned properties that define pa
504. x opens The illustration below shows a complete example of a BufferPrep recipe in the New Recipe dialog box m Buller A Stock cone Buffer substances i 003 M Hydrogen phosphate Pliage Correction factors 003 M Foumate x Fron 3 To 75 0 06 M acetate Mf x M x r cid Base Sar r Component defirsion tler substance empo oxe Th een Sat e p 537 E How to create and edit BufferPrep recipes E 1 How to create a BufferPrep recipe Step Action Select buffers from the Buffer substances droplists and type stock concentrations in the corresponding Stock conc box See How to define a new buffer substance below if the desired substance is not available Select either HCI acid or NaOH base from the Acid Base droplist and type the required stock concentration typically 0 1 M Select a salt from the Salt droplist and type the maximum outlet concentration of the salt for 100 B typically 1 0 M See How to define a new salt below if the desired salt is not available Type the desired pH range minimum and maximum values in the From and To boxes See How to select the pH range below this table e Click the Notes button optional e Type your notes about the recipe in the displayed dialog box e Click OK to return to the New Recipe dialog box Click Save as to save the recipe under a new name Note A warning message will appear if
505. you want to export e Click the Export button Result The Export Peak Table to File dialog box opens Select the export file format from the Save as type drop list e ASCII files asc e Lotus 1 2 3 files wks e Excel files xIs e XML files xml e Select a destination folder e Type a file name e Click OK Note Peak tables are exported as text strings in ASCII format and numerical values in the Lotus 1 2 3 formats All possible columns in the peak table are exported e p 323 11 How to edit results 11 9 How to import and export results 11 9 2 How to export results How to export methods docu mentation and evaluation logs Copy to the clip board 03 0014 90 ep 324 The table below shows how to export methods documentation and evaluation logs Step Action Select the data you want to export e Select options in the dialog box e Click the Export button e Select a destination folder and type a file name e Click OK You can also use the Windows clipboard to copy the contents of the active window and paste it into other programs e g Microsoft Word Curves and documentation are copied as Windows enhanced metafiles emf and peak tables are copied as text Only the peak table columns that are selected in the spreadsheet will be copied How to edit results 11 11 10 How to sign results electronically Instruction Result files can be signed electronically t
506. ype a name for the new salt and click OK to return to the Define salt dialog box e Type the appropriate charge of anion value in the corresponding Value cell Example The value for CI is 1 The value for SO is 2 e Type the appropriate charge of the cation value in the correspond ing Value cell Example The value for Na is 1 The value for Mg is 2 Click OK Result The new salt is added to the list of available salts ep 541 E E How to create and edit BufferPrep recipes E 2 How to edit a BufferPrep recipe E 2 How to edit a BufferPrep recipe Introduction This section describes how to edit a BufferPrep recipe in the Method Editor How to edit a re The table below describes how to edit a BufferPrep recipe cipe Step Action Choose Edit BufferPrep Recipes Result The BufferPrep Recipes dialog box opens Select a recipe and click the Edit button Result The Edit Recipe dialog box opens Recipe name Acetate pH 4057 m Butler 2 H ances _ pH range Conrecton factor p mmm Fom 4 tof 57 E SS a Component det Acid Base io if ij umpa SSC 100 B C eE a Sat sete caes e Change the substances and parameters as desired and click the Save button or the Save as button to save the new recipe Changes to recipes If a recipe has been selected and saved in a method and the recipe is later changed in methods the corresp
507. ys open flow paths in color Monitor signals can be displayed numerically See the illustration below pane The Logbook pane The Status bar Toolbar icons in the System Con trol wastel frot asn omn we C f4 Eo F5 a ua TN re 7 rs The Logbook pane displays all actions during a separation run e g method start and end base instruction method instructions and manual instructions such as Pause or Hold See the illustration below 52 mia ColhamnPosition Position Bypass t 15 min Wavelength 260 254 215 63 min Averaging Timell JV 2 56 2 min Pause 227 min ButterPrep_pH 7 000 2 35 min Cornue 2 83 min Gradient 0 000 0 000 213 mia End The Status bar in the bottom of the System Control module displays the current status of the separation run See the illustration below CESR RS ra pe E The current system status is represented by the colored dot e A green dot represents a running system e A red dot represents a system in Pause state e A yellow dot represents a system in a Hold state e A white dot represents a system in an End state The table below describes the toolbar icons in the module Icon Function The Run icon opens the Run dialog box which shows all available methods If a method is loaded Run Setup opens Run ep 33 2 UNICORN concepts 2 2 The UNICORN user interface 2 2 3 The System Control module 03 0014 90 ep 34 Icon nd 3
508. ystem H E Air sensor 9 Auto Sampler Frac 950 pH Cond Pump 4 General ra Specific 3 Pump P 950 a UV Valve 1 9 Valve 2 Valve 3 Valve 4 9 Valve 5 Valve 6 Hardware version 56 3045 944M K318 i General software version 1 03 oe Module software version V1 40 Serial number 55119594 K4 002102 re t como 0 0 0 How to display Click a component in the list to display the component information component inform i ation You can choose two different views e General e g serial number version number etc e Specific e g how many hours a pump has been run etc e p 465 15 System maintenance and error reporting 15 1 System maintenance functions How to setupa The table below describes how to set up a maintenance warning maintenance warning Step Action Click the Warning tab Select a component e Choose Warning New or e Right click the component and select the New option on the shortcut menu e Type the appropriate value in the Periodicity field e Type a warning text in the Pop up text box e Type a name for the warning type in the Name text box e Click the Save button Repeat steps 2 and 3 to set up more warnings Click the Close button How to view the The component that has been set up for a maintenance warning is marked by an warning paramet icon and the name of the warning ers and
509. ystem but e Check that the system is turned on have no system contact e Check that all the cable connections Indications In the System Control are intact e the option Connection in the Run e Ifthe above actions do not help data pane says Yes try to restart both the computer e the option Instruments says Scan and the system ning e there is no contact with the system after a period of waiting Flow scheme not The table below describes a problem and its solution displayed properly Problem description Solution The flow scheme is not displayed Choose Settings Control Panel Dis properly play Settings in the Windows Start menu to check that you have selected 65536 colors 03 0014 90 ep 488 Evaluation functions and instructions B B Evaluation functions and instructions Introduction This appendix describes the functions that are implemented in the Evaluation module In this appendix This appendix contains these sections Topic See Smoothing algorithms Baseline calculation theory Peak table column components Procedure instructions B 4 ep 489 B Evaluation functions and instructions B 1 Smoothing algorithms B 1 Smoothing algorithms Introduction This section describes how the smoothing functions are calculated Choose Operations Smooth in the Evaluation module to view and edit the options Moving Average The table below describes the process when

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