Making your first run - GE Healthcare Life Sciences
Contents
1. KTA design Making your first run Begin here with KTApurifier 10 100 18 1120 02 z Amersham o Biosciences Important user information A M eaning Consult the instruction manual to avoid personal injury or damage to the product or other equipment WARNING The Warning sign is used to call attention to the necessity to follow an instruction in detail to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met CAUTION The Caution sign is used to call attention to instructions or conditions that shall be followed to avoid damage to the product or other equipment Be sure not to proceed until the instructions are clearly understood and all stated conditions are met N ote T he N ote sign is used to indicate information important for trouble free or optimal use of the product Should you have any comments on this instruction we will be pleased to receive them at Amersham Biosciences SE 751 84 Uppsala Sweden Trademarks AKT A and UNICORN are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham Biosciences is a trademark of Amersham plc Windows is a trademark of Microsoft Corp Terms and Conditions of Sale All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions of sale is av2. Creating a method 3 11 Select File Save Enter a name Store the method in the directory of your choice by double clicking on a directory Click on OK Comment The method name followed by two consecutive numbers starting with 01 will then be used as default name for the result file of your method after runs 12 You have now created a method 13 Click on the Main menu icon at the bottom of the screen Your saved method appears in the window to the left Now you are ready to start a run Go to chapters 4 and 5 You can also go to chapter 8 to learn how to vary any variables systematically and automatically in repeated runs This is know as scouting and is a convenient easy to use function Comment If you want a more flexible method once you are used to the basic template select File New in the method editor and one of the more advanced templates The template man_f_gr is similar to the basic_gr template but allows more flexibility such as segmented gradients and flexible start of fraction collection in the gradient Some of the more advanced templates are named according to the following abbreviations XXX_y_ZZ Note Some of the functions indicated below are unic for a specific system and may not be included in the templates supplied with your system The first three letters xxx identify the type of sample application used man the sample loop or Superloop is filled manually with a syringe sys the sample is applied
3. 1 onset re m a C AlarmskMon Inlet amp 2 Other OFF z Close MethodBase inieiB1 __ Chose nle Help i InletB2 OFF 9 Connect an injection fill port or a union luer female 1 16 male to port 3 on the injection valve and apply the sample manually with a syringe Making your first runs 18 1120 02 Edition AC 19 5 Starting a run 5 Starting a run Click on the System Control icon if it is not open 2 Select File Run Select the method to start Click on Run the method will not start yet A Start protocol appears consisting of a number of pages The first page you see is Variables This is the same page you were working on in the method editor Here you can fine tune the method before starting it This is very convenient when repeating runs with minor adjustments Comment When starting run no 2 immediately after run no 1 with the same method but for example a different flow rate you simply 1 Click on the Run button in System Control 2 Change the flow rate on the Variables page 3 Continue through the start protocol by clicking on Next and then start the run You do not need to change the method in the Method editor Yariables Ed Start_Conditions_GR Pressure_limit Flow UV _Averaging_time Start_ConcB Column_E quilibration Equilibrate_with Flowthrough_Fractionation Flowthrough_FracSize Sample_Injection Empty_loop_with o o MPa 0 00 5 00 1 00 ml min
4. A method file contains a series of instructions for controlling a run In the Results window to the right all result files are displayed A result file is the result from a run including all documentation e g the method used and the generated chromatogram Making your first runs 18 1120 02 Edition AC r 2 The system and the software In general UNICORN consists of 4 different modules of which the Main menu is one The other modules are represented by icons in the Toolbar These modules are e New method e System control e Evaluation e Instant run e Logon Logoff e about UNICORN opens a dialogue window for creating new methods opens a dialogue window for controlling the system and running your methods opens a dialogue window for evaluating your results A module is opened by clicking on its icon An additional three buttons are provided in the Toolbar These are opens a dialogue window where you directly can choose a template method to run This is handy for starting routine runs instantly opens a dialogue window to control the logon logoff process opens an information window about the UNICORN version and how to contact Amersham via the world wide web internet Making your first runs 18 1120 02 Edition AC The system and the software 2 Help and Adviser Comprehensive on line help is available Click on the Help menu in the upper right corner of each module and select Help for
5. be collected To end the fractionation earlier than the gradient end set End Frac_at to the desired ConcB value Gradient segment 1 3 The target concentration of eluent B and the length of the gradient is set here A linear gradient is developed from the initial ConcB value Start_ConcB to the Target_ConcB_1 value default 100 for the duration of Length_of_gradient_1 in column volumes Two additional gradient segments can be defined Clean after gradient The concentration of eluent B necessary to clean the column after the separation is set here usually 100 The length of cleaning is also set here Reequilibration GR The number of column volumes necessary to reequilibrate the column is set here If zero is entered no reequilibration takes place Making your first runs 18 1120 02 Edition AC 15 3 Creating a method 9 Click on the Gradient page to view the method graphically The length of each block is marked at the bottom of the graph Click in the graph The name of the block at that position is shown in the upper part of the chromatogram Click on the x axis to view the method in time volume or column volumes Gradient Block name 35 10 Gradient Maximise the method notes field with the button to the right of the field The method notes contain comments and information about the method e g how sample injection fractionation and elution are performed 16 Making your first runs 18 1120 02 Edition AC
6. directly through the pump The letter in the middle y identifies the type of fractionation used f fraction collection of fixed volumes p fraction collection of peaks v valve fractionation of fixed volumes The last two letters zz only identify the technique for which the template is written of gel filtration ac affinity chromatography ax anion exchange with BufferPrep cx cation exchange with BufferPrep gr gradient without BufferPrep A CIP Cleaning in place template is also available It enables automatic cleaning of the column with up to 4 different solutions All xxx_y_ax and xxx_yy_cx templates are pre defined for BufferPrep automatic buffer preparation which allows pH scouting see section 8 for further details In AKTApurifier 10 there are also basic templates for each one of the _zz templates These templates are sligthly simplified versions of the more comprehensive xxx_y_Zz versions Making your first runs 18 1120 02 Edition AC 17 4 Preparing the system for a run 4 Preparing the system for a run System connection Connection NO 3 1 Connection YES If the text NO is written in the Connection panel in the Run Data window follow the instructions in the comment below If not go directly to General system preparation Comment Before you can start a run you must always connect to the system Connecting means that the System Control window is set up for
7. it and its capabilities Below is a list of operations and descriptions that you may find of interest they are cross referenced to other manuals in the AKTApurifier manual package To learn about Read section Purifying E coli proteins 2 in the Method Handbook Purifying synthetic peptides 3 in the Method Handbook Purifying oligonucleotides 4 in the Method Handbook Different sample application options 2 2 2 5 in AKTApurifier System Manual Different fraction collection options 2 8 in AKTApurifier System Manual BufferPrep details 2 7 in AKTApurifier System Manual Columns and recommended tubing 2 1 in AKTApurifier System Manual Changing tubing kits A 5 in AKTApurifier System Manual Calibrating monitors and pumps 6 6 in UNICORN 3 0 User Manual Comparing chromatograms 9 4 in UNICORN 3 0 User Manual Integrating curves 10 1 in UNICORN 3 0 User Manual Measuring HETP and resolution 10 1 8 and 10 1 10 in UNICORN 3 0 User Manual Exporting curves and data to 10 4 in UNICORN 3 0 other programs User Manual Finding information about a certain Click on the Help button in menu instruction in UNICORN the dialogue box that appears or look in the index in UNICORN 3 0 User Manual Making your first runs 18 1120 02 Edition AC 33 9 Going further 34 Controlling Pump P 900 Monitor UV 900 and Monitor pH C 900 from the dials on the instruments themselves Details about each component Security features Controlling the system
8. not be collected Flowthrough Fractionation Flowthrough_ FracSize o mi 0 00 50 00 e Eluate FracSize in the block Start Fractionation Enter a suitable fraction size The fraction collection starts at the beginning of the gradient Zero means that fractions will not be collected Start Fractionation Fluate FracSize 0 00 50 00 Making your first runs 18 1120 02 Edition AC 13 3 Creating a method 14 Comment Each method template is unique but they are all built up with the same principle Below is a description of all the blocks in the man_f_gr template Main The column selected is shown here Start conditions GR Enter the wavelengths at which the run should be monitored The UV averaging time is automatically set to a default value for the column selected The pressure limit is automatically set to a default value for the column selected Initial eluent condition GR Select the inlet for eluent A usually A1 Select the inlet for eluent B usually B1 Select the start concentration of eluent B usually 0 If a BufferPrep template is used xxx_yy_AX or xxx_yy_CX see Section 8 the pH can be entered here Otherwise it should be Zero The flow rate is automatically set to a default value for the column selected If you manually alter default values and thereby exceeding the recommended values for the selected column eventually you will get a warning when you save your method Please note that
9. the total setting range shown within brackets for the method variables is for your information only to state the valid range for the variables Column equilibration The number of column volumes CV necessary to equilibrate the column is set here If you are using AKTApurifier 10 and zero is entered no equilibration will take place If you are using AKTApurifier 100 a sub block called System_volume_compensation is included This block adds 8 ml of equilibration to compensate for the system gradient delay volume This facilitates to accomplish comparable results when scaling up from small columns Flowthrough fractionation The flowthrough fractionation starts at sample injection and continues to the start of the gradient If not fractionated the flowthrough will be collected in position F3 of the Outlet valve Sample injection To apply all the sample onto the column empty the sample loop with a volume 5 x the volume of the sample loop Wash out unbound sample To wash out unbound sample with the starting buffer enter the length of the wash here Eluate fractionation By default the fraction collection will start at the beginning of the gradient To start eluate fractionation later than the gradient start set Start_Frac_at to the desired ConcB value Making your first runs 18 1120 02 Edition AC Creating a method 3 Start fractionation Set the fraction size used during elution Zero means that fractionations will not
10. 0 00 20 00 2 60 ha 0 00 B 0 00 100 00 5 0 Cw 0 00 999993 00 0 00 ml 0 00 50 00 jas ml 0 00 999999 00 a Help Cancel 5 Go through the Variables page to check that the method is OK this is not necessary if this was done in the method editor 6 Click on the Next button at the bottom of the window this takes you to Questions Enter the answers to the questions The answers will be saved in the result file Some questions may 20 have been defined as mandatory mand These must be answered before the run can be started Making your first runs 18 1120 02 Edition AC Starting a run 5 Questions Ed Sample volume and type Chrom Column Chrom Eluent Chrom Eluent B Chrom Remarks Chrom 7 Click on Next This takes you to Notes You can write your own comments in the Start notes Method Notes Start Notes Here you can write your own comments and notes A Run Notes Evaluation Notes Jo 8 Click on Next This takes you to Evaluation Procedures Evaluation Procedures are automated evaluation operations that are performed after the run Mark Print_Chromatogram The chromatogram will then automatically be printed after the run Evaluation Procedures Ed Selected Evaluation Procedures will run at the end of the method Integrate and Print Making your first runs 18 1120 02 Edition AC 21 5 S
11. a particular system If you are not connected the text NO is written in the Connect panel in the Run Data window Once you are connected the text changes to YES Click on the System Control icon The System connect dialogue window appears System connect ZAK TAPPLE AK TAexplorer 100 AKT Apunfter 10T Process OF Cancel Help Select a system from the list If you are not connected to a network only one system will be shown Click on OK When connected the text YES is written in the Connect panel in the Run Data window You only have to connect once If you do not select System Disconnect you will be automatically connected to the system the next time you login to UNICORN General system preparation 1 The correct tubings 0 25 0 50 or 0 75 i d mm for the column you intend to use must be installed See Section 2 1 in AKTApurifier System Manual for an overview over columns with recommended tubings For most columns the tubings mounted from the factory can be used Comment If tubing with too large inner diameter are used the peaks will become broader than necessary If tubing with too small inner diameter are used the backpressure from the tubing might become higher than the max pressure for the column and the run will stop immediately after it is started Immerse inlet tubing Al or A2 if you changed this in the method in buffer A and inlet tubing B1 in buffer B 3 Put the w
12. ailable on request Adresses Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England H P7 9NA Amersham Biosciences SE 751 84 Uppsala Sweden Amersham Biosciences 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences 1998 All rights reserved Contents The system and the software O O N OO OF FP QO N Short instructions on back page Making your first runs 18 1120 02 Edition AC About this Quide ccccccccssseesesseseees Creating a method ssssssssnnnnnnnnnnnnnns Preparing the system for a run Starting a run 20 cceeceeeseeseeneeeeeeneeseeees Viewing a run sssssssssnnnnnnnnnnnnnnnnnnnnnnnnan Viewing and printing the result BufferPrep and Scouting 0000 Going further cccceeseseeseeeseeeeeees Contents Contents il Making your first runs 18 1120 02 Edition AC About this guide 1 1 About this guide This guide is written for users who are not familiar with UNICORN software and KTA purifier Here you will learn the basics of UNICORN and how to operate AKTApurifier from UNICORN UNICORN is a software package for control and supervision of the AKTApurifier chromatography system It runs on an IBM compatible PC under Windows NT and includes hardware for interfacing the controlling PC to the chromatography liquid handling parts of AKTApurifier In this guide you will learn h
13. am The current chromatogram is the one you have open on the screen In the next step you will select which curves to print Remember that the term chromatogram refers to the whole chromatogram window and not to individual curves Comment Select All chromatogram if you have many chromatogram windows on the screen and want to print them all 28 Making your first runs 18 1120 02 Edition AC Viewing and printing the result A Generate Report x Report formats Modified Report contents Select items IY Header O Curent chromatogram All chromatograms Method i T Documentation e Evaluation log gt W Chromatogram Quantify and Mol size Akee Chromatogram layout Indicates that items for Chromatogram are shown to the right l Use thick curve lines Print curves in landscape format Define Curent Report title Preview Help Define button Current button 6a To print the current chromatogram as it looks on the screen click on the Current button below the list to get the current layout 6b If you want to change the layout click on the Define button The Chromatogram Layout window is opened and you can select curves and change the layout of the chromatogram just as you did in the previous section Mark which curves to print Click on the Header button The information selected here will be printed on top of the chromatogram Select Variables and or Questions Click on OK Clic
14. aste tubing from port 1 of the outlet valve into a waste 18 Making your first runs 18 1120 02 Edition AC Preparing the system for a run 4 bottle Check that the tubing from port 2 of the outlet valve is connected to the fraction collector if used The flowthrough will be collected via the tubing F3 from port 3 of the outlet valve or if you enter a value for Flowthrough_FracSize in the fraction collector 4 If there is air in the inlet tubing or if you suspect air in the pump purge the pump with a syringe as described in section 2 8 in Pump P 900 User Manual 5 Calibrate the pH monitor optional if required Refer to Section 6 6 in UNICORN 3 0 User Manual or Section 3 6 in Monitor pH C 900 User Manual 6 Connect the column between port 1 of the injection valve and the top of the UV flow cell Use a suitable length of 0 25 mm PEEK tubing blue supplied with your system 7 Insert a sufficient number of tubes into Frac 900 optional and place the arm at the first tube 8 Double click on the System Control icon Fill the inlet tubing with the correct solutions by selecting Manual Pump Then select instruction PumpWash Select ON for Inlet A1 and set Inlet A2 to OFF Select ON for Inlet B1 and OFF for Inlet B2 Click on Execute to fill the inlet tubing The injection valve will switch to waste WINCORN_LAB2 Pump Instructions Ea Instructions Parameters Pump Inlet amp
15. c 300 Flowthrough andor elution Elution Linear segmented or step gradient CHECKLIST BEFORE METHOD START sys_f gr ELUENTS a For column Select column RESOURCE G 1 mi Global Press OK Cancel Help 2 Select a system Then select a chromatographic technique for example Anion_Exchange 3 A list of available templates will appear By clicking on a template an explanation for the template appears to the right in the Method Notes field Select the template Man_f_gr which is suitable for the first run Comment The other templates are briefly described at the end of this chapter 4 Select the column you intend to use The correct column volume the recommended flow rate and the correct pressure limit for that column will then be automatically implemented in the method Comment If you manually alter the default values and thereby exceed the recommended values for the selected column you will get a warning when you save your method Making your first runs 18 1120 02 Edition AC 11 3 Creating a method Method variables 12 If you want to perform a test run without a column you should still select a column a small one is recommended to get suitable default parameters in the method Then in the method use a peice of tubing to replace the column Comment If you do not find your column in the list you can add one See section 5 9 in UNICORN User Manual 5 Click on OK The method t
16. defined all the runs you require Use the horizontal scroll bar to see more runs Making your first runs 18 1120 02 Edition AC 31 8 BufferPrep and Scouting 14 Click on the buttons at the top of the scheme to toggle between Run and Excluded for the different runs Those marked Excluded will not be run A scouting scheme is now defined 15 Click on the Variables page Change and check the values for the same variables as in the man_f_gr see point 8 in Section 3 16 Select File Save 17 When the method is started all the runs in the scheme will be performed automatically and the set pH for each run will be prepared automatically Each run in the scouting scheme will generate a separate result file which are all stored in a special scouting directory 18 Prepare the system and start the run as described in section 4 and 5 When filling the inlet tubing with the correct solutions using the instruction PumpWash in System Control Manual Pump select the correct inlet A1 for Inlet A1 and set Inlet A2 to ON Select ON for both Inlet B1 and Inlet B2 Click on Execute to fill all the inlet tubing Click on Close The sample should if possible have a pH close to the highest pH in the scouting run for anion exchange and close to the lowest pH for cation exchange 32 Making your first runs 18 1120 02 Edition AC Going further 9 9 Going further Once you are used to the system and software you may want to learn more about
17. e run if selected you need not alter anything described in this section However if you want to alter the chromatogram layout this section will teach you the basics of the evaluation module Viewing 1 After a run you can view the result Click on the Main menu icon Double click on a result file icon in the list to the right 2 The Chromatogram window is opened automatically in the Evaluation workspace when you open a result file The Chromatogram window contains all the curves Note that the term chromatogram is used here when talking about the whole window containing all the different curves Evaluation e1p203 1 File Edit View Integrate Operations Procedures Quantitate Mol Size Adviser Window Help Data Salo e1p203 1 No header information selected or available E1P203 1_U 81P203 1_Cond E1P203 1_Cond E1P203 1_Cone E1P203 1_pH E1P203 1_Pressure E1P203 1_Flow E1P203 1_Temp E1P203 1_Fractions E1P203 1_Inject j l oy No peak table selected or available Chromatogram window Temporary jim E Ready S ee ee Astar 45 UNICORN Main Menu lt 4qMethod Editor untitled Evaluation e1p203 1 S Paint Shop Pro The result file from a run holds a complete record of the run E including method system settings curve data and run log It is accessed by clicking the Documentation button Comment Original raw data curves can never be modified renamed or deleted from a re
18. e at the top of the Curve Data window to shift to a scale for another curve The colour of a curve its Y scale and its name are always the same Click on the X axis to shift between time and volume 10 The Logbook is shown at the bottom The Logbook shows exactly when the instructions in the method are executed during the run The Logbook is stored in the result file 11 You can make manual changes during the run Select Manual Pump The Instructions box is opened WINCORN_LAB2 Pump Instructions Ed m Instructions Parameters ante s Insert Pump FlowR ate 0 100 Frrieccscasted G radient Flowpath BufferPrep_pH 0 000 ml min Ki gt SampleFlow C Alarms amp Mon Pumpwash SystemWash Other SamplePump wash MethodB ase 12 If for example you want to change the flow rate select Pump and then Flow Enter a new flow rate under Parameters and then click on Execute The new flow rate will be used until the end of the run or until a new flow rate instruction is reached in the method Close the box by clicking on Close All manual interactions are recorded in the Logbook 13 If you want to stop the run before it is finished click on the End button at the top Run _Pause_ Continue End alale 24 Making your first runs 18 1120 02 Edition AC Viewing and printing the result r4 7 Viewing and printing the result If you are satisfied with the automated printout obtained after th
19. e correct method for preparing buffers in the Notes field Accuracy of preparation is essential When preparation is finished connect them to the correct inlets 8 Click on the Scouting page 9 Click on Define A list of all the variables will appear Mark the variable BufferPrep_pH and any other variable you wish to alter e g flow rate Click on OK Comment Values for variables selected for scouting are greyed on the Variables page and cannot be changed there Each run column represents one run Evaluation Procedures Method Infgfmation Result Name Start Protocol Variables Scouting Questions Notes radient BufferPrep Columns Reference Curves Initial Eluent_Conditions IX Run 1 Run 2 Run 3 Run 4 Excluded Excluded Scouting variable BufferPrep_pH 6 0 7 0 a0 jad a KE Define Clear All Delete Insert Help 10 Click on any cell in the column under Run 1 in the scouting scheme This inserts the default values for the scouting variables 11 Make any changes you require in the variable values Comment For variables with text values e g column position double click on the variable field and select the required value from the list that appears 12 Click on the next run column in the next run and click on any cell in that column to copy the values from the preceding run and change the values as required 13 Repeat step 12 until you have
20. emplate will now be opened as an untitled method 6 Select View Run setup may already be checked Click here to select page 7 Run setup consists of a number of pages You will only look at two of them now Variables and Notes You select a page by clicking on the respective tab at the top of the run setup screen 8 On the Variables page the method is presented by a number of blocks name in blue The blocks represent the typical steps in a chromatographic run Start conditions Column equilibration Flow through collection Sample injection Wash out unbound sample Fluate fractionations Making your first runs 18 1120 02 Edition AC Creating a method 3 Gradient Clean after gradient Reequilibration Each block contains a number of method variables name in black with suitable default values The values are easily changed to suit your requirements Click on the scroll bar to see the next part of the Variables page The only values you must change in the man_f_gr template are for e Empty_loop_ with in the block Sample_Injection Enter a value of 5 x the volume of the sample loop to apply all the sample onto the column Sample Injection Ernpty loop with ml 0 00 999999 00 e Flowthrough_FracSize in the block Flowthrough and Fractionation Enter a suitable fraction size The fraction collection starts at injection and continues until gradient start Zero means that fractions will
21. emplates not only basic_ax A special template is not required for scouting Scouting can be performed with any template for any technique 3 Select View Run setup in the Method editor may already be checked Click on the BufferPrep page 5 The radio button is ON when BufferPrep is selected These stock solutions should be prepared and connected to the correct inlets Evaluation Procedures Method Information Result Nami Start Protocol Variables Scouting Questions Notes Gradient BufferPrep Columns Reference Curves BufferPrep Stock solutions ON OFF Inlet 41 418 Buffer 0 05 M 1 methyl piperazine 0 05 M Bis Tris 0 025 M Tris 100 B 1M pH Range krata lla N Notes BUFFER SOLUTION 2000m a Inlet A2 Acid Base 1 methyl piperazine 0 02g Mw 100 16 0 1 M HCI Note Volatile liquid Place a beaker with water on the balance and add 1 methyl piperazine Bie Trit Inlet B1 Water 20 929 Mw 209 2 Tris 6 06g Mw 121 1 Inlet B2 Salt ACID SOLUTION 1000mi 2 M Nef Use ampoule 0 1M HCI x Corr Factors Help Making your first runs 18 1120 02 Edition AC BufferPrep and Scouting 8 6 The recipe 5 0 9 5 pH AIEX is selected With this buffer recipe any pH between 5 and 9 5 can be prepared on line 7 The required solutions and the inlets to which they should be connected are displayed to the right on the BufferPrep page You find th
22. from a remote computer 3 in the User Manual for each instrument found in the binder AKTAdesign Components See each individual manual in the binder AKTAdesign Components 11 in UNICORN 3 0 User Manual 6 5 in UNICORN 3 0 User manual Making your first runs 18 1120 02 Edition AC November 1998 Short Instructions When you have read through the complete Making your first run booklet for AKTApurifier you can use these short instructions as a check list for creating a method and starting a run 1 Select File New Method in the Main menu or click on 2 Select System Technique Template and Column Click on OK 3 Select File Save in the Method editor and give the method a name Click on OK 4 Click on 5 Select File Run Select the method and click on Run 6 The start protocol will appear Check the method on the Variables page and change values as you require 7 Click on the Next button and go through all the other pages 8 On the Evaluation procedures page select Print_Chromatogram to get a printout automatically after the run 9 Click on the Start button on the last page and the run will start Amersham Biosciences TC information ab Uppsala Printed in Sweden by T K i Uppsala AB
23. ght mouse button and select Properties Now you can select which parameters you want to see in the Run Data window For eaxample select Acc time Block time flow and pressure Click on OK Run Data Properties Ea Run Data Layouts Colour Setting New layout Delete layout Cancel Help 4 The Curves window shows the curves during the run Position the cursor in the Curves window Click on the right mouse button and select Properties Here you can select which curves to show during the run All curves are stored in the result file Curves Axis Y Axis Curve Style and Colour m Display curves Select All Clear All Cancel Help Making your first runs 18 1120 02 Edition AC 23 6 Viewing a run 6 By clicking the different tabs in the Curve Properties window you can set the properties for the different curves Normally the curves are scaled with auto scaling i e the scale is adjusted continually to the highest and lowest values for each curve 7 To fix the Y axis scale for a curve mark the curve click on Y axis click on Fixed and enter the max and min values You can repeat this for other curves Click on OK 8 To maximise the Curve Data window Position the cursor in the Curve Data window Click on the right mouse button and select Maximize Go back to normal size by clicking on Restore 9 Click on the Y axis scale or click on the curve nam
24. k on OK at the bottom of the Chromatogram Layout window Comment The changes made here in the Chromatogram Layout window will only affect the printout and not the presentation of the chromatogram on the screen Report Chromatogram Layout x Curve Style and Colour Edit Texts Save and Open Layouts Header Curve Names Y Axis AK S Curve Peak Table Include in header window l Scouting variables IV Variables 7 Click on Preview to view the report on the screen Click on Print and the report is printed Making your first runs 18 1120 02 Edition AC 29 8 BufferPrep and Scouting 8 ButferPrep and Scouting 30 BufferPrep ON Selected recipe pH range Notes field The BufferPrep function allows a buffer of any pH to be prepared on line from four stock solutions The pH can be varied automatically between scouting runs to find the optimal pH for the separation A pH electrode is not necessary to obtain correct pH using BufferPrep For more details about BufferPrep see Section 2 7 in AKTApurifier System Manual Scouting allows any run parameters e g pH to be systematically varied automatically in repeated runs Below is a description of how to perform a pH scouting 1 Select File New Method in Main menu Select system Then select Anion exchange as technique 2 Select template man_f_ax Select a column and click on OK Comment BufferPrep can be used with all xxx_yy_ax and xxx_yy_cx t
25. n the injection valve 5 After the injection valve the flow is directed to the column and then forward to the UV cell and the conductivity cell which are located inside the cell holder on Monitor UV 900 6 The flow path continues to the flow restrictor and through the pH flow cell if fitted optional and further to the outlet valve which is used to switch the outlet flow between waste fraction collection and flowthrough 7 The flow path can continue to a fraction collector optional if desired Making your first runs 18 1120 02 Edition AC 5 2 The system and the software UNICORN overview 1 Switch on the computer Log in to Windows NT 4 by first F E pressing Ctrl Alt Del and then clicking on OK After a while the ar Windows NT 4 desktop appears Unicom 3 0 2 Start UNICORN by double clicking on the UNICORN icon 3 Select a user from the Users list and enter the password If you log in for the very first time select user default and enter the password default Click on OK Note You should enter users and individual passwords before starting using AKTApurifier on a regular basis Logon EI Users Password 4 An information window appears during start up About UNICORN 3 00 x k UNICORN Version 3 00 04 lind Amersham Pharmacia Biotech Copyright 1998 http ww apbiotech com b UNICORN y Making your first runs 18 1120 02 Edition AC The system and the softwa
26. nly learn how to operate the chromatography system from UNICORN Switch on the chromatography system with the ON OFF button located on the front of the base platform to the bottom left 4 Making your first runs 18 1120 02 Edition AC The system and the software 2 Comment The flow path between the different components in the system is shown and described below It is not necessary to go through this in detail to make your first runs Look to the right hand side of the system if you want to follow the description System pump Column Monitoring Fractionation lt SSS ey ri Injection T J t F ih NJ Outlet valve alll cil On line filter estretor INN ME NS al 6 265 pH monitor Optional Mixer Sample UV monitor Conductivity monitor Switch valves ls Flowthrough ti collector Waste 1 The pump has 4 pump heads two for pump A and two for pump B Pump A is the one closest to the front 2 The buffer solutions are pumped through switch valves and further to a mixer Inlet A1 and B1 are placed in buffer A and B respectively Inlet A2 and B2 are used when buffers are prepared automatically by BufferPrep 3 The flow path continues from the pump to the mixer and forward via an on line filter to the injection valve 4 A sample loop is connected between ports 2 and 6 on the injection valve The sample loop is filled manually using a syringe for this procedure connect a fill port to port 3 o
27. ow to e create methods e prepare the system for runs e perform runs e make simple evaluations e make reports e perform automatic method optimisation Scouting e prepare automatically buffers of any pH BufferPrep Follow the guide from page to page in front of the computer The time will be well spent Note To follow the instructions it is not necessary to read the comments written with smaller font containing additional information Making your first runs 18 1120 02 Edition AC 1 1 About this guide Pre requisites The system and the software must be installed and functioning and the monitor and pump calibrated as described in the separate Installation guide IMPORTANT Before using AKTApurifier read all the safety information in Section 1 2 in KTApurifier System Manual Typographical conventions Menu commands and dialogue box prompts are identified in the text by bold text A colon separates menu levels thus File Open refers to the Open command in the File menu Z Making your first runs 18 1120 02 Edition AC The system and the software 2 2 Ihe system and the software AKTApurifier AKTApurifier is a fully automated liquid chromatography system designed for method development and research applications The separation unit of the chromatography system has three main modules which are stacked on the left hand side of a base platform They are e Pump P 900 a family of binary high performance g
28. radient pumps In AKTApurifier 100 the flow rate is up to 100 ml min and the pressure up to 10 MPa pump designation is P 901 In AKTApurifier 10 the flow rate is up to 10 ml min and pressure up to 25 MPa pump designation is P 903 e Monitor UV 900 a multi wavelength UV Vis monitor for simultaneous monitoring of up to 3 wavelengths in the range 190 700 nm e Monitor pH C 900 a combined monitor for on line conductivity and pH monitoring Fraction collector Frac 900 optional is placed to the right of the system Up to 175 fractions can be collected SS S ESED 3 SSE SS ae SSeS Making your first runs 18 1120 02 Edition AC 3 2 The system and the software Components such as the mixer column and different valves are mounted in the section to the right The separation unit is controlled from UNICORN software Column holder Column Injector valve INV 907 Box 900 Monitor pH C 900 Outlet valve PV 908 On line filter Monitor UV 900 gt To Fraction collector is valve Flow restrictor a Mixer chamber Mixer M 925 Pump P 900 P 90 1 903 To waste flask W1 W2 and W3 ON OFF button Conductivity cell below lid Switch valve A Switch valve B UV flow cell SV 903 SV 903 Pump P 900 Monitor UV 900 and Monitor pH C 900 can also be controlled individually from the modules without UNICORN software In this guide however you will o
29. re 2 5 Eventually the UNICORN Main menu window appears on the screen At delivery a Toolbar guide is displayed providing a quick guide on how to use the toolbar items The Toolbar guide is inhibited by unchecking the square at the bottom left Click on Close e lt 5 Methods c default basicac basicgf kal basicgr z 901 tz insttest iz manfgr E manpac manpgf manpgr Ready 4 UNICORN Main Menu File View Administration MethodQueue Logoff Adviser Window Help Aktafple Aktafple Aktafple Aktafple Aktafple Aktafple Aktafple Aktafple Aktafple Siz Tuna Toolbar Guide 249KE 248KE 249KE 302KE 177KE 250KE 250KE 249KE 251KE Begin your work directly by using the Easy Start buttons el x Iof x Results Iof x a io i Modified 2 3 98 2 50 PM 2 3 98 5 17 PM 2 3 98 4 05 PM u nn nnn en 2 4 98 12 29 PM UNICORN Main Menu 2 3 98 4 59 PM 2 3 98 4 59 PM 2 4 98 2 50 PM 11 19 97 11 2 2 3 98 4 48 PM 2 3 98 4 54 PM 2 4 98 12 05 PM 2 4 98 12 26 PM 2 4 98 6 47 PM yee the 2 3 98 4 13 PM Evaluation module 2 3 98 4 29 PM immediately start a new run Control your system Create a new method User default 6 The Main menu window is the central part of UNICORN displays from which you navigate through the control system It is mainly used for file handling In the Methods window to the left in Main menu all method files that you create are displayed
30. sult file Making your first runs 18 1120 02 Edition AC 25 r4 Viewing and printing the resut gt gt S S 26 Highlight curves to view 3 Maximise the Chromatogram window by clicking on the larger square in the upper right corner 4 All changes regarding the presentation of the curves are done in the Chromatogram Layout window Position the cursor in the Chromatogram window Click on the right mouse button and select Properties or select Edit Chromatogram layout to activate this window Chromatogram layout 1 Curve Style and Colour Edit Texts Save and Open Layouts Header Curve Mames YT Asis Axis Curve Feak Table Select curves to display TTTEs Es FvIICO aU 7 pe2 Esra aE Dz Batch E21 UV Onm 03 Batch R21 US Onm 04 05 06 Batch kK2 1_ Cone Dr 08 BatchiK2 1 _ Pressure 09 Batch 2 1 Flow 12 Batch1K2 1 Inject 13 Apply to all chromatograms Cancel Spp 5 Highlight the curves to view under Curves Curves are named as Resultfile 1_ curve where a curve can be for example UV_wavelength Cond pressure etc De select all curves except for example the UV Cond and Conc curves Click on OK at the bottom of the Chromatogram Layout window You can easily zoom in on the curves Place the cursor in the chromatogram click on the mouse button and holding it pressed down move the mouse A rectangle appears on the screen When you release the mouse but
31. tarting a run 9 Click on Next This takes you to Method Information Here you see information about the run The approximate volume of buffer used A B is shown as well as how long the method will take 10 Click on Next This takes you to Result Name Here you name the result file and define in which directory the result should be stored A default name the method name followed by 01 and a directory are suggested But you can change the result name and directory click on Browse if you so wish 11 Click on START The run will start lt gt System Control 1 Systemi Method Result X File View Manual System Adviser Help Run Hold Pause continue End Connection Run Status Acc volume Block volume Block time U4 _260nm U 2_254nm U 3_215nm Pressure Flow Run Data Temp Flow Scheme f Injection alye CAS 0 00 min Flow 1 000 ml min Manual 0 08 min End 10 37 Manual For Help press F1 O End Controlled By default 22 Making your first runs 18 1120 02 Edition AC Viewing a run 6 6 Viewing a run When the system pump is running the text Run is shown in the Run Status Run Status panel in the Run Data window 1 2 Select View Windows Check Rundata Curves and Logbook Click on OK The Run Data window at the top shows current values for running parameters Position the cursor in the Run Data window Click on the ri
32. to get general help about the current module and find new help topics or Help Index for a specific topic Double click on green text to get more help In any box click on the Help menu to get help on how to use the current active box Click on Adviser in the menu bar and choose the appropriate system to get help about separation optimisation protocols Method Adviser detailed information about chromatographic columns Media Adviser and information about AKTApurifier System Adviser Fo vise Help m Hle View Manual System Making your first runs 18 1120 02 Edition AC 2 2 The system and the software 10 Making your first runs 18 1120 02 Edition AC Creating a method 3 3 Creating a method UNICORN is supplied with a number of almost ready made methods called method templates Different method templates are available for different chromatographic techniques The method templates can be run as they are or you can easily modify them to design your own method in a very short time Let s start 1 Click on in the Main menu Toolbar The New Method window will appear For system Select system purifier Z T No template Template selection Technique Method notes Select technique Anion E change TEMPLATE man_f_aor version 1 03 A Seeda lat Temolat Gradient techniques without BufferPrep elect template emplate Injection Manual Loop or Superloop filled before method start Fractionation Fra
33. ton the part within the rectangle will be enlarged You can zoom further on the enlarged part Click on the right mouse button and select Undo or Reset zoom to return to the complete chromatogram Click on the Y axis scale to change to a scale for another curve The style and colour of a curve its Y scale and its X scale can all be changed Making your first runs 18 1120 02 Edition AC Viewing and printing the result r4 8 Open the Chromatogram Layout window again Click on the Y axis and X axis tabs to set the scale for the different curves Normally the curves are scaled with auto scaling i e the highest and lowest values for each curve set the scale 9 To fix the Y axis scale mark a curve click on Fixed and enter the max and min values for that curve You can repeat this for other curves 10 To fix the X axis scale click on Fixed in the X axis field and enter the min and max values for the X axis Click on OK 11 Click on OK at the bottom of the Chromatogram Layout window to execute all the changes Comment When you have made the necessary changes in the Chromatogram layout box they can be saved as a Layout Click on the Save as button at the top of the Chromatogram layout box to save the layout Give the layout a name and click on OK Layouts can be selected in Apply layout at the top of the box and all your saved selections will apply Saved layouts can be applied to any result file 12 Minimise the chromatogram
34. window by clicking on the smaller squares in the upper right corner 13 Click on the Documentation button A number of pages appear 3 as in the Run Setup in the Method Editor All documentation about the run is stored here e g the method answers to questions variables logbook etc For example click on the Notes and Logbook pages to check the contents Close the Documentation window by clicking on the X in the upper right corner Documentation x Text Method Questions Motes Columns Reference Curves Evaluation Procedures Method Information Result Hame Start Protocol Settings Calibration Logbook E valuation Log ml Run 98 01 28 11 11 56 Method Apis Result qhansfolesp24F pfrsde res Filter 0 00 ml Base Volume iml dl 0 00 ml ColummPosition Position erat 0 00 ml Gradient OO 2B O 00ml I Method 0 00 ml Flow 3 20 rolemin on 0 00 ml AveragingT imel 0 64 M Manual 400 ml AutozeroLl y 420 ml Injection ale Inject M Errors 4 50 ml Qutletyalve F2 M System 470 ml Injectionyalve Load F 00 ml Qutlety alve F3 6 50 ml Qutletyalve Fi 6 00 ml Gradient 90 0 8 10 00 ml 6 00 ml Peak Fractionation 2 000 ml 6 00 ml Peak_FracParameters 15 000 m min 10 000 m min 0 100 min 18 07 ml Gradient 30028 5 00 ml 22 50 ml Peak_FracStop 23 00 ml Outlet ale F2 23 50 ml Gradient 0 0 6 0 00ml 24 50 ml Outletyalye F7 ml End 11 21 17 Base Time Volume m ok ewes Making
35. your first runs 18 1120 02 Edition AC 27 r4 Viewing and printing the result Printing and making a report 1 The quickest way to print out the chromatogram as it appears on the screen is to select File Print 2 To include more information in the report select File Report Under Report contents select which main items to include For example select only Header and Chromatogram Generate Report Report formats Indicates that items for fa _Saveas _ pees Dete Aae Header are shown to Modified the right Report contents Select items I Header 5 Current user Aur user It Main items to include Method i gt Curent date amp time E ad i Aun date amp time in the report I Documentation o Report title in Header Result fle name Evaluation log s Method file name Chromatogram e Fage number Quantify and Mol size C E Print curves in landscape format l Use thick curve lines Report title Preview Close Help 4 Check and select the contents in Header by clicking on the radio button beside Header Select what you want to include from the panel to the right Comment You can mark items in the right hand panel regardless of whether the corresponding Report Contents heading is selected ticked or not Only the selected items for which the Report Contents headings are ticked will be printed 5 Click on the radio button beside Chromatogram and select Current Chromatogr
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