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P/ACE™ MDQ User`s Guide

1. E 5 5 2 8 7 7 O V ES Ed 7 9016951 Figure 2 6 Tray and Cartridge Covers 2 Cartridge Cover 1 Tray Cover 32 Karat 8 0 PN A33115AB 10 System Overview 2 UV Detector Optics 2 6 UV Detector Optics The UV optics includes an ultraviolet light source selectable wavelength filters aperture capillary and a single photodiode detector as shown in Figure 2 1 The UV source is a deuterium lamp with a wavelength range of 190 to 600 nm Two lenses focus and direct the output of the lamp through one of the wavelength selecting filters located in a rotating wheel behind the capillary cartridge The beam continues through the aperture in the cartridge plug and through a section of the capillary that has been treated to remove the polyimide coating the detection window The non absorbed beam then continues through a fiber optic cable to a photodiode The light signal is converted to an electrical signal digitized and sent to the 32 Karat workstation for processing by the software This signal is also available as an analog output through a connection on the left side of the instrument The design of the instrument insures that the optical system remains in alignment No user alignments are required There are eight positions on the UV filter wheel
2. Actual Vials Used For Actual Vials Used for Run Repetition Event Inlet Outlet 1 1 Rinse 1 BI A1 BO A1 1 1 Rinse 2 1 BO A1 1 1 Inject SEA1 BO A1 1 1 Wait 1 BO C1 1 1 Separate BI C1 BO C1 2 1 Rinse 1 BI A2 BO A2 2 1 Rinse 2 BI B2 BO A2 2 1 Inject SI A2 BO A2 2 1 Wait BI C2 2 2 1 BI C2 BO C2 3 1 Rinse 1 BI A3 BO A3 3 1 Rinse 2 BI B3 3 1 Inject SI A3 BO A3 3 1 Wait BI C3 B0 C3 3 1 Separate BI C3 4 1 Rinse 1 BI A4 BO A4 4 1 Rinse 2 BI B4 BO A4 4 1 Inject SI A4 BO A4 4 1 Wait 4 4 4 1 4 4 From Sequence Table 2 run results can see that those results are identical to results from Sequence Table 1 run Therefore one can conclude that no matter how the sequence table is organized multiple repetitions of one method in one run or separate runs each run consisting of one repetition of same method the vial incrementing results are the same E 6 32 Karat 8 0 PN A33115AB Appendix E E Examples of Use Example 2 Vial incrementing also works when there are different methods interspersed with one another in sequence table Consider 2 methods methods A and B that alternate in the sequence table Method Time Program A 5 Initial Conditions amp PDA Detector Initial Conditions C Time Program Summary 17 Min ramp normal polarity In Qut vial inc 2
3. 22 Creating a New Instrument 22 Configuring the New 1 5 23 Auto Gontiguratiom 26 visa Lice BORE era e n 25 Configuration 27 Instrument 26 Commguranont 29 Configuration 5 30 Manual Configuration of the PDA and LIF Detectors 30 User s Guide PN A33115AB Section 4 Direct Control t tra e te re ER ER RR ART Rr dts 31 Direct Control Window 32 Exercise Conditioning the Capillary 35 Voltage Setting Parameters 38 Section 5 Creating and Editing a Method REC E 41 Creating and Editing a Method 41 Initial Conditions Tab 42 Auxiliary Data Channels 42 Mobility channels 43 Temperature oea
4. 4 4 As one can see from the Sequence Table run results above when method time program event calls for incrementing every one cycle of method the vial positions are shifted by one vial after each repetition is run e g from BEAI to BI A2 from BEA2 to BI A3 and so on for Rinse 1 event Remember one vial position is incremented in the same column to the adjacent vial position of the next row Incrementing is also done from the vial position of the highest row of one column to the lowest row of the next column For example Al A2 A4 A5 A6 B2 etc 32 Karat 8 0 PN A33115AB Appendix E E Examples of Use Alternatively the user can generate the same Sequence Table 1 run results by doing a Single Run of method B using 4 repetitions Single Run Acquisition Calibration level The user then generates another sequence table Sequence Table B2 with 4 separate runs each run consisting of only one repetition of the same method B lii Sequence Sequence Table B2 seq Unknown 1 08 0 5 0 7 methodf met Unknown Eo eth met thd met e User s Guide PN A33115AB E 5 Appendix E Examples of Use The results of instrument sequence run of Sequence Table E2 are as follows Table E 2 Sequence Table Run Results
5. 102 Modifying a Report Template 102 Saving Report as Template 105 Report Templates cence eset ELS A 105 Method Custom Report Templates 105 Sequence Custom Report Templates 105 Appendix A 32 Karat Online 1 Installation and Maintenance Manual 1 Beckman Coulter Service 1 on Accra eot er trt d e eat ix a ua Oh pus aestus 1 Oiler RelerenGes ee tu sawn Mes button itx dw cb datei M a BA at 1 Appendix B EPIIT 1 AW te tt deeds pe A E 1 Corrected Migration Time 1 Velocity Corrected Area 1 Vn ENTER 2 Appendix C AVES oce ma pde cedes Ud UR Cu da E 1 Appendix D qx cce IUE D Mu E IE 1 Appendix E Introduction S 6 d raros d scho a d asbestos aod Ger 1 Example Tiu 41 acea edad oec ee Neat ees o D Mos ole a eua als 3 N us lote ea quA tue a d 7 e
6. although other factors such as pressure assisted migration could also be considered here In this discussion we will limit apparent mobility to the sum of electrophoretic mobility and EOF Papp eof The 32 Karat Software has the capability to use mobility as an alternate to migration time for peak identification Used in this way mobility increases the robustness of a capillary electrophoretic method by correcting for small changes that may be present from run to run The software is also capable of displaying electropherograms with the X axis scaled in terms of mobility rather than time In this exercise you will review these features of the software User s Guide PN A33115AB 93 Mobility Mobility Markers 10 2 Mobility Markers 94 Every run that will incorporate mobility calculations must include a mobility marker This is a component for which the mobility has been defined under a given set of run conditions Reproducing these run conditions in subsequent runs is critical in using mobility as a parameter for peak identification Buffer factors such as ionic strength and pH are especially important See the resources in Appendix A for more information on this topic Assigning Mobility Values Assigning a mobility value to the mobility marker involves a series of steps NOTE The following assumes that the method development work to create an effective separation technique for the samples to be analyzed has alrea
7. Review C Results review pause each Calibration review pause efter each calibration set Printing Print sequence reports Figure 8 5 Process Sequence dialog 5 From the menu bar select Method Review Calibration A window will open showing the results of the calibration for the first component Alpha The data looks reasonable but the line fit could be better 6 Select Beta in the upper right corner of the window The curve fit here is certainly not acceptable The lower right corner of the window indicates that this fit has been made point to point Perhaps another type fit would be better Re open the Peaks Groups table Scroll left to the column headed Fit Type For line one Alpha select Quadratic 10 For line 2 Beta select Linear 11 In line one select the box in the Zero column to force the line through the origin 12 Do not click Zero in line 2 13 Save the method and look at the calibration results again MDQ User s Guide PN A33115AB 87 Calibration Analyzing Unknowns 8 4 88 This time the curve fit should be better for both Alpha and Beta In practice you would select and vary the curve fit parameters to determine the most appropriate fit for your data set Saving the method will save the calibration It is not necessary to reprocess the sequence Analyzing Unknowns Creating a standard curve is only useful if
8. m Sample Trays n3 c5 e 3 Jo Enable Trav Definition Temperature Control Height fi fi fim Available Figure 3 7 P ACE Instrument configuration for UV Detector Each P ACE MDQ connected to the computer must have a unique device ID The default setting is 1 If multiple MDQ instruments are installed select a unique ID 1 2 3 or 4 for each instrument 9 After the device address is selected click Set Bus Address to send this information to the instrument The type of buffer tray and sample tray present is detected at startup Tray type is detected each time the tray cover is opened and closed The instrument autodetects three types of trays 36 vial buffer trays large reservoir buffer trays and 48 position sample trays 96 well plates are not autodetected by the instrument and must be manually configured from this window Tray configuration may be manually changed at any time 10 When a 96 well plate is selected as the sample tray type the Enable Tray Definition check box will become active If this check box is selected the user has the option to define the height and depth of the plates installed This feature allows the use of deep well and other non standard plates If this check box is not selected only standard Beckman Coulter 96 well plates may be used If a Laser Induced Fluorescence Detector has been detected t
9. Help by e mail A special mailbox has been created for users of 32 Karat Software Send your questions and comments to 32karat beckmancoulter com A team of specialists will review your message and contact you Messages received outside of regular business hours Monday through Friday 8 a m to 5 p m Pacific time will be reviewed the next business day Other References There are many publications dealing with applications of capillary electrophoresis methodology routine operations and related techniques Your local research library is the best place to begin Beckman Coulter web site http www beckmancoulter com The online source for the latest information on CE from Beckman Coulter P ACE Setter Newsletter A quarterly newsletter for scientists employing capillary electrophoresis P ACE Setter features tips and techniques and product updates Each issue features a specific application for CE Free subscriptions are available through the Beckman Coulter web site or contact your local Beckman Coulter representative Your Local Beckman Coulter Representative The best source for product information Contact your representative for information on the latest products or to place an order 1 Appendix A Additional Resources 32 Karat 8 0 A 2 PN A33115AB Appendix B B CE Calculations Appendix B B 1 CE Calculations Introduction Processing of electrophoretic data is similar to the processing of chromatographic
10. In Method Time Program A line 1 indicates a separation step that increments inlet and outlet vials applied after every second cycle of method By combining Method Time Program A and Method Time Program B from Example 1 the user can generate the following sequence table Sequence Table to run lii Sequence Sequence Table AB seq Run Reps Sample Inject Inlet Sample Inject Outlet Sample Inject Duration Method The results of instrument sequence run of Sequence Table AB are shown below For simplicity only the separation event is considered User s Guide PN A33115AB Appendix E Examples of Use Table E 3 Sequence Table AB Method Actual Vials Actual Vials Run Repetition Used Event Used Inlet Used Outlet Cycle Used 1 1 BED1 BO D1 1 2 1 B Separate BI C1 BO C1 1 3 1 A Separate BED1 BO D1 2 4 1 B Separate BI C2 BO C2 2 5 1 A Separate 2 BO D2 3 6 1 B Separate BI C3 BO C3 3 7 1 A Separate 2 BO D2 4 8 1 B Separate BI C4 4 4 Sequence Table AB run results show that vial incrementing is followed through for each method even when there are other methods interspersed in between within the sequence table In cases where not all of the sequence table run lines are to be executed by the user vial incrementing follows the number of method cycles e
11. Method Filename Rep Inject Inject Inject Other Other Rinse Rinse Separate Separate In Out Time Inject In Inject Out VialsIn Vials Out Vials In Vials Out 1 overnitePD A met MD Gpda3 4in 1 of BI C1 BO B1 BEAT BO A1 01 BO D1 stwHPLC 8hz 100 BI B1 BO 42 2 4qhz 050 603 001 Rep 1 dat 2 overnitePD MD Gpda3 4in f BI D1 stwHPLC 8hz 2 4ghz 050 603 001 Rep 2 dat 3 overnitePD A met MD Qpda3 4in BI D1 atwHPLC 8hz 2 4ghz 050 503 001 Rep 3 dat To print any Sequence Vial Report Confirmed right click on the report and then select Print Sequence Vial Report Confirmed Zoom Print Print Preview v Header Footer User s Guide PN A33115AB F 11 Appendix F Sequence Vial Report Confirmed Alternatively one can select Sequence Custom Report under Print from the Reports menu Reports Window Help View Advanced Report Properties 1st Dimension Report 2nd Dimension Report Area Current Baseline Check Current System Check External Standard Internal Standard Mormalization The Print Sequence Report dialog window pops up and it looks exactly the same as the View Sequence Report dialog window The user can select the desired Sequence Vial Report Confirmed and click Print to print it Sequence Records Report Template Sequence Summary 2 C 32Karat Projects D efault T emplate S equence Vial Sumr Sequence Summary 3 3 C 32Karat Pr
12. When PDA data is selected the drop down box in the tool bar will contain a list of the available data channels Before data can be integrated or analyzed the desired channel must be selected from this list Each data channel will have a unique Peaks Groups table and Integration Events table that will be available from the Method menu only when the data channel has been selected More information about using PDA data is available in the resources listed in Appendix A 32 Karat 8 0 PN A33115AB Appendix D D System Administration Appendix D D 1 System Administration Overview User s Guide PN A33115AB The System Administration functions include adding and configuring instruments which has already been discussed in this manual It also includes features for defining users and projects Projects definitions include defining default folders in which data and methods will be stored specifying what instrument s can be used for a project defining which users can work on a project and what privilege level each user will have These administrative functions are handled through software wizards which are pre defined step by step processes It is NOT necessary to implement this function in order to use the system System Administration is managed from the main 32 Karat Software Screen From the Tools menu select Options Click on the Enterprise tab Clicking on Enable user login and project management will activate the
13. 3 Highlight the new title and select Bold and Italic from the toolbar The information below the title that is highlighted in gray represent fields Fields allow you to insert blocks of information without tedious code writing Fields and text may be used together in this template each field is preceded by a text descriptor These fields may be easily repositioned 4 Click after the Acquired field and press the Delete key This will move the Print Time field to the same line as the Acquired field Press the Tab key twice to place space between the two fields Click in the blank line below these two fields and type User ID then press the Tab key 7 Right click and select Insert Field then User Name A new field will appear with the current User Name the default name System will appear if no user name has been defined 8 Usethe keyboard just as you would a word processor to align the field and the text in the desired manner 32 Karat 8 0 PN A33115AB Creating Reports Creating a Method Custom Report Below the header information is a graph displaying the electropherogram By default the graph is scaled to show the full range of data In the case of CE Data Sample 3 the area of interest is only at the right end of the electropherogram As the next step we will adjust the electropherogram display so that the peak region is more easily seen 9 Double click on the graph to display the Graph Properties box Data
14. Method Save As from the menu bar 2 Give the method a name with a title and date for future reference For example TestMethod 061106 Click Save The default path at installation is C 32Karat Projects Default Methods Your system administrator may have assigned you to a different default path The method is now ready to run In the next section you will run the method you just created For now practice writing and editing methods to explore the various parameters and how they interact The resources in Appendix A and the 32 Karat Online Help describe the events and parameters not covered here 32 Karat 8 0 58 PN A33115AB Running the System Single Run and Sequence Runs Running the System 6 1 Single Run and Sequence Runs In the last section you created an instrument method In this exercise you will run that method in Single Run mode In this mode the method must be manually started by the user prior to every run Single mode is useful in method development where the results of one run will suggests modifications to the method or other procedures In the second part of this section you will create a Sequence to do multiple runs You must be online to run samples and acquire data If you are not online offline will appear in the title bar of the instrument window Close any offline windows before proceeding From the main 32 Karat Software screen double click on an instrument icon to go online
15. ae ad 43 Peak detect parameters 43 Trigger settings o m Era me R ETSI S 43 Inlet Outlet trays 43 Detector Initial Conditions 44 UV Detector Initial 44 Electropherogram channel 44 Filtet uns omia osa Pelosi sco aiios BOAT ers UO Mate eh dalles candidi c 44 Relay and Delay Aute Nome 45 Absorbance Signal 45 PDA Detector Initial Conditions 46 Electropherogram Scan Data 46 EREE RA E a ed 46 Relay 1 and Relay 46 Reference Channel 46 Absorbance Signal 46 Electropherogram Channel Data 47 LIF Detector Initial Conditions 48 Electropherogram Channel 1 2 48 e Lc 49 Data Rate tore Hot Set ut cuu C xd S
16. appropriate boxes and inputting the desired number of cycles between changes For more information about vial incrementing refer to Appendix E Tray Selection Figure 5 8 Tray Selection dialog Pressure Direction Specifies whether pressure or vacuum will be applied to the inlet or outlet end of the capillary Pressure but not vacuum can also be applied to both ends of the capillary at the same time At Time Specifies that this will be a timed event at the time specified Separation is usually a timed event External Adapter Changes the way the instrument manages the power supply Select this box only if the External Adapter accessory is in use See the Installation and Maintenance Manual for more information 32 Karat 8 0 PN A33115AB Creating and Editing a Method 5 Time Program Rinse Dialog The rinse event is used to clean the capillary and to load fresh buffer or other separation media Pressure Type r Values Pressure Pressure 20 0 psi i C Vacuum Duration 1 00 min Cas ER Hel r Tray Positions Pressure Direction e Inlet Forward Outlet B0 a1 Gece Increment Inlet Outlet Increment Every fi Cycles Trays p 00 min At Time Figure 5 9 Rinse dialog This dialog has the following parameters Pressure Type Selects the mechanism to be used to move fluid through the capillary Tray Positions Fun
17. instrument to execute multiple cycles of one or more methods throughout a set of runs The execution of multiple cycles for the instrument system is called repetitions Although the terms are similar do not confuse repetitions with cycles The cycles term is used in SETTING UP vial incrementing through themethod The repetitions term is used to describe the EXECUTING of those cycles For instance a repetition of 5 for a single method will mean the instrument will execute 5 cycles of the same method A set of repetitions makes up a run A set of runs makes up a sequence Repetitions Sequence 32 Karat 8 0 PN A33115AB Appendix E E Examples of Use When Single Run running a single method is selected the repetitions term is used Runs and repetitions terms are used in the sequence table and Sequence Wizard In summary the method drives the vial incrementing And the sequence table plans and organizes the vial incrementing activities for the user E 2 Examples of Use The following examples illustrate how vial incrementing works Example 1 Method Time Program B 5 Initial Conditions 45 PDA Detector Initial Conditions C Time Program Summary 20 0 psi 10 15 min forward In Out vial inc 1 n 7 Out vial inc 1 In Method Time Program B the following lines explain the incrementing scheme Rinse 1 Increment inlet and outlet vials after every cycle of method Rinse 2 Inc
18. this menu is used to insert codes that will be automatically inserted into the filename when the sequence is run In this example Date and Time has been selected NOTE The Open File choice is used to reprocess existing data files and not for data acquisition Number of Unknown Runs in Sequence will determine how many lines there are in the Sequence Table For this exercise input 3 Repetitions per Run will determine how many times each line in the Sequence Table will be run An identifier will automatically be added to the file name for each repetition when this option is used For this example input 1 2 Click Next Sequence Wizard m First unknown vials of sequence Inlet Bci Trays Ady Outlet Bom Trays Advance m First calibration vials of sequence 3 Inlet pas Trays Iv Advance Dutlet po cs Trays Advance Injection duration 4 0 sec Advance direction Row major C Column major BI Cancel Finish Figure 6 7 Sequence Wizard Vials dialog User s Guide PN A33115AB 65 Running the System Programming a Sequence If the method being used allows vial position override see Appendix E the desired starting position can be input here If override is not allowed or if auto incrementing has been set any input here is ignored A set of calibration vials can also be identified in this window See the resou
19. 8 0 PN A33115AB 32 Karat 3 Creating and Configuring an Instrument This selection will be reflected on electropherograms and in reports 1 Click to exit and save the module configuration 2 Click OK to return to the Opening Menu Manual Configuration User s Guide PN A33115AB Manual Configuration of the UV Detector Manual Configuration may be used to create an instrument when the hardware is offline or in use NOTE Many of the exercises in this manual require that a virtual UV instrument be configured Use this procedure to create a UV instrument regardless of the type of detector you have installed 1 From the main 32 Karat Software screen create a new instrument and open the configuration window as previously described in Creating a New Instrument on page 3 22 Name the instrument UV Detector 2 Forthis exercise a UV Detector will be configured Select the UV Detector in the left pane and click on the green arrow The P ACE MDQ UV Detector icon is added must now be configured Beckman CE P ACE MDQ Configuration Available modules Configured modules 2 B UY Detector PDA Detector UY Detector LIF Detector Stand Alone No Detector 5 External Event Detector Configuration Options Auto Configuration Cancel Help Figure 3 11 UV Detector as configured Module 3 Right click on the new icon and select Open The P ACE MDQ Instrument Configurati
20. A33115AB Creating and Editing a Method Introduction Creating and Editing a Method 5 1 Introduction In the section on Direct Control the exercise on regenerating a capillary required user input at each step of the process A method combines a series of steps into a logical process The steps in a method are executed automatically by the instrument This section will describe the basic steps in creating an instrument method for data analysis There are many more features such as data analysis and reporting that can be incorporated into a method Some of these are covered later in this manual For more information see Appendix A Creating and Editing a Method To create or edit a method you must be in the Instrument Window 3 To open this window go to the 32 Karat main screen Right click on the icon you created to represent your instrument system and select Open Offline The instrument window will open after a few seconds When the window opens the Instrument Wizard will appear It is possible to open the method editing dialogs by selecting Create or Modify a Method from this dialog For now just click OK to close the Instrument Wizard We will write a new method to run an instrument test sample 1 User s Guide PN A33115AB To begin select File Method New from the menu bar The name of the method in the Instrument Window title bar will change to untitled met To access the instrument control and data
21. Do not close the Sample Run Type s dialog window yet 17 18 19 Clear All Calibration Clear Calibration at Level Print Calibration Report amp verage Replicates Clear Replicates Mi Select Sequence Report Template 1 Template End Calibration SysSuit brp Summary brp Summary tpl Vial Summary tpl Sequence Vial Summary CE Sequence Report Template brp tpl Select End Summary as the run type in the Sample Run Type s dialog window There is no need to open any report template for End Summary There should now be two run types selected Begin Summary and End Summary Highlight Begin Summary to assure the correct report template has been opened Click OK to close the Sample Run Type s dialog window and return to sequence table Shutdown Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability Begin Summary Summary Run MEnd Summary Vial Summary OAC Check Standard Sequence Vial Summary Appendix F Sequence Vial Report Confirmed F 10 Sequence table should reveal SMB SME notations that refer to Summary Begin and Summary End as the run type for the single sequence table run line that consists of one or more repetitions to be executed The user can repeat steps 1 through 3 for the other sequence table run lines if Sequence Vial Report Confirmed is des
22. ER DIE D RR RF E EA EE ERES 9 Appendix F peat eae 1 vi 32 Karat 8 0 PN A33115AB Preface Overview Preface Overview The 32 Karat Users Guide is a self paced introduction to the operation of the Beckman Coulter Inc PPACE MDQ and the 32 Karat Software It contains a brief introduction to the technique of capillary electrophoresis an overview if the MDQ hardware tutorials for the most frequently used software features and an appendix of useful information The 32 Karat Users Guide has been designed to assist users who are new to CE or the 32 Karat Software in beginning to use both the software and the instrument It is largely built on exercises that are designed to take you step by step to a point where you can begin to create your own methods generate and analyze data and report results It does not attempt to cover every possible feature in the system or every aspect of capillary electrophoresis The on instrument activities contain specific instructions for users with ultraviolet photodiode array and laser induced fluorescent detector systems Follow the instructions for the detector s installed on your system The activities in this manual are progressive Each builds on the previous ones To get the most from this manual work through all the activities in order The activities that teach data reprocessing calibration and reporting are all
23. Inc lasers the test mixture used in this exercise may not perform as described Acquisition enabled Checked to enable the collection of data in this channel Dynamic range Specifies the upper limit of the signal range Higher values allow for the collection or larger peaks without truncation but at the cost of sensitivity Lower values give greater sensitivity but large peaks may be truncated This value should be slightly larger than the largest peak expected 32 Karat 8 0 48 PN A33115AB Creating and Editing a Method Time Program Signal Select Direct when separating fluorescence samples peaks in a non fluorescent electrolyte background electrolyte If a fluorescent background electrolyte is used non fluorescent samples may be detected Selecting Indirect in this case will invert the signal so that negative peaks appear as positive peaks Data Rate Specifies the rate at which data will be acquired Both LIF data channels will have the same data rate They cannot be specified separately Relay 1 and 2 See the UV Detector Initial Conditions section for information on this topic For this exercise setup the LIF Detector Initial Conditions to match Figure 5 4 5 6 Time Program User s Guide PN A33115AB The Time Program window is arranged like a spreadsheet An event is entered into each line Events will be executed in order top to bottom Each line in the window contains these columns imi Instrument S
24. OK Pressure 25 psi Forward ok Duration E min C Reverse Cancel Tray positions Pressure type Help Inlet Pressure Trays Outlet 1 Vacuum Figure 4 2 Pressure Settings Dialog This dialog is used to set up the conditions for a rinse or a pressure separation It has the following options Pressure Input the desired pressure in psi The valid range for positive pressure is 0 1 to 100 psi for vacuum is 0 1 to 5 psi Duration Specifies how long the pressure will be applied Direction Designates whether the flow of fluid will be Forward from inlet to outlet or left to right or Reverse from outlet to inlet or right to left Pressure Type Specifies whether positive pressure or vacuum will be used This choice defines the valid pressure range Tray Positions Specifies which vials or wells will be located at the capillary ends during the Operations l Toselect the positions click the Trays button to open the Tray Selection dialog shown in Figure 4 3 2 Clickon the appropriate positions to select them For this exercise click B1 on the left tray and Bl on the right tray to select the Methanol and Empty vials 3 Click OK to return to the Pressure Settings dialog box The tray positions area should now indicate Inlet BI B1 and Outlet BO B1 4 25 in the Pressure box and 1 0 in the Duration box Pressure direction should be Forward and Pressure Type should be Pressu
25. UV detector systems are shipped with four standard filters 200 nm 214 nm 254 nm and 280 nm 10 nm bandwidth These are installed in positions 2 3 4 and 5 respectively of the filter wheel position 1 is opaque and serves as a shutter for the detection system Additional wavelengths are obtained by placing the appropriate filters in positions 6 7 and 8 the standard filters can also be replaced if desired If the instrument will be used with a PDA detector position 8 should always be left open no filter The filter wheel will accommodate widely available 4 inch 12 7 mm diameter filters with wavelengths from 190 to 600 nm User s Guide PN A33115AB 11 System Overview UV Detector Optics Figure 2 7 UV Optics Layout 901706L AI Filter Position i e 214 nm 10 Fiber Optic Cable Fiber Optic Connector 1 Capillary Aperture 2 Fused Silica Lenses 3 Deuterium Lamp 4 Lamp Power Supply 5 Photodiode 6 Fiber Optic Connection 7 Motor 8 8 Position Filter Wheel 9 12 Capillary 12 32 Karat 8 0 A33115AB 2 1 User s Guide PN A33115AB System Overview The Photo Diode Array PDA Detector The Photo Diode Array PDA Detector The Photo Diode Array detector like the UV detector uses the absorbance of light to detect the presence of samples as they pass through the detection window Unlike the UV detector
26. Vial incrementing is an automation process in which the inlet or outlet vials may be programmed to advance after a pre determined number of cycles This behavior of shifting to adjacent vial positions occurs after the instrument executes a certain number of cycles of a method Vial incrementing eliminates the need to devise new methods whenever different vial positions are desired during the course of a set of instrument runs Therefore one method can be used to allow the instrument system to change to and utilize new vial positions automatically in those runs Users may incorporate vial incrementing in their applications as a powerful tool For example vial incrementing may be used to perform efficient separation of biological or chemical compounds without constantly replacing or replenishing separation media solutions during instrument system runs Vial incrementing is controlled by the software s method and sequence Incrementing for either or both of the inlet outlet vials may be selected under the Tray Positions dialog for each of the following events in the method time program Rinse Inject Separate Wait r Tray Positions Inlet 1 Dutlet 0 41 Increment Inlet Outlet Increment E very 1 Cycles Trays Cycles are the number of repeats of a method before vial incrementing occurs So selecting Increment Every 2 Cycles will cause the instrument system to run the method two times before changing vial positions
27. a potential hazard to your personal safety exists from a high voltage source A A AS BIOHAZARD Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a biological source LASER LIGHT Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from a laser source User s Guide PN A33115AB xi Safety Information Safety Symbols SHARP OBJECTS Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from unblunted corners or other appendages on the outside or inside of the equipment HOT SURFACE Paragraphs marked by this symbol indicate that a potential hazard to your personal safety exists from heated surfaces or other appendages on the outside or inside of the equipment PROTECTIVE EARTH OR GROUND TERMINAL This symbol identifies the location of the protective earth or ground terminal lug on the equipment OFF POSITION OF PRINCIPAL POWER SWITCH This symbol graphically represents the equipment main power push button switch when it is in the off position ON POSITION OF PRINCIPAL POWER SWITCH This symbol graphically represents the equipment main power push button switch when it is in the on position Chemical and Biological Safety WARNING Normal operation of the system can involve the use of solvents and reagents that are toxic flammable or biologicall
28. acquisition sections of the method select Method Instrument Setup from the menu bar A window will open that contains three or four tabs depending on your detector type Select the tab marked Initial Conditions to bring it to the front The dialog in Figure 5 2 will appear This dialog is used to set instrument parameters at the start of a method before the separation process begins 41 Creating and Editing a Method Initial Conditions Tab 9 2 42 Initial Conditions Tab In the previous section Direct Control the exercise on regenerating a capillary required user input at each step of the process This section shows how to combine a series of these steps into a logical process called a Method The steps in a method are executed automatically by the instrument This section will describe the basic steps in creating an instrument method for data acquisition There are many more features such as data analysis and reporting that can be incorporated into a method Some of these are covered later in this manual For more information see Appendix C In this exercise you will write a method to run an instrument test mixture To write a new method select File Method New from the menu bar 2 the instrument control and data acquisition parts of the method select Method Instrument Setup from the menu bar A window will open that contains three or four tabs depending on the detector configuration 3 S
29. activities and instrument status in real time NOTE Additional information regarding Direct Control features can be found in the 32 Karat Online Help MDQ User s Guide PN A33115AB 31 Direct Control Direct Control Window 4 2 Direct Control Window To access the Direct Control window go to the Control Menu and select Direct Control View nm Absorb 0 0 00000 Voltage Current Power Pressure 901699L Al Figure 4 1 Hot Spots on the Direct Control window 1 Capillary Temperature 2 Tray Up Down 3 Label dialog 4 Tray Up Down 5 Lamp On Off 6 Detector dialog 7 Autozero 8 Trays to Home Position 9 Trays to Load Position 10 Injection Dialog 11 Stop Current Processes 12 Sample Storage Temperature 13 Voltage Dialog 14 Current Dialog 15 Power Dialog 16 Pressure Dialog 17 Vial Position Dialog 32 Karat 8 0 32 PN A33115AB Direct Control Direct Control Window The UV Detector is shown above The PDA and LIF Detectors are similar The functions are defined in Table 4 1 Table 4 1 Direct Control Functions Control Functions in Direct Control Capillary Temperature Hot Spot Opens a dialog to set the coolant Temperature temperature Storage Temperature Hot Spot Opens a dialog to set the sample storage temperature Tray Motion Up Down Hot Spot Toggles trays up and down Vial Positon Hot Spot Open
30. based on data collected with an ultraviolet detector system The steps in these procedures are very similar regardless of the detector type used so these exercises are important for users of all detector types Data files for the post run exercises are installed with the 32 Karat Software If your instrument does not have a UV detector you can manually configure a virtual UV instrument in the software so that you can do these exercises Additional information can be found in the 32 Karat Online Help This can be accessed at any time while using the 32 Karat Software through the Help menu located on the menu bar by pressing the F1 key while working within the software or any time you see a Help button displayed within an application window This manual was created by a team of technical writers scientists and engineers at Beckman Coulter Inc We value your feedback Please let us know how we can improve future editions of this manual Send your comments to 32karat beckmancoulter com and include Users Manual in the subject line Questions concerning the exercises can be sent to the same address User s Guide PN A33115AB VII Preface Instructions for using the Data Samples Instructions for using the Data Samples In some of the exercises in this manual you are asked to open specific data and method files These are located in the Data Samples folder which is a sub directory to the 32Karat folder that was created when
31. can be rotated around the X and Y axes by selecting the command from the right click menu and manipulating the 3D plot with your mouse or by typing in Elevation and Rotation parameters within the 3D Data Graph Properties window Zooming is accomplished by setting limits of time wavelength and absorbance The Apply button causes the changes to be made to the 3D plot without closing the properties window This allows you to test changes without having to close and re open the dialog 3D and Contour Plot shows only these two plots in a two pane window The individual 3D Contour Electropherogram and Spectrum plots display the individual pane as a full window The limits of the Electropherogram and Spectrum plots are those set on the Contour window even if the Contour window is not being displayed The remaining choices in the PDA View menu are not related to these four items The Max Plot view is an electropherogram that does not represent a particular wavelength It is built by taking the maximum absorbance across the wavelength scan range at each point in time This plot is useful for finding peaks that might otherwise go undetected This data may be analyzed and the results reported C 3 C Appendix C 3D Plot C 4 The Multi Electropherogram view is only available if multi electropherograms are defined in the Instrument Setup component of the method Like the Max Plot these traces are synthesized from the 3D data set To set up these
32. channels open the PDA Options window from the Method menu and select the Multi Electropherogram tab Three items must be specified for each channel The channel can be checked On or Off Channels which are Off will not be displayed and cannot be analyzed Wavelength refers to the central wavelength of the data to be incorporated into the multi electropherogram channel Bandwidth specifies how many adjacent wavelength bands will be combined to form the new data set These bands are weighted with bands more distant from the central wavelength contributing less to the final result than do bands closer to the midpoint Using too narrow a bandwidth will result in a noisy data file Using too wide a bandwidth will cause a loss of spectral sensitivity in the data set These data may be analyzed and the results reported If many multi electropherogram channels have been defined each will occupy a small area of the display The frame around each channel can be moved by clicking and dragging with the mouse Channels can also be temporarily disabled by unchecking them in the Multi Electropherogram tab of the PDA Options window Ratio opens a three pane window Two windows will display the electropherograms at specified wavelength The third displays an electropherogram that is the arithmetic ratio of the two Abs 1 Abs 2 on a point by point basis The parameters for this window are set on the X Y tab of the PDA Options window available from the Method menu
33. column Change the level in line 1 from 0 to 1 13 Use the down arrow to move to the same column in line 2 and change the 0 to 2 The Level entered in this column of the sequence table corresponds to the Level column in the Peak Group table 14 Continue down the column changing the remaining zeros to 3 4 and 5 respectively As you change the level to a non zero value the sample type automatically changes to Calibration 15 Click in the Run Type column of line 1 The dialog shown in Figure 8 3 will open The items here are the available run types 16 Click on Clear All Calibration and click OK The run type for line one will change to CAL CCA Selecting this type will cause any previous calibrations of this data set to be cleared before the new calibration is performed User s Guide PN A33115AB 85 Calibration Creating a Calibration 86 Sample Type s Clear All Calibration Clear Calibration at Level Print Calibration Report JAverage Replicates Clear Replicates Begin Loop End Loop Shutdown Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability Begin Summary Summary Run End Summary Vial Summary OOC Check Standard Begin Calibration End Calibration Figure 8 3 Sample Run Types dialog 17 Save the sequence with the name Calibrate seq The right and left sides of the window sho
34. data but there are some differences Some CE Specific calculations are built into the 32 Karat Software This appendix describes those along with some other useful calculations Ohm s Law V IR The most important relationship to know in capillary electrophoresis is Ohm s law This simple relationship defines the interaction between Voltage V Current and Resistance R For example if Voltage is held constant change in Current must be due to a change in Resistance Information of this type is invaluable in developing and troubleshooting CE methods Corrected Migration Time tcorr t tref tref t migration time of peak of interact tef actual migration time of reference peak lig expected migration time of reference peak from peak id table Velocity Corrected Area 4 v velocity capillary length to detector t migration time A uncorrected peak area In CE early peaks migrate through the detector window faster than do later peaks This creates a peak area bias which is eliminated by using velocity corrected peak area MDQ User s Guide PN A33115AB B 1 Appendix B CE Calculations Mobility Mobility is calculated as the apparent mobility minus the contribution of electroosmotic flow ueof Once the mobility of a reference analyte has been determined the mobility of related analytes can be calculated 1 1 u Ya Vreftre uref AE V average appli
35. direction become available Maximum vacuum is 5 psi Vacuum can be applied to the inlet Forward or outlet Reverse end of the capillary but not both 32 Karat 8 0 PN A33115AB Direct Control Exercise Conditioning the Capillary External Adapter This box must be selected when the external adapter accessory is in use For additional information see the Installation and Maintenance Manual Polarity Determines the sign of the charge on the electrodes The graphic will indicate the selection For this exercise use the Trays button and select A1 on the inlet side and A1 on the outlet side Both ends of the capillary will be in Run Buffer A Set the other parameters as follows Voltage Duration 2 minutes e Ramp time 0 5 minutes 9 Click OK The trays will move to position the selected vials at the ends of the capillary The voltage will begin to ramp up to 30 kV Observe the Direct Control screen during this process taking particular note of the Voltage Current and Power displays The Current should reach between 27 and 33 mA when the Voltage is at 30 kV At the end of two minutes the voltage will return to zero and the Status will return to idle This completes the Direct Control exercise It may be helpful to experiment with the other Direct Control functions before proceeding to the next section MDQ User s Guide PN A33115AB 39 Direct Control Exercise Conditioning the Capillary 32 Karat 8 0 40 PN
36. installed Cover Status Graphic Indicated if the door is open or closed Cartridge Temp Text Graphic Hot Spot Current coolant temperature is displayed Storage Temp Text Current storage temperature is displayed Storage Status Graphic Indicates presence of trays in the storage unit Lamp Laser Status Graphic Hot Spot Indicates Lamp Laser s on or off Voltage Text Graphic Button Indicates present target and maximum voltage Current Text Graphic Button Indicates present target and maximum current Power Text Graphic Button Indicates present target and maximum power Pressure Text Graphic Button Indicated present target and maximum pressure Pressure Type Direction Graphic Indicates positive pressure or vacuum and capillary end to which the pressure is applied Polarity Graphic indication of electrode potential 34 32 Karat 8 0 PN A33115AB Direct Control Exercise Conditioning the Capillary 4 3 Exercise Conditioning the Capillary This exercise will use the Direct Control functions to condition a bare fused silica BFS capillary This capillary will be used for additional exercises in this manual NOTE This procedure is intended for bare fused silica capillaries only Internal capillary coatings may be destroyed by this procedure Materials needed 75 micrometer I D capillary 60 cm total length 50 cm to the detector installed in cartridge appropriate for detector type configured See the Installati
37. instructions Creating a New Instrument Up to four P ACE can be controlled from a single computer with 32 Karat Software An unlimited number of virtual instruments can be created for offline method editing or data analysis Use the following exercise to configure a new instrument Select the detector type s appropriate to your hardware configuration l Right click within the right pane and select New Instrument from the drop down menu 2 Anew instrument will be created with its icon placed in the right pane Enter the name of the instrument in this example we will refer to Instrument 1 in the highlighted 32 Karat 8 0 PN A33115AB 32 Karat Creating and Configuring an Instrument name field This name will be used throughout 32 Karat Software in the Instrument window data reports and the Instrument logs A 32 Karat Software i OF x File Edit View Tools Help je Beale ie ws SB Current location group is The Enterprise Figure 3 2 32 Karat Software Enterprise Screen Main Menu Configuring the New Instrument User s Guide PN A33115AB Configuring the instrument is a process in which the software is prepared to control the hardware and process the data It consists of first identifying the P ACE MDQ as a unique instrument and second identifying the hardware components that are present Each physical instrument should be represented by at least two icons One
38. min Tray positions Inlet B1 Help Extemal adapter Pressure With pressure Direction Forward Outlet 80 41 With vacuum pue Polarity prs Normal Pressure psi C 1 C Reverse Figure 4 4 Voltage Settings Dialog Voltage Setting Parameters 38 Voltage The voltage at which the separation will be run 0 1 to 30 kV Duration The total time the voltage will be applied 0 1 to 999 9 minutes Ramp Time The amount of time the power supply will take to reach the voltage 0 10 to 999 9 minutes Ramp time cannot exceed the Duration time Voltage Max The maximum voltage the system will be allowed to reach This must be greater than or equal to the set voltage Current Max Maximum current the system is allowed to reach If this value is reached at a voltage lower than the set voltage the set voltage will not be achieved Maximum allowed current is 300 pA Tray Positions This is the same as previously described under Pressure With Pressure Allows the simultaneous application of pressure and voltage When selected additional parameters of pressure level and direction become available Maximum pressure is 100 psi Pressure can be applied to the inlet Forward outlet Reverse or both ends of the capillary With vacuum Allows the simultaneous application of vacuum and voltage When selected additional parameters of vacuum level and
39. molecules such as drugs 32 Karat 8 0 2 PN A33115AB System Overview 2 Main Components System Overview 2 1 Main Components The main components of the P ACE MDQ instrument include trays that hold vials of sample buffer and other solutions an interface block a high voltage power supply and electrodes a source optics module and detector temperature control hardware and a sample injection mechanism PIACE MDQ o I c BECKMAN COULTER te E 901697L AI Figure 2 1 P ACE MDQ System MDQ User s Guide PN A33115AB 3 System Overview Main Components W SECK q COULTER GO OO J Figure 2 2 Instrument 901704L AI 1 Coolant Fill Ports 2 Inlet Buffer Tray 3 Inlet Sample Tray 4 High Voltage Power Supply inside 5 High Voltage Electrode 6 Interface Block 7 Insertion Bar 8 Source Opt
40. name in line 1 to Alpha and the name in line 2 to Beta 3 Use the scroll bar at the bottom of the window to review the column headings in this table We will only use some of these columns in this exercise See the resources in Appendix A for more information 83 Calibration Creating a Calibration 84 The items that can be changed here include The defined migration time and allowed window for a component The data channel to be used for multichannel detector types The type of curve fit to be used for the calibration data set e Up to 10 calibration levels We must input the concentration values from the table above 4 Scroll right until the columns headed Level 1 through Level 5 are visible 5 Input the known values for Alpha and Beta Level 1 is the lowest value Level 5 is the highest For now we will accept the defaults for the other columns When all the values have been entered the table should look like Figure 8 2 Peak Group Tables UV 214nm i OF x Named Peaks Groups Figure 8 2 Peak Group Tables 6 Save your method 7 Right click on the electropherogram and select Annotations 8 Select parameters so that only Name and ESTD concentration are in the Show box 9 Click OK 10 Analyze the sample again The name of the peak will appear on the electropherogram ESTD concentration will be 0 000 because we have not yet completed the calibration process A
41. of the data set we are willing to extrapolate to find the quality of an unknown For the Y axis we can use any name we choose Because our quality is the number of bases comprising the oligonucleotide enter Bases A Linear Y axis scale will be appropriate for our data 32 Karat 8 0 PN A33115AB Qualitative Analysis Data Analysis and Reporting There is a two column spreadsheet in this dialog box Figure 9 2 The Left column contains data for the Y axis The heading of this column will contain the text entered for the Y axis label in this case Bases The Right column contains data for the X axis The heading is that selected for the X axis In this case we are using Migration Time l You must manually enter the X and Y values into the spreadsheet One way to obtain this data in tabular form is to open an Area 96 report by selecting Reports View Area from the menu bar This may be printed if you have a printer connected to the system or the report can be displayed as a window alongside the Qualitative Analysis window 2 For each peak enter the base number from 40 60 and the corresponding migration time As you enter the data it will be graphically displayed in the lower right corner of the dialog box 3 When all the data has been entered select Fit Type to determine the best curve fit to the data set In this example the highest Goodness of Fit values is obtained using a Cubic fit When done do a Save As rena
42. of these will be used for control and data acquisition the other is used for offline data reprocessing while the first is being used for acquisition The following example will use a UV detector as a model NOTE If there is more than P ACE MDQ connected to the computer the configuration process must be repeated for each instrument Only the instrument that is being configured may be ON during configuration All other instruments must be turned OFF Once all instruments are configured they may all be turned back on 3 Right click on the Instrument 1 icon and select Configure from the drop down menu 23 32 Karat Creating and Configuring an Instrument 4 Select P ACE MDQ from the Instrument type drop down menu input a name to identify the instrument Instrument Configuration P ACE MDO 2 HPLC Sustem P ACE MDO Figure 3 3 Instrument Configuration dialog 5 Click Configure The Configuration Module Configuration window is launched with the modules available for configuration listed in the left pane P ACE MDQ Configuration UY Detector PDA Detector E LIF Detector Stand Alone No Detector 1 e External Event Detector Configuration Figure 3 4 MDQ Configuration dialog 32 Karat 8 0 24 PN A33115AB 32 Karat Creating and Configuring an Instrument Auto Configuration 32 Karat Software can automatically configure all M
43. page 2 20 for more information A fiber cable transmits excitation light from the laser to the capillary in the cartridge Substances in the capillary which fluoresce at the laser wavelength are detected The LIF Detector measures and records this fluorescence which appears as a peak on the computer screen or printed electropherogram The LIF Detector can also be used in the indirect mode In this mode a fluorescent buffer system is used to detect non fluorescent components See Figure 2 9 and Figure 2 10 The initial installation of the LIF Detector will be performed by a Beckman Coulter Field Service Engineer The P ACE MDQ can easily be converted between LIF and UV PDA modes as detector components are modular User s Guide PN A33115AB 15 System Overview Laser Induced Fluorescence Detector LASER ON CURRENT LIMIT rm al Ht Figure 2 9 Instrument with LIF 1 LIF Plug 2 LIF Detector Module 3 Cartridge Cover 4 LIF Interconnect Module 5 488 nm Laser Module 6 635 nm Laser Module 16 32 Karat 8 0 PN A33115AB System Overview 2 Laser I
44. points per second Narrow peaks require a higher data rate Data rate interacts with the Peak width parameter in Filter settings below Scan data collected at high data rates can result in very large data files Scan range species the wavelength range over which data will be acquired limit 190 600 nm Filter See the UV Detector Initial Conditions section for information on this topic Relay 1 and Relay 2 See the UV Detector Initial Conditions section for information on this topic Reference Channel See the sources in Appendix A for information on this feature Absorbance Signal See the UV Detector Initial Conditions for information on this topic 32 Karat 8 0 A33115AB Creating and Editing a Method PDA Detector Initial Conditions Electropherogram Channel Data Up to three independent data channels can be collected simultaneously with the scan data For each channel the following must be input Acquisition enabled Check this box to enable the collection of data in this channel Reference channel See the sources in Appendix A for information on this feature Wavelength This parameter specifies the central wavelength for the data channel Bandwidth This parameter specifies the width in nanometers of the data collection channel Larger values will give higher signal to noise ratios than will low values Low values will be more specific for molecular structure than will higher values Peak detect See the source
45. software The principles of integration are the same regardless of detector type Configure a UV instrument for offline analysis From the Instrument window select File Method New Close the instrument setup window when it appears as it will not be needed for this exercise NOTE Review the instructions in the Preface on page viii before using the sample files Work from a copy of the data files and not the originals 4 From the Instrument window select File Data Open Right click on the electropherogram and select Annotations The following dialog will appear Trace Annotation Properties x Annotation Trace fi Current Data Channel A UV Show the following annotations Available Annotations Name Pk Area Percent Height Area rh Migration Time Height Percent ESTD concentration ISTD concentration NORM concentration Width Start Time Decimals 3 r Other MT Window M Show undetected peaks Cancel Apply Appl Toan Help Figure 7 2 Trace Annotations dialog Navigate to your copy of the Data Samples folder and select the file named Data Sample 1 dat The data will open in the Instrument window Click the electropherogram window to select it then type Ctrl Z to zoom the data to maximum size Only Channel A data will be used Auxiliary data current voltage power pressure can not be integrated 32 Kar
46. steps below 1 Open Windows Explorer To open this utility right click on the Start button in the Windows toolbar and select Explore Navigate to the Data Samples folder It is a sub directory of the 32Karat folder In the left hand pane of the Explorer window click on the Data Samples folder to open it From the menu bar select File New Folder A new folder will be added to the Data Samples folder The New Folder will be highlighted which indicates that the name may be edited Type a new name The name of the individual who will be using the tutorial exercises in the Users Guide is a good choice 5 Select all of the data files listed above but do not select the new folder There are several ways to do a multiple file select Use any one of the following Select the first file in the list by clicking on it Hold down the Shift key and click on the last file in the list the files between these two files will be selected Select a file by clicking on it Hold down the Ctrl key and click each individual file in the list Using the mouse you can click and drag a box around the files to be selected 6 When all the files are selected highlighted select Edit Copy from the menu bar Double click on the new folder to open it Then select Edit Paste from the menu bar You have now created a copy of the sample files in a new folder When the manual asks you to open a file open the copy you have just created and not th
47. such as sodium dodecyl sulfate SDS can be added to the electrolyte solution Neutral molecules with differing affinities for the charged micelles will separate during electrophoresis under these conditions Capillary Gel Electrophoresis CGE In the techniques described above the analyte molecules are moving in a low viscosity liquid By increasing the viscosity of the matrix to the point where the migration of molecules is physically impeded it is possible to separate different species based on size and shape as well as charge A variety of gel solutions may be employed in this technique Capillary Isoelectric Focusing In cIEE a mixture of special buffers called ampholytes is used to create a pH gradient along the length of the capillary During separation molecules will migrate to the point in the pH gradient at which they have no net charge At the end of the process the various analytes are located in discrete zones in the capillary In a separate mobilization step the bands are moved past the detector for analysis This technique is most often used for protein and peptide analysis Capillary Electrochromatography CEC CEC uses a capillary packed with porous particles similar to those used in HPLC Driven by EOF analytes partition between mobile and stationary phases as they migrate down the capillary Under some conditions electrophoresis may also participate in the separation CEC is primarily employed to separate small
48. system administration function The effects will be seen the next time the program is started WARNING If the System Administration function is activated a System Administrator must be defined immediately If no System Administrator has been defined there will be no user with the privilege to disable the System Administration function should that be necessary Depending on the privilege levels assigned to users not having a System Administrator may make some or all software features inaccessible When the Enable option is selected the User list becomes available Clicking on Add user opens a dialog in which the user s name and password are entered A password is not required If a password is used it must be recorded elsewhere as there is no feature in the software for generating a list of user passwords When all the names are entered at least the name of the administrator user privileges must be assigned New users do not have any privileges until privileges are assigned IMPORTANT A user with no privileges cannot use the software Click to close the Options dialog From the Tools menu select System Administration Wizard The wizard will only be available if System Administration Mode has been selected checked A screen will open with three options for Users Instruments and Projects If there are secondary administrators they may be given access only to some of these wizards The Users Wizard allows the assignment of
49. the PDA detector can provide spectral analysis of samples Spectral signatures obtained in this way can be useful in identifying unknowns The PDA detector utilizes the same cartridge configuration as the UV detector Please refer to the section on the UV detector for a description of the cartridge and to the 32 Karat Installation and Maintenance Manual for instructions on rebuilding the cartridge In PDA detection the full spectrum of light from the deuterium lamp illuminates the capillary see Figure 7 Light that is not absorbed by samples is delivered by a fiber optic cable to a grating that breaks the light into a spectrum This spectrum is projected onto an array of 256 photodiodes With this arrangement the absorbance profile of the sample can be measured The PDA detector also allows the simultaneous measurement of light at different discrete wavelengths By collecting spectral data over time 3 dimensional plots of absorbance can be created The photo diode array converts the light signal into an electrical signal This is digitized and sent to the P ACE MDQ computer for processing by the software The PDA detector always uses filter wheel position 8 It is essential that no filter be in position 48 when the PDA is in use The PDA detector is calibrated in wavelength using the discrete emission bands generated by a mercury lamp The mercury lamp is an integral part of the detector system Calibration is performed automatically by the so
50. values as those under the Sample Inject Inlet Outlet columns in the sequence table These values are used when the user chooses to inject from the sequence table The Inject Time column contains the same injection duration time values in seconds as those in the sequence table under Sample Inject Duration column These values are used when the user chooses to inject from the sequence table The Inject In Out and Inject Time columns will contain values from the Inject time program in the method by default if the user does not input values in the Sample Inject Inlet Outlet and Sample Inject Duration columns of sequence table This is the case for users who choose to inject from the sequence table However the sequence will not run since no values are entered in the Sample Inject Inlet Outlet and Sample Inject Duration columns of sequence table The Other Inject In Out columns contain the inlet outlet vial positions for the injection event when the user chooses to inject from the method these values do not appear in the sequence table The Rinse Vials In Out columns and the Separate Vials In Out columns contain the inlet outlet vial positions for the rinse and separation events programmed in the method these values do not appear in the sequence table the vial position values presented in the report except the Inject In Out columns correspond to each method s time program including incremented vials to be used in the sequence run if vial increm
51. your software was installed The following information is also contained in the Readme doc file located in the Data Samples folder viii IMPORTANT Make sure to read and understand the following before using the sample files This folder contains the following files CE Data Sample 1 dat CE Data Sample 2 dat CE Data Sample 3 dat CE Level 1 dat CE Level 2 dat CE Level 3 dat CE Level 4 dat CE Level 5 dat CE PDA Data Sample dat CE Calibrate met CE Quantitate met CE Quality met CE ssDNA 40 60 dat Purpose of these files The Data Samples folder contains data and method files to supplement the exercises in the Users Guide for the 32 Karat Software The files were created solely for the purposes of this tutorial They are intended as training tools only and are not to be used for system validation or any other purposes 32 Karat 8 0 PN A33115AB Preface Instructions for using the Data Samples Using the files These files are referred to by name in the 32 Karat User s Guide In performing some of these exercises you will be asked to open one or more of these files By using these files the images you see on the computer screen and the images in the training manual will be the same IMPORTANT Duplicate these files before use Processing these data files and methods in the 32 Karat Software will cause changes to the files To keep the original files unaltered and available for later use in these exercises please follow the
52. zero as expected The mobility of ALPHA is the value we defined it to be The mobility of BETA has now been calculated relative to the mobility of ALPHA We can now set up to collect data for a mobility plot if we wish 1 Open the Instrument Setup window and click on the Initial Conditions tab 2 Select Mobility see the figure above After saving the method and running the sample again we have two electropherograms One is the UV electropherogram which looks very similar to the run we used to measure the mobility of the marker The second appears VERY different as shown here Mobility PDA 214nm D x Mobility 4 66715e 005 Mobility Amplitude 0 000734 AU Mobility 214nm 0 0150 0 0125 0 0100 0 0075 0 0050 0 0025 0 0000 D 00030 0 00025 0 00020 0 00015 0 00010 0 00005 0 00000 Mobility The order of the peaks has been reversed because the latest eluting peaks have the most negative mobility The relative spacing between the peaks has also changed User s Guide PN A33115AB 99 Mohility Peak Identification by Mobility 10 4 Peak Identification by Mobility 100 In the exercise on mobility we used migration time and a migration time window to establish the identity of peaks in a data file In the same fashion we can use mobility and a mobility window to identify peaks based on the mobilities of standards The choice of migration time or mobility appl
53. 000 ALPHA marker 0 00034763 0 00000000 BETA 0 00032093 0 00000000 As expected the faster moving EOF marker has the higher apparent mobility The units of mobility are cm V 1secl We can calculate the Electrophoretic Mobility of ALPHA according to H Papp eof substituting yields 0 00034763 0 00060227 0 00025464 the mobility of ALPHA under these separation conditions The mobility value is a negative number indicating that ALPHA s electrophoretic migration is toward the anode and is opposite the direction of the electroosmotic flow NOTE In practice the above run should be repeated several times to verify that the system is giving consistent results before defining the mobility value for the mobility marker We now re open the Peaks Groups table and insert the value of the mobility marker as described in steps 9 and 10 The table now looks like this Peak Group Tables PDA 214nm Named Peaks Groups ID Mobility Marker Mobility Window EOF marker z 0 D 00000000 We save the method and analyze the data file again The results are now Peak Name Apparent Mobility Mohility EOF 0 00060227 0 00000000 ALPHA marker 0 00034763 0 00025464 BETA 0 00032093 0 00028134 32 Karat 8 0 98 PN A33115AB Mobility 1 0 Demonstration Using the Mobility Features The electrophoretic mobility of the EOF marker is close to
54. 1 1 Description User s Guide PN A33115AB An electropherogram is a graphical depiction of the passage of molecules through the detector window The area under the peak is directly related to the amount of the analyte present in the sample The process of determining this area is known as Integration In practice integration includes steps that determine the points at which peaks start and stop and the shape and position of the baseline If sample components are not fully separated from one another resolved it is also necessary to estimate where to divide the area of the two peaks A very simple data set is shown in the figure below Figure 7 1 Typical Data set In this example there are four components Two are well resolved from the others Two other components are only partially resolved The four components differ in peak area This may indicate that they are present at different levels that they produce different responses in the detector or both Quantitation will be covered in the next section This exercise will focus on peak detection and baseline construction 7 Integration Integrating Data 1 2 72 Integrating Data There are two required parameters for peak integration width and threshold More complex separations will require the use of more parameters The test mix separations used in the previous exercises yield very simple electropherograms In this exercise we will use examples that are installed with the
55. 4 LED Indicators System Overview 2 LED Indicators The front panel of the instrument contains LED indicators for Power UV and High Voltage Figure 2 5 LED Indicators BECKMA COULTER 901700L AI 1 High Voltage Indicators 3 Instrument Power Indicator 2 UV Lamp On Indicator MDQ User s Guide PN A33115AB System Overview Cartridge and Tray Cover Interlocks 2 5 Cartridge and Tray Cover Interlocks The double hinged doors of the P ACE MDQ have been designed with interlock sensors that will prevent unsafe access to the interior of the instrument The first lower door is called the tray cover the second upper door is called the cartridge cover Opening the tray cover Stops any tray movement immediately Prevents execution of any programmed events which require tray movement e Aborts a method when a step requiring tray movement is encountered Opening the cartridge cover Shuts off the high voltage if it is on Turns off the pump circulating the capillary coolant Moves the detector filter wheel to the closed position
56. A BECKMAN COULTER P ACE MDQ User s Guide 32 Karat 8 0 A33115AB November 2009 Beckman Coulte 0 S Kraemer Blvd Bre Mn 92821 e e opyright 2009 Beckman Co ulter Inc Copyright Licenses and Trademarks Copyright Beckman Coulter Inc 2009 rights reserved No part of this publication may be reproduced transcribed transmitted or translated into any language in any form by any means without the written permission of Beckman Coulter Inc Licenses and Trademarks Beckman Coulter is a registered trademark of Beckman Coulter Inc System Gold is a trademark of Beckman Coulter Inc 32 Karat M Software is a trademark of Beckman Coulter Inc Microsoft amp and Windows are trademarks of Microsoft Corporation Table of Contents Section 1 Introduction to CE night S Bae ee are ci ed het E G 1 General Description 1 Common Modes of 515 1 Capillary Zone Electrophoresis CZE 1 Micellar Electrokinetic Capillary Chromatography MEKO 2 Capillary Gel Electrophoresis CGE 2 Capillary Isoelectric Focusing 2 Capillary Electrochromatography CEC 2 Section 2 System Overvie
57. CCheckStd Spike e SysSuit They are located in the 32 Karat Software Sequence directory and have the BRP extension 32 Karat 8 0 PN A33115AB A 1 Software Validation Pack PN A33115AB Appendix A Additional Resources Appendix A Additional Resources 32 Karat Online Help The Online Help is your primary software reference for the 32 Karat Software It describes all of the features used in this manual In addition it describes all of the advanced features which are not covered here To access the Help select Help Contents from the instrument window menu bar Context sensitive help is available by selecting the Help button located in dialog boxes or by pressing the F1 key Installation and Maintenance Manual The first place to look for answers to questions on hardware and maintenance is the Installation and Maintenance Manual This document includes detailed descriptions of routine operations such as rebuilding a capillary cartridge and of non routine operations such as minor repairs A printed copy of this manual is shipped with the instrument An electronic copy is available on the Manuals CD ROM shipped with the 32 Karat Software Beckman Coulter Service If you have determined that a problem exists with the instrument that can not be fixed by following the procedures in the Installation and Maintenance Manual contact your local Beckman Coulter Inc Service Representative There may be a charge for this service
58. DQ modules in the Instrument The instrument must be connected to the PC and turned ON 6 Click the Auto Configuration button at the bottom of the window and listen for tray movement within the MDQ This indicates that the software is communicating with the instrument The window below will appear during this process Auto Configuration Status B 9090 909 909 Figure 3 5 Auto Configuration Status 7 The installed detector will appear in the configured modules window P ACE Configuration Available modules Configured modules 2 B UY Detector PDA Detector E mou LIF Detector Stand Alone No Detector O 5 External Event Detector Configuration Options Auto Configuration Cancel Help Figure 3 6 MDQ Configuration dialog with installed UV Detector 8 Right click on the new icon and select Open Verify that the instrument was properly detected and configured 25 32 Karat Creating and Configuring an Instrument 26 P ACE MDQ Instrument Configuration x r GPIB Communication Sa Board GPIBO Device ID H Set Bus Address ancel p Inlet trays DF Galibration Wizard Help Buffer 36 vials halter i e rime Sample No tra y 2 200 n ition Bl 1 Trays Home position 214 nm 7 320 am r Outlet trays 4 254 IT 340 fim Buffer 36 vials Sample No tray rm Units Home position 80 1 Trays Pressure units psi
59. Graph Properties x Trace 1 Data source Cuentas Trace hand O Scale to OOO Y min Y max Units Absorbance X offset A scale Y offset p Y scale Annotations Hide Details Reset Scaling Cancel Apply Help Figure 11 2 Data Graph Properties The Trace Setup tab is where we select the data to be displayed We can select the Current Data the file currently open in the Instrument window or a data file or files stored on the computer For example we may want to display the results from analysis of a standard along with results from an unknown We can display multiple channels from a common data file by selecting the same Data Source but selecting different channels in the Trace column For example the UV and electrical Current traces could be displayed simultaneously The X and Y offset and scale features can be used to position the traces so that the pertinent details are visible These features are described in the resources listed in Appendix A Units is a label which can be adjusted to match the data displayed For an LIF detector this could be changed to Fluorescence by selecting in the Units option and entering the desired text MDQ User s Guide PN A33115AB 103 Creating Reports Creating a Method Custom Report 104 10 Click on the Annotations button 11 Because this will be a Qualitative report remove any existing annotations from the ri
60. If the instrument software window is not already open open it as described in Starting the Software on page 3 21 32 Karat 8 0 60 PN A33115AB Running the System 6 Single Run 6 2 Single Run Select Control Single Run from the menu bar or click on the Single Run icon to open the Single Run dialog shown below r Run information Sample ID Calibration level 1 Method Clear all calibration Cancel Data path C N32KaraProjectsNDefaulD ata Clear calibration for level Help Data file Print calibration report A Glear replicates Number of reps fi Print method report Average replicates r Amount values Sample amount Internal standard amount Multiplication factors 1 Begin run Dilution factors r Sample inject override Inlet vial Inlet Tray Duration sec Dutlet vial Dutlet Tray Description Immediately Figure 6 1 Single Run Acquisition dialog You must provide at least a Sample ID Method and Data File The Data File will be stored in the directory identified in the Data Path Sample ID can be any text string that identifies your sample This ID will appear on all subsequent reports The method file can be selected by clicking on the Open icon and navigating to the desired method For this exercise select the method you created in the Method Editing sectio
61. NOTE The detector lamp or lasers must be ON prior to starting a method The status of the lamp or lasers is available from Direct Control The UV lamp may be started there Lasers may be started from Direct Control if the power switch on the laser module is already ON and all safety interlocks are closed Materials needed Cartridge prepared in Direct Control exercise 75 micrometer id capillary 60 cm total length 50 cm to the detector installed in cartridge appropriate for detector type configured See the Installation and Maintenance Manual for instructions on building a new cartridge or installing a capillary e Capillary Regenerator Solution A 1M Sodium Hydroxide PN 338424 e Run Buffer PN 338426 Distilled or deionized water HPLC grade e 2mLl vials and red caps 36 position buffer trays For UV or PDA Detection Test Mix B PN 501333 For LIF detection using a 488 nm argon ion laser LIF Detector Test Mix PN 726022 MDQ User s Guide PN A33115AB 59 Running the System Single Run and Sequence Runs Load the trays as follows Left inlet buffer tray Right outlet buffer tray Test Mix C1 Regenerator Solution A D1 Water E1 Run Buffer A1 A2 A1 Empty Vial B1 PN 501333 for the UV or PDA Detector PN 726022 for an LIF Detector with a 488 nm laser Install the loaded trays and the capillary cartridge into the instrument as described in the Installation and Maintenance Manual
62. able from 0 5 to 32 Hz data points per second Narrow peaks require a higher data rate Data rate interacts with the Peak width parameter set under Filter Settings in this dialog box Filter This filter refers to an algorithm for filtering the collected data and does not refer to the wavelength filters Data filtering is necessary to remove extraneous noise that might interfere with data analysis Filter setting requires two steps the selection of the filter type High Sensitivity Normal or High Resolution and the setting of the Peak width High Sensitivity increases signal to noise at the expense of resolution 32 Karat 8 0 44 PN A33115AB Creating and Editing a Method Detector Initial Conditions High Resolution increases resolution at the expense of signal to noise Normal is a compromise setting that is suitable for most analyses The filtering algorithm is most efficient if it has information about the number of data points that make up a peak The optimum number of points is from 16 25 There are also options for less than 16 and more than 25 data points per peak The Data rate setting in the Electropherogram channel dialog can be adjusted so that a typical peak has from 16 25 data points The other Peak width settings should be used only if changing the Data rate is not desirable NOTE Peak Width here is not the same as Peak Width in Data Integration Relay 1 and Relay 2 The two relays incorporated into the P ACE MDQ can
63. analysis is to identify the expected migration times of the two peaks To do this open an instrument window continue to use the offline UV instrument from the previous exercise l Select File Data Open and navigate to the Data Samples directory 2 Open your copy of the file CE Level 3 dat Two channels of data are present Channel A UV 214nm and Channel C current You may minimize the Channel C window as it will not be used in this exercise Channel A is the electropherogram from the analysis of the middle concentration of the calibrators The smaller of the two peaks between 3 5 and 4 5 minutes is Alpha and the larger of the two is Beta previously determined in runs of the individual components Developing a Calibration Method We will build a new method to calibrate our data set Select File Method Open 2 Navigate to your copy of the Data Samples directory and open the method CE Calibrate met 3 Select Analyze to integrate the data set The integration parameters have already been optimized for you The two large peaks will be well integrated with the default parameters 4 Right click on the electropherogram and select Graphical Programming then select Define Peaks 5 Click in the electropherogram to bracket the large peaks approximately 3 7 and 4 2 minutes 32 Karat 8 0 82 PN 11 User s Guide PN A33115AB Calibration Creating a Calibration The following dialog will appe
64. ant flows through the cartridge via two openings in the bottom of the housing located between the ends of the capillary This fluid removes the heat generated by electrophoresis 2 2 Syringe Pump P ACE MDQ is capable of generating pressures using an internal pump mechanism This pump can supply 0 1 to 25 psi to perform pressure injections or low pressure mobilizations It can also apply up to 100 psi to move fluids through the capillary Vacuum injections can be performed from 0 1 to 5 0 psi Pressure can be applied to both ends of the capillary simultaneously to facilitate methods such as capillary electrochromatography 2 3 High Voltage Power Supply The high voltage power supply is capable of delivering a maximum of 30 kV with a maximum current of 300 pA The voltage range is from 1 to 30 kV selectable in 100 V increments The polarity is selectable from the software The current is selectable from 3 0 to 300 pA in 0 1 pA increments The software allows the user to select current voltage or power operation During operation the system will ramp the voltage or current up to the programmed value Limits for voltage current and power may also be entered to protect the capillary For example if the user programs a voltage setting for 30 kV but the setting for current is only 3 0 pA the system may reach the limit set for current before reaching the voltage setting and regulate voltage to maintain that current 32 Karat 8 0 PN A33115AB 2
65. ar NOTE Your time values may differ slightly from those shown r Define peaks in range DK Start time Minutes Stop time Minutes Came r Migration time window ep Relative C Absolute 0 092 Minutes Units x Quantitate peaks on Area Minimum peak area o Add all peaks to table C Replace existing peaks in table Figure 8 1 Define Peaks dialog The Define Peaks functions allows you to place any peaks in the specified window into the method s Peaks Groups table Only peaks that have been identified during Analysis can be entered into a method Start and Stop times define the part of the electropherogram that contains the peaks we wish to add Migration time window allows us to specify how much variability in migration time is allowed before a peak is no longer considered to be the component of interest This can be defined as either a percentage of the defined time or as an absolute time interval We can add all the peaks in the window to an existing table if any If we chose to Replace existing peaks any currently defined peaks that fall within the newly defined time windows will be deleted For this exercise accept the defaults l From the menu bar select Method Peaks Groups A table will open listing the two peaks just added As these peaks have not yet been named the system will create a name based on the migration time 2 Click in the Name column and change the
66. arker Mobility Window W EDF marker EM S d 0 00000000 0 00000000 PD x ALPHA 0 00025464 0 00000000 0 00028134 0 00001407 PD When we click on Analyze the peak identification will be by mobility To verify this change the Mobility entry for BETA to some other value like 0 00025227 and Analyze again Because the mobility of the peak is now outside the window it will be integrated but not associated with the ID BETA 32 Karat 8 0 PN A33115AB Creating Reports Introduction Creating Reports 11 1 Introduction User s Guide PN A33115AB The ultimate purpose of operating a CE or any other instrument system is to answer a question This answer is only useful if it can be communicated The 32 Karat Software includes a powerful suite of utilities for creating printed reports It also allows you to export data and results to other software packages Full integration into the Windows operating system environment allows you to copy and paste electropherograms and other information from 32 Karat Software directly into a wide variety of other software packages such as word processors This section of the manual will describe how to use the built in report generating features of the software For information on other ways of moving data to other applications see the resources listed in Appendix A In other sections of this manual you have been introduced to some of the
67. aser is turned off the cartridge cover opened or until the fiber cable coupler is disconnected from the LIF Interconnect Module The yellow light is a current limit indicator A sensor in the laser module measures the amount of laser light being produced Circuits in the laser module adjust the amount of current being drawn to maintain a stable 3mW output The yellow indicator light will come on when the system begins to draw excessive current This light indicates that the laser tube should be replaced If it comes on contact Beckman Coulter Customer Service for assistance CLASS 1 LASER PRODUCT THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE DATE OF MANUFACTURE MANUFACTURED 270 726024 A 900710L AI Figure 2 11 488 nm Laser Module Back Panel 32 Karat 8 0 PN A33115AB System Overview 2 The Laser Modules 635 nm Laser Module The power switch is located on the back panel of the 635 nm laser housing However the laser will not light up unless the MDQ system is on and all doors and interlocks are in place A red light on the front panel of the 635 nm laser module indicates that the laser is on Refer to Figure 2 9 User s Guide PN A33115AB 19 System Overview Using Other Lasers with the LIF Detector 2 10 Using Other Lasers with the LIF Detector Laser units other than the Beckman Coulter Laser Modules 488 and 635 can b
68. at 8 0 PN A33115AB Integration 7 Integrating Data This dialog allows the selection of items that will be displayed in the electropherogram window l Select the desired items Peak Area and Migration Time by clicking the item in the left box then using the green arrow to move the item to the right box 2 Verify that Baseline and Show undetected peaks are selected checked 3 When the dialog looks like the one above click OK NOTE Some items may not appear on the electropherogram display until the data is integrated 4 From the Method menu select Integration Events This will open the Integration Events spreadsheet There are two items in this table by default Width 0 2 and Threshold 50 Every Integration Table must contain at least one Width and one Threshold value 5 initially integrate the data use only the default parameters Select Analysis Analyze from the menu bar or click on the Analyze The integration process takes a few seconds larger data files and files with more peaks will take more time to process During integration the Analyze button will turn into a Stop button Clicking the Stop button will abort the analysis When integration is complete approximately 26 peaks will appear Above or below each peak the peak number area and migration time will be displayed The default parameters will have identified major peaks but also captured a number of baseline artifacts that are not of i
69. ata 7 Setthe Threshold value to 1000 and analyze the data again You should observe that the minor peaks are no longer integrated and the baseline below the large peaks has shifted dramatically Before examining the effects of the Width setting restore the Threshold value to 100 Click Analyze again 10 Zoom in on the base of the major peaks 11 Change the Width value to 0 1 and Analyze The tail of the second major peak should now be split off as a separate area 12 Change the Width to 0 5 and analyze The tail is again cut off but in a different place that it was with the smaller value for the window The question will inevitably arise which is the correct integration The answer is it depends You the analyst must use your knowledge and judgement to determine the correctness of the integration results In this example the peak tailing is a separation artifact and the area of the tails should be included in the area of the peak To obtain this result with THIS data file a Width of 0 2 and a Threshold of 100 seem to be appropriate Integration Parameters 74 There are many other integration parameters besides the required Width and Threshold that can be used to generate an integration result that properly reflects the components of the sample These are discussed at length in the resources listed in Appendix A In the next part of this exercise we will use some of these additional parameters to aid in the integration o
70. ata files From the point of view of data analysis the Y axis scale is the primary difference between UV data and LIF data PDA data consists of multiple components These are Channel data each channel is equivalent to a UV trace Up to three channels can be collected simultaneously 3D spectral data the three axes of this data set are time wavelength and absorbance Multi Electropherogram data similar to channel data this is a trace of absorbance versus time extracted from the 3D spectral data There are no limits to the number of data sets that can be created Channel data and multi electropherogram data are processed in the same manner as UV data all the procedures described in this manual apply The remainder of this appendix will describe some of the features of PDA data If you wish to re create these displays on your computer screen you will need to open an instrument configured for a PDA detector offline mode is satisfactory From a copy of the Data Samples directory open the data file CE PDA Data Sample dat In the figures in this manual the background color of the windows have been changed from the default color scheme to facilitate printing The multiple components of the PDA data set are available by choosing View PDA View from the menu bar O view Method Data Sequence Analysis Control Reports i Full Unzoom m Display All Data Tile Data Overlay Data Preferences PDA Vi
71. ate 4 4 4 From the above instrument run results of the selected run range the user can see that vial incrementing resets itself when the sequence run is initiated on run line 5 of Sequence Table AB The vial positions used in the results of the selected run range are the same as those of the sequence run when all lines of the sequence table are executed sequentially from run line 1 to 8 see Example 2 However the selected run range now yields that each run line number being executed by instrument corresponds to a different set of vial positions from those in the previous sequence run see Example 2 Important Notes When there is an Injection event in the method and the user chooses to inject from the method vial incrementing is enabled The vial positions for the incrementing in the run range are dictated in the same fashion as the Separate event above However if the user chooses to inject from the sequence table enabling Allow Override option in method time program vial incrementing is disabled That being the case the instrument runs will execute and utilize the vial position values inputted by the user in the Sample Inject Inlet Outlet columns of the sequence table with each run line having specific injection vials MDQ User s Guide PN A33115AB E 9 Appendix E Summary E 3 Summary E 10 The examples show that the method drives the vial incrementing and the sequence table plans an
72. ation fle Car TestRuns D DaT W Number of calibration levels Repetitions per level n i O Clear all calibration at start of sequence Create a separate row in the sequence for each repetition Multiple calibration sets sets calibration vials with unknown vials Reuse calibration vials ham frst set QI Frish Figure 6 8 Sequence Wizard Calibration dialog 32 Karat 8 0 66 PN A33115AB User s Guide PN A33115AB Running the System 6 Programming a Sequence In this example we will not be doing calibration Calibration is covered in the resources listed in Appendix A 1 Input 0 in the Number of calibration levels box and click Next to continue Sequence Wizard Reports Eirstosatlorat ame GT UP t Allicalipration sets Figure 6 9 Sequence Wizard Reports dialog This screen allows you to have reports generated automatically at the end of the sequence run We will not generate a report in this exercise the other choices should be blank 2 Click Finish A window similar to this one will appear 1 Sequence untitled seq Figure 6 10 Sequence Table This screen contains many more columns than can be displayed at one time Most of this information can be edited 67 Running the System Programming a Sequence 3 Scrollleft and right to r
73. ature Fraction Collection Timed Fraction Collection Capillary Temperature Sample Storage Temperature Fraction Collection Wavelength Timed Fraction Collection Autozero Autozero Lamp Lasers Stop Data Stop Data End End UV amp PDA LIF Figure 5 6 Time Program Event List The following will describe only those events that are used in this exercise For information on the other event types see the resources in Appendix A or refer to the 32 Karat Online Help 50 32 8 0 A33115AB Creating and Editing a Method 5 Time Program Separate Dialog The separate dialog is used to control the conditions under which the separation process takes place Every method must have at least one separation step The step at time 0 00 is usually a separation x r Values Pressure Direction 4 Voltage Voltage 1 0 KV Forward C Current Pressure for psi Reverse C Power Duration ro min C Both Pressure Ramp Time 017 min C Vacuum r Separation Type At Time Tray Positions 34 000 J min Options Inlet Bat With Pressure Outlet 1 with Vacuum Increment Polarity Inlet Outlet Normal Increment Every 1 Cycles C Reverse 7 Trays Extemal Adapter Figure 5 7 Separate Dialog This dialog has the following param
74. be used to control or signal other devices This dialog allows you to set the state of the relays at the start of the method to Off open or On closed These relays may also be controlled from the time program See the Installation and Maintenance Manual for more information on using the relays Absorbance Signal This parameter has two options Direct and Indirect Direct is used when the analytes have higher UV absorbance than does the background electrolyte In this case upward deflecting peaks are produced when analytes pass through the detector window In some modes of CE the background electrolyte has a higher absorbance than the analytes In this type of separation the passage of analytes creates negative peaks valleys in the electropherogram By selecting Indirect the entire electropherogram is inverted so that these negative peaks appear as positive peaks For this exercise set up the UV Detector Initial Conditions dialog to match Figure 5 3 MDQ User s Guide PN A33115AB 45 Creating and Editing a Method PDA Detector Initial Conditions 9 4 46 PDA Detector Initial Conditions E Instrument Setup Figure 5 3 Instrument Setup dialog PDA Detector Initial Conditions tab Electropherogram Scan Data This dialog controls the parameters for the collection of 3D data The Acquisition enabled box must be selected in order for data to be collected and saved Data rate is selectable from 0 5 to 32 Hz data
75. ctions exactly as described in the Separation event Values Specifies the magnitude of pressure to be delivered and for how long Pressure Direction Specifies whether pressure will be applied to the inlet or the outlet end of the capillary At Time Functions exactly as described in the Separation event MDQ User s Guide PN A33115AB 53 Creating and Editing a Method Time Program 94 Inject Dialog The inject event is used to deliver a precisely measured amount of sample into the capillary This step is always untimed and usually precedes the first separation step TT x Injection Type r Values Voltage Pressure 05 psi Pressure Duration 5 0 sec Cancel C Vacuum r Tray Positions Help Polarity Inlet Bat Normal a Outlet 80 41 Gites m Increment Inlet m Pressure Direction Forward Increment E very fi Cy C Reverse Trays r Sequence T able cles Allow Override Figure 5 10 Inject Dialog This dialog has the following parameters Injection Type Sample can be delivered to the capillary by positive pressure vacuum or by the application of voltage electrokinetic injection Polarity Specifies the charge on the electrodes during a voltage injection Pressure Direction Specifies whether pressure or vacuum will be applied to the inlet or the outlet end of the capillary Values Specifie
76. d 79 Section 8 Calibration de ot heels das eu ds 81 Analysis Components seas tica hte n RE Gus tts heo Meret nies GA het ete 81 Qualitative 515 81 Quantitative Analysis 81 Creating a Calibration 82 Developing a Calibration Method 82 Generating the Calibration Curve 85 Analyzing UNKNOWNS 88 Section 9 Qualitative Analysis Data Analysis and Reporting 89 Section 10 Mobility F RCHOHS OPIMODIEV eid ut ode dovete est abeat op S e raa Sir 93 Mobility Markers er accu orm uem tci t ERR die ie Re nS Edo eb bee e Pond 94 Assigning Mobility 94 eiee CTS 96 Demonstration Using the Mobility Features 97 Peak Identification by Mobility 100 Section 11 Creating Reports ntrod ction wines te Dre fo oS aasad 101 MDQ User s Guide PN A33115AB Creating Method Custom
77. d organizes the incrementing for the user Vial Incrementing in Runs Submitted to Instrument Run Queue Vial incrementing is reset whenever the user adds new runs single or sequence to the run queue In single runs of one method the vial positions will be incremented within the repetitions of each individual single run The incrementing will not be carried over to the next single run of the same method submitted to the run queue In sequence runs the vial positions will be incremented within each individual sequence runs The incrementing will not be carried over to the next sequence run of the same sequence submitted to the run queue The following examples illustrate Type Name Status Begin Single Run C 32Karat Projects DeFault Data methodB 001 dat Processing Immediately Single Run C 32Karat Projects Default Data methodB 002 dat Pending Immediately Single Run C 32Karat Projects Default Data methodB 003 dat Pending Immediately Status Begin hb Sequence Run All Records C EdMac Sequence Table Bl seq Processing Immediately Sequence Run All Records C EdMac Sequence Table Bl seq Pending Immediately Sequence Run All Records C EdMac Sequence Table B1 seq Pending Immediately In the first example of run queue that includes single runs of Method Time Program B the first second and third single runs are independent of each other Vial incrementing is not car
78. d the separation of nucleic acids by number of base pairs The 32 Karat Software allows you to construct a standard curve based on parameters such as these In the case of DNA for example a series of fragments of known length are separated in a gel filled capillary A plot of migration time versus chain length is constructed and this plot is used to determine the length of an unknown fragment In this exercise we will create a standard curve based on the base number of single stranded DNA A sample of ssDNA is injected into a capillary containing a polymer network Smaller molecules can negotiate through the polymer strands more easily than can larger fragments resulting in a separation based on DNA chain length The migration time of peaks of known lengths of ssDNA is used to create a standard curve that can be used to estimate the length of an unknown fragment Channel Time 30 075 Minutes Amplitude 0 001666 AU Channel Untitled 0 014 Quality 0 012 8 0 010 e 5 iD g 0 008 Ta i5 3 e cu 89 0 006 N 0 004 0 002 25 26 27 28 29 30 Minutes Figure 9 1 Electropherogram of Single stranded DNA 89 Qualitative Analysis Data Analysis and Reporting 90 l Openan offline instrument configured for a UV detector 2 From the Data Samples directory open the data file ssDNA 40 60 dat and the method CE Quality met The data file represe
79. default reporting options Here you will learn how to customize a default report to include the information you want to present We will continue to use a UV instrument as a prototype LIF reports are essentially identical to UV reports PDA data can be reported in the same formats as UV data additionally other PDA specific report types are available such as PDA Spectrum Library reports The most simple approach to reporting is to use pre defined reports These were used in earlier sections of this manual From the menu bar select Reports View to display the menu shown below which lists the default set of pre defined reports Reports Method Custom Report Ctrl Shitt F5 Print amp Sequence Custom Report Area X External Standard Internal Standard Library Search Results Normalization POA Library Report Figure 11 1 Reports View selection In this exercise we will use the data file and method from the exercise on Qualitative Analysis A review of the reports menu will show that there is no default report for qualitative data so one will have to be created We will create a Method Custom Report which will become part of the method We will also save the report as a standard template that will be added to the reports list 101 1 Creating Reports Creating a Method Custom Report 11 2 Creating a Method Custom Report 102 1 Open your Virtual UV instrument from the main 32 Karat window 2 From your working fo
80. der field type Bases Click OK We now have a column that contains the number of bases that the software has calculated for each peak Mem der D Right click in the table and select Report Properties We are not interested in Height or Height so remove them from the right side box using the red arrow 8 Click OK Our table is now too wide for the remaining three columns By clicking on the vertical dividers between columns we can drag the column widths to whatever we desire At any point during editing we can click on the Print Preview icon to see how the report will look on our default printer Save the method The new report is now part of the Quality method and will be available whenever the method is used by selecting Method Custom Report 32 Karat 8 0 PN A33115AB Creating Reports Creating a Method Custom Report Saving a Report as a Template In order to use the report in another method we can save the it as a template 1 From the File menu select Report Template Save As 2 TypeQuality as the filename and click By default the template will be saved with the extension rep 3 Select File New Method 4 Open Method Custom Report by selecting it from the Method menu if not already open The report will be blank because a new method has no defined Custom Report 5 From the File menu select Report Template Open and open the Quality template that you created When the new met
81. dy been developed 1 Create a sample containing the proposed marker and an EOF marker The sample matrix should be a close approximation of the unknown samples to be analyzed The EOF marker is a small uncharged molecule that can be detected at low concentration It is used to calculate the contribution of electroosmotic flow to the apparent peak mobility If running under conditions where EOF is minimal such as a neutral coated capillary the EOF marker can be omitted as is approximately equal to zero under these conditions Standards for other peaks expected in the unknowns should be included in this run E dvanced Method Options UY ETT ioj x Export Custom Parameters Capillary Performance Files Advanced Reports r Capillary Information Capillary length o C meters iem Capillary length to detector fo C meters Capillary lot number NL29c8624485 Capillary installation date 26 Juy 200 Capillary description i micrometer fused silica uncoated TT Calculate performance parameters for this channel Caleulation metas USP JEMG DAB BP EP ASTM 1A OH JP Figure 10 1 Advanced Method Options dialog 32 Karat 8 0 PN A33115AB Mobility 1 0 Mobility Markers Run the separation and integrate the results Adjust all integration parameters to their final form Add the mobility marker peak and the EOF marker peak i
82. e an individual to the possibility of severe electrical shock and or mechanical injury For this reason any service requiring removal of a panel or otherwise overriding or disabling safety interlocks must be done by Beckman Coulter personnel only Sharp Objects Moving Parts Keep loose clothing and hair away from the plate area Never attempt to physically restrict movement of the plate assembly Laser Safety WARNING Since laser light is not accessable to the user during normal operation of the system the overall classification of these lasers is Class 1 i e lasers which are safe under reasonably foreseeable conditions of operation To prevent eye damage from potentially harmful laser light observe all safety warnings User s Guide i PN A33115AB XIII Safety Information Safety Symbols Waste Management and Recycling It is important to understand and follow all laws regarding the safe and proper disposal of electrical instrumentation WARNING The symbol of a crossed out wheeled bin on the product is required in accordance with the Waste Electrical and Electronic Equipment WEEE Directive of the European Union The presence of this marking on the product indicates that the device was put on the European Market after August 13 2005 A is not to be disposed via the municipal waste collection system of any member moes state of the European Union For products under the requirem
83. e buffer systems a voltage of 12 kV will generate a 32 Karat 8 0 PN A33115AB Creating and Editing a Method Initial Conditions Tab current of 10 pA In this case the voltage will not exceed 12 kV as the current limit will be the determining factor Mohility channels This topic is covered later in this manual Temperature Sets the initial temperature of the cartridge coolant and the sample storage unit if installed These settings can be changed by the time program Peak detect parameters These parameters are used to trigger specific events such as fraction collection They are not used in peak integration or data analysis peak integration is discussed later in this manual Trigger settings The P ACE MDQ can be forced to wait until certain conditions are met before beginning a run These are selected by clicking the appropriate box If Wait for external trigger is selected the MDQ becomes a slave device and will not start until an external signal is received The two Wait for temperature options assure that the system has reached the correct operating temperature before beginning a run These options delay only the start of the time program Parameters set in initial conditions will occur without a wait Inlet and Outlet trays The types of trays that will be used when a method is run must be specified here When the method is run this information will be compared to the tray types that are configured in the instrument I
84. e original file WARNING DO NOT USE THE ORIGINAL FILES Save all methods and sequences you create as part of the tutorial in this new folder A backup set of files is included in case the originals become altered These are stored in the file Backup Files exe Running this executable file will restore the original set of sample files When running the backup files program you may be prompted to over write existing files Select Yes The contents of the Data Samples file will be replaced by this action including any changes you have made to the original set of data and method files User s Guide PN A33115AB Preface Important Information in this Guide Important Information in this Guide The following information describes the notes and warnings used in this document Beckman Coulter recommends that you review this information before using the MDQ System WARNING Paragraphs marked by WARNING alert you of a potential hazard to your personal safety if you do not adhere to the information stated within the paragraph CAUTION Paragraphs marked by CAUTION indicate that there is a potential danger of equipment damage software program failure or that a loss of data may occur if information stated within the paragraph is not adhered to or if procedures are executed incorrectly IMPORTANT Paragraphs marked by IMPORTANT contain information about a possible software program failure draw attention to a specific software setting or poi
85. e used with the LIF detector as long as the guidelines below are strictly followed If the LIF detector was purchased without a Beckman Coulter Laser Module an Interlock Fiber Optic cable PN 360671 will have been provided The custom interlock connector plugs into the LIF Interconnect Module The fiber optic termination of the cable is the standard SMA 905 The laser unit must be fitted with a fiber launcher which is compatible with this connector The two electrical wires in the cable connect to the interlock switches inside the detector and instrument These switches close when it is safe for the laser to come on These wires must be connected to the lasers interlock circuit The connectors and switches in the detector are rated for low voltage TTL level signals NOTE To avoid damaging the interlock connector and switches in the LIF Detector Module do not connect interlock wires in the laser unit to a voltage greater than the rated voltage of the interlock connector and switches 5V 0 1 A U S Federal regulations require that this connection be made to limit access to laser energy when the cable is disconnected from the LIF Interconnect Module 32 Karat 8 0 20 PN A33115AB 32 Karat Starting the Software 32 Karat 3 1 Starting the Software To start the 32 Karat Software select Programs 32 Karat 32 Karat Software from the Windows Start Menu button on the Windows Tool Bar 32 Karat Software opens to the current locat
86. ed by using the mouse to rubber box the desired area Right click and select Full Unzoom to restore the view C 4 Spectrum The spectrum detected at a specified point in time This is selected by moving the horizontal slider in the Contour or Electropherogram plots to the desired time point It represents a slice 32 Karat 8 0 PN A33115AB Appendix C Electropherogram through the 3D data This plot can be zoomed by using the mouse to rubber box the desired area Right click and select Full Unzoom to restore the view C 5 Electropherogram The electropherogram at a specified wavelength This is selected by moving the vertical slider in the Contour plot or the horizontal slider in the Spectrum view to the desired wavelength It represents a slice through the 3D data in a direction that is perpendicular to the Spectrum view This plot can be zoomed by using the mouse to rubber box the desired area Right click and select Full Unzoom to restore the view C 6 3D Plot User s Guide PN A33115AB A three dimensional representation of the data The presentation of this window is not tied to the Contour view To adjust the display right click on the 3D plot and select properties to open the 3D Data Graph Properties window PDA 3D Gf x IE Views Minutes The plot can be displayed as a one color surface or color can be used to specify absorbance ranges Colors are user selectable The image
87. ed to your system right click on the report and select Print User s Guide PN A33115AB 79 Integration Integration Results and Reports 32 Karat 8 0 80 PN A33115AB Calibration 8 Introduction Calibration 8 1 Introduction The previous exercise demonstrated how the 32 Karat Software can be used to determine peak parameters such as migration time area and height This information is of limited utility unless it can be translated into terms that are descriptive of the components in the sample There are two basic questions that may be asked What is this component and How much is present in the sample 8 2 Analysis Components Qualitative Analysis The first question What is this component refers to QUALITATIVE analysis It can be answered in two different ways First if a known substance is used as a standard the migration time or mobility of the resulting peak possibly combined with other information such as a PDA absorbance spectrum can be taken as evidence that a peak in an unknown sample represents that known substance Second if the migration time or mobility varies in some linear way with a molecular property such as molecular weight or number of base pairs a standard curve can be created that can be used to determine the defined quality such as molecular weight based on a series of standards In this method the unknown need not be present in the standard mixture Quantitative Analysis Thi
88. ed voltage up to the migration time of the peak of interest Ly capillary length to detector L total capillary length tef migration time of reference peak in the current run defined mobility for the reference peak Viep average applied voltage up to migration time of reference peak t migration time of the peak of interest Apparent Mobility is the sum of true mobility and the mobility caused by the electroosmotic flow Apparent mobility can be calculated directly for any peak The apparent mobility of a neutral marker one that has a true mobility of zero is called the electroosmotic mobility uap v LdLt apparent mobility capillary length to detector L capillary total length V average voltage up to migration time of peak t migration time of the peak v velocity Mobility calculations depend on an accurate measurement of the average applied voltage up to the point that the component passes the detector The output from the MDQ voltage monitor is used to calculate average applied voltage as follows n Vi _ p z n n data point number at peak migration time V voltage value at data point i 32 Karat 8 0 B 2 PN A33115AB Appendix C C Understanding PDA Data Appendix C C 1 Understanding PDA Data Overview Most of the exercises in the manual have used example data from an ultraviolet detector system LIF data files appear nearly the same as UV d
89. elect Initial Conditions The dialog in Figure 5 1 will appear E Instrument Setup of x I Initial Conditions UY Detector Initial Conditions Time Program Auxiliary data channels Temperature Peak detect parameters max 30 0 kV Cartridge 25 0 T Threshold 2 Current 300 0 pA Sample storage 25 0 ME Peak width 3 Power m Trigger settings Pressure Wait for external trigger p Mobility channels Wait until cartridge coolant temperature is reached Mobility Wait until sample storage temperature is reached Apparent Mobility efter voltage ramp Inlet trays Outlet trays Analog output scaling Buffer 36 vials Buffer 36 vials J Factor fi Sample tray Sample tray Figure 5 1 Instrument Setup Dialog Initial Conditions tab Auxiliary Data Channels The 32 Karat Software gives you the option to collect any one or all instrument parameters voltage current power and or pressure Select the channels you wish to record by clicking the appropriate box es The max kV and max pA boxes are used to set the allowable limits for these parameters Voltage and current are interrelated by the expression V IR The system will limit both parameters whenever one limit is reached For example assume a voltage set at and a current limit set at 10 pA With som
90. ent of WEEE directive please contact your dealer or local Beckman Coulter office for the proper decontamination information and take back program which will facilitate the proper collection treatment recovery recycling and safe disposal of the device 32 8 0 A33115AB Introduction to CE Overview Introduction to CE 1 1 Overview This section describes the common modes of capillary electrophoresis and how these processes are performed by the P ACE MDQ instrument using the Ultraviolet UV Photodiode Array PDA and Laser Induced Fluorescence LIF detectors 1 2 General Description The P ACE MDQ instrument separates sample components within fused silica capillary using one of several modes of electrophoresis of these modes are generally referred to as Capillary Electrophoresis CE In the P ACE MDQ sample is injected into the capillary using either pressure vacuum or voltage Under the influence of high voltage sample components migrate differentially through the capillary There are two basic methods for detecting samples using CE The first is the absorbance of light This method is utilized in UV and PDA detection As these components pass a window in the capillary a single wavelength UV detector or a multiwavelength photodiode array detector PDA measures absorbance and transmits the signal to the computer The signal can also be transmitted to an external recorder integra
91. enting is enabled in the method Any possible tray collision vial collision between the inlet tray and the outlet tray due to invalid vial combinations between the inlet vials and the outlet vials is indicated by a red asterisk mark next to the specific vial position in the report as illustrated below Rinse Rinse Separate Separate Vials In Vials Out Vials In Vials Out BI B1 BO D6 BI B2 BO E1 BI B3 BO E2 BI B4 BO E3 5 BO E4 32 Karat 8 0 PN A33115AB Appendix F E Sequence Vial Report Confirmed Important Note Users are not aware of possible vial collisions after the sequence table is set up with one or more methods included The instrument system will encounter these invalid vial combinations that lead to vial collisions when the sequence is run In the course of a sequence run the instrument system detects for invalid vial combinations and aborts execution of any method s repetitions that can lead to those collisions Other repetitions of the same method that have valid vial combinations are successfully executed by the instrument system It is the recommended that the user view the Sequence Vial Increment Preview report before running any sequence F 3 Sequence Vial Report Confirmed The Sequence Vial Report Confirmed is a sequence summary report that is generated after running a sequence It is a confirmation report of the specific vials that have been successfully utilized by the instrument sys
92. ers for ten 2mL vials Sample trays hold either forty eight vials or a 96 well plate Each tray is numbered from the front to the back beginning with the number 1 and lettered from left to right beginning with the letter A see Figure 2 3 User s Guide PN A33115AB 5 System Overview Main Components OJLL C2 QOOOOOGOO OOQOOO OOGOOO CODOOOOQ0 QOQUOQUO e QOOOOOOO J QOODOO L cT N OOOOO0O00 m o r OOO OOOO B 901694L AI Figure 2 3 Sample Trays 1 Buffer Tray 2 48 Vial Sample Tray 3 96 Position Sample Tray 4 Large Reservoir Tray WARNING The P ACE is not intended to accommodate volatile materials in 96 well plates Volatile solvents may release hazardous or flammable vapors leading to a risk of fire or explosion Solvent vapors may also damage the instrument Do not use volatile solvents in 96 well plates WARNING The 2 mL vials are pressurized during rinse and separation with pressure events To reduce the risk of breakage and expelled glass particles use only Beckman vials PN 144980 and inspect each vial for damage prior to use Do not use any vial that appears to be cracked or damaged in any way Wear safety glasses when opening the sample cover while the vials are pressurized 32 Karat 8 0 PN A33115AB System Overview 2 Main Compone
93. eters Separation Type Electrically driven separations can be done at controlled Voltage Current or Power When one of these is selected the other two will float to a value determined by the resistance of the capillary contents Voltage and Current cannot exceed the limits sets in the Initial Conditions window Separations can also be programmed to use pressure or vacuum to move the fluid in bulk through the capillary Voltage Current or Power can be combined with Pressure or Vacuum so that two processes are at work simultaneously Polarity Determines the direction of the current The charge on the electrodes is indicated by the graphic in this dialog Values Allows for input of the set points for the Separation Type parameters The available options will change depending on the Separation Type selected Ramp time is only valid for electrical separations It determines how long it will take for the Voltage Current or Power to change from the present to the programmed level MDQ User s Guide PN A33115AB 51 Creating and Editing a Method Time Program 92 Tray Positions Can be selected graphically by clicking on the Trays button The type of trays shown are determined by the settings in the Initial Conditions dialog When a method is used in a Sequence Table it may be desirable to change the vial positions after a specified number of cycles The inlet outlet or both positions can be incremented automatically by selecting the
94. etup x 25 Initial Conditions Uv Detector Initial Conditions Time Program Figure 5 5 Instrument Setup dialog Time Program tab Time the point after time zero at which the event will occur Event the action that will occur see below Value this will vary depending on the action selected Duration time the event will last Inlet and Outlet Vial where the capillary ends will be during the event 49 Creating and Editing a Method Time Program Summary a system generated description of the event Comments a user generated annotation of the event Time is not a required event Events that have no time associated with them will be run in the listed order top to bottom and each will be finished before the next event begins Timed events must be grouped together a group of timed events cannot be interrupted by an untimed event Untimed events can only occur before a group of timed events Some events do not have a time option others can be timed or untimed Data acquisition begins with the first timed event time 0 00 it ends when the method ends or when a STOP DATA event is reached To program a line click in the EVENT box Click on the down arrow to open a menu of events Select an event to open a dialog box for that event Separate Rinse Separate Inject Rinse Relay on Inject Wait Relay on Message wait Message Capillary Temperature Sample Storage Temper
95. eview the entire screen Not all of the columns will be used in this exercise The important ones are described below Run Type Because we have not selected any summary reports all of the runs are of the unknown type See the resources in Appendix A on page 1 1 for more information Reps tells the system how many times to run the line 4 Click in the Reps boxes for the second line and change the Reps to 2 Line two will now execute twice giving a total of four runs in this three line sequence 5 Scroll to the right until the window resembles the one shown here Sequence untitled seq olx Test Mix 001 Test Method of 5 16 2000 met Test Mix 002 My Test Method of 5 16 2000 met Test Mix 003 My Test Method of 5 1 6 2000 met F Test Runs lt D gt DAT Test Runs lt D gt DAT Test Runs lt D gt DAT Figure 6 11 Sequence Table cont d Sample ID is based on the Sample ID text you entered in the Wizard The line number has been added to the Sample ID you input 6 Click in the Sample ID box for Run 2 and change it to read Test 002 Twice Method shows the method you entered in the Sequence Wizard To change the method click in the Method box and click on the green icon NOTE For this exercise do not change the method Filename is based on the File Name Text you entered in the Sequence Wizard The filename still shows the Date and Time symbol l
96. ew Mixed View Spectral Library Search Spectrum Electropherogram Max Plot Multi Electropherogram Ratio Plot 3D Contour 3D amp Contour User s Guide PN A33115AB C 1 Appendix C Mixed View Each item in the menu represents a different way of presenting the data on the screen Only one of these selections may be made at one time C 2 Mixed View This option is shown below This image illustrates several of the other options as well PDA Mixed View 200 views E Synchronize 34 Actions E Options Time 5 41841 Minutes Wavelength 200 nm Time 0 Minutes Amplitude 0 mAU P ACE MDQ 200 nm ini x Wavelength 295 587 nm Amplitude 2 231 mAU This window shows four different views of the same data set The sample is the same mixture of Alpha and Beta that has been used throughout this manual This window is divided into four panes By clicking and dragging the bars that separate the panes the relative areas assigned to each pane can be changed The views available here are Left Right Upper Contour Plot Spectrum Lower Electropherogram 3D Plot C 3 Contour Plot The Contour plot is an overhead view of a three dimensional data set The X and Y axes are Time and Wavelength respectively Absorbance at any given wavelength and at any given time is indicated by color or by a shade of gray This plot can be zoom
97. f a somewhat more complex data file To begin return the Width value to 0 2 and the Threshold value to 50 the default values Open the data Data Sample 2 dat Use Ctr1 Z if necessary to view the full file This data file differs from the previous example in several ways There are multiple major peaks distributed throughout the run There is a long stretch from about 12 to 21 minutes where there are many features that may be peaks or may be artifacts Close examination of the peak between 24 and 25 minutes will reveal that it is in fact at least three separate peaks 32 Karat 8 0 PN A33115AB Integration Optimizing Integration that are not fully resolved The major peaks are also taller and wider than the peaks in the previous example After examining this file zoom out Ctr 1 Z and analyze with the default Width and Threshold values A large number of peaks will be identified The big peak at the end of the run is not included The analysis appears to end at around 28 minutes Zoom in on the area around 27 to 28 minutes Many peaks which appear to be quite small have been identified and the last peak is number 150 There is a limit of 150 peaks built into the software which accounts for the end of the data being ignored Not all 150 components are of interest Some parameters need to be changed to reduce the number that are identified In this part of the exercise graphical programming will be performed to build the i
98. f there is a tray type mismatch the method will not run NOTE Additional information on these and other parameters of the Instrument Setup window can be found in the 32 Karat Online Help User s Guide PN A33115AB 43 Creating and Editing a Method Detector Initial Conditions 5 3 Detector Initial Conditions For this exercise set conditions in the Initial Conditions tab to match Figure 5 2 NOTE The next tab will be used to set up the initial conditions for the detector Select the option below that corresponds to the detector type in your instrument UV Detector Initial Conditions Click on the UV Detector Initial Conditions tab Instrument Setup x Initial Conditions UV Detector Initial Conditions e Time Program Electropherogram channel Filter Acquisition enabled High sensitivity Wavelength 214 nm C High resolution Data rate 4 Hz Peak width points 1625 7 Relay 1 Relay 2 Absorbance signal amp Off amp pff Direct C On C On C Indirect Figure 5 2 Instrument Setup dialog UV Detector Initial Conditions tab Electropherogram channel The Acquisition Enabled box must be selected in order for data to be collected and saved The Wavelength option allows the selection of one of the filters installed in the filter wheel The available selections will be those set up during instrument configuration Data rate is select
99. f used to the Peaks Groups table The method must contain the correct dimensions for the capillary length These are entered in the Capillary Performance tab of the Advance Methods Options window accessible from the Method menu The capillary length and length to the detector are required 5 Annotate the electropherogram with Apparent Mobility and Mobility 6 Analyze the data again The Apparent Mobility and Mobility values will be displayed At this point the mobility values should all be zero 7 Calculate the electrophoretic mobility of the mobility marker according to the following Papp Heof where Happ is the apparent mobility of the reference peak and eof is the apparent mobility of the EOF marker Scroll right in the Peaks Groups table to the Mobility column In the row containing the mobility marker enter the value of determined in step 4 10 Select the in the Mobility Marker option column of that same row 11 Save the method 12 Analyze the sample again and examine the electropherogram The mobility value of the marker should now be non zero as should the mobility values for all other peaks in the electropherogram except the EOF marker The EOF marker should have a value very close to zero variation from zero at this stage is the result of rounding errors If the results are acceptable the method can now be edited to allow the generation of an electropherogram with mobility as the X axis in place of ti
100. for any single sequence table run line that consists of one or more repetitions to be executed the process is similar 13 Begin by clicking on the blue arrow in the Run Type box of an initial sequence table run line where the sequence run will start it does not have to be run number 1 The Sample Run Type s dialog window will open 14 Select Begin Summary as the run type User s Guide PN A33115AB F 7 Appendix F Sequence Vial Report Confirmed IMPORTANT Do not select Vial Summary as the run type This run type is not used to generate the Sequence Vial Report Confirmed Sequence Sequence TableA seq Status Level Conc Override Reps E uem Sample Run Type s Clear All Calibration Clear Calibration at Level Print Calibration Report JAverage Replicates Clear Replicates Begin Loop End Loop Shutdown Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability Begin Summary Summary Run BC Check Standard 15 Click on the folder icon in the Run Type Parameters region to open up a report template 32 Karat 8 0 PN A33115AB User s Guide PN A33115AB Appendix F E Sequence Vial Report Confirmed 16 Select Sequence Vial Summary CE brp as the report template and click Open The report template name should be in the Report Template field after opening
101. for the specified time program event e g rinse separate On the other hand selecting Increment Every 5 Cycles will cause the instrument to run the method five times before changing vial positions for the specified time program event The Inject event unlike Rinse Separate and Wait can be programmed through both the method or sequence table Inject event has the option of allowing the user to introduce samples from the vial positions stated in the method time program or in the sequence table Vial incrementing is enabled if user chooses to inject from method If the user chooses to inject from the sequence table vial incrementing is disabled giving sequence table priority with the indicated Allow Override enabled box E Appendix E Vial Incrementing Te m Injection Type r Values C Voltage Pressure 5 Pressure Duration 50 sec Cancel C Vacuum r Tray Positions C Reverse Polarity Inlet Normal 29 Outlet 1 Reverse Increment Outlet Pressure Direction Forward Increment Every fi Cycles Help Trays r Sequence Table Allow Override After vial incrementing has been set up in the method the method can be executed as a single method run using Single Run command Or the method can be incorporated into the sequence table for running a sequence using Sequence Run command The user can direct the
102. ftware when requested by the user System Overview The Photo Diode Array PDA Detector Figure 2 8 Dioade Aray Optics Layout 901716L AI 1 Capillary Aperture 2 Fused Silica Lenses 3 Deuterium 4 Lamp Power Supply 5 Concave Holographic Grating 6 256 Element Diode Array 7 Motor 8 8 Position Filter Wheel 9 Monochromator Entrance Slit 10 Fiber Optic Connector 11 9x 200 Fiber Array Slit 12 Fiber Optic Connector 13 Mercury Calibration Fibers 14 Mercury Lamp Power Supply 15 Mercury Lamp 16 Y fiber Optic Cable 17 Filter wheel in open position 8 18 Fiber Optic Connector 19 Capillary 14 32 Karat 8 0 PN A33115AB System Overview 2 Laser Induced Fluorescence Detector 2 8 Laser Induced Fluorescence Detector The LIF Detector consists of the LIF Detector Module the LIF Interconnect Module and a Laser Module Refer to Figure for more information A MDQ capillary cartridge with an LIF Detector plug installed is required for use with this system The LIF detector uses a laser light source A 488 nm argon ion laser and a 635 nm diode laser are available from Beckman Coulter Other lasers can be adapted The LIF detector can use dual lasers and dual photodetectors making it a true dual wavelength system Refer to the section titled Using Other Lasers with the LIF Detector on
103. g is not intended to be a hands on exercise but rather a step by step example of the process described above If you wish you can attempt to reproduce these results on your instrument This example utilizes a UV detector and Beckman Coulter Test Mix B PN 501333 We have previously determined the optimum conditions for the separation of a mixture of two components ALPHA and BETA see the exercises earlier in this manual ALPHA has been chosen as our mobility marker Our method has considerable EOF so we will need to use an EOF marker We have chosen to use Ethanol it is readily soluble in our sample creates a detectable peak and does not interfere with our analysis all previously determined in practice this must be shown by experiment A final concentration of 596 Ethanol has been chosen The sample is prepared and run After step 6 above the resulting electropherogram looks like this PDA 214nm Jol x Time 5 99722 Minutes Amplitude 0 00077 AU PDA 214nm Migration Time Apparent Mobility Mobility 5 408 0 00032093 0 00000000 5 004 000034763 000000000 950 000060227 0 00000000 3 0 3 5 4 0 45 5 0 55 6 0 Minutes 97 Mohility Demonstration Using the Mobility Features Our ethanol marker gives a small negative peak but it is properly detected by the software The values obtained are listed in the table below Peak Name Apparent Mobility Mohility EOF 0 00060227 0 00000
104. gative Mobility toward the cathode negatively charged electrode is defined as positive and mobility toward the anode positively charged electrode is defined as negative Mobility is not a constant like molecular weight as it can change depending on the separation conditions employed As an example consider the amino acid glycine At alkaline pH this molecule has a net negative charge NH5 CH COO and a negative mobility At acidic pH it has a net positive charge NH5 CH COOH and positive mobility Near pH 7 it has no net charge NH4 CH5 COO and a mobility of zero In practice mobility is defined for a given molecule under a given set of conditions Variations in separation conditions that affect all species equally such as variations in electroosmotic flow or the voltage delivered by the power supply and variations that have similar effects on closely related groups of compounds such as the effect of small pH changes on a series of basic drugs can be accounted for by including in the analytical run a standard of defined mobility There are two different mobility values to consider electrophoretic mobility which represents the movement of the molecules in the electrical field and apparent mobility which is the algebraic sum of the electrophoretic mobility and any other forces which drive the sample through the capillary The most common additional force is electroosmotic flow EOF sometimes referred to as
105. ght side panel 12 Add Quality and set the number of decimal places to zero 13 Click OK to return to the main dialog Now we can change the X axis so that the peak area fills the graph window 14 Click on the Axis Setup tab 15 With X axis selected click on Use this range 16 Enter a minimum of 24 and a maximum of 30 to bracket the 24 30 minute time interval 17 When done click OK The part of the electropherogram containing the 21 peaks should fill the graph and each peak should be properly labeled with the Quality value The graph can now be resized by dragging the handles on the right and bottom edges of the image Moving the graph requires that it be treated as a text object 18 To center it on the page single click the graph to select it a narrow dotted border will appear and use the centering icon from the toolbar Space can be added above and below the graph by inserting blank lines with the Enter key Additional charts can be inserted by right clicking on an empty line and selecting Insert Graph You might for example want a second graph showing the full time range of the data set The new graph is then formatted as described above Below the graph is a table with a variety of peak parameters Some of these we will keep others we will change for our new report Right click on the column header Area and choose Change Parameter Select Quality as the new parameter and change Decimals to one In the Column Hea
106. he LIF Calibration Wizard will become active This feature is described in greater detail in the Installation and Maintenance Manual The Filter dialog is used to define the specific filters that are installed in a UV detector The data must be entered manually It is important that the values entered match the installed filters exactly If a PDA detector is installed position 8 must be empty no filter installed indicated by a 0 value 32 Karat 8 0 PN A33115AB 32 Karat 3 Creating and Configuring an Instrument Pressure units may be defined in psi pounds per square inch or in mbar millibars The units selected will be used only for this instrument Temperature control indicates the installation of a sample storage unit If installed the storage unit may be disabled by selecting Unavailable from the drop down list box 11 Click to return to the Instrument Configuration window Available modules Configured modules 8 UV Detector PDA Detector p LIF Detector Stand Alone No Detector O External Event Detector Configuration Options Auto Configuration Cancel Help Figure 3 8 Instrument Configuration screen for the P ACE MDQ Configuration Options Clicking the Options button will open the Configuration Option dialog See Figure 3 9 Analysis Options Analysis options define what software functions will be available when data is reprocessed Configuration Options x Genera
107. hen you designate a run as a Begin Summary run for example you will be required to designate a template name for the summary report 105 1 Creating Reports Creating a Method Custom Report 106 Other run types requiring templates include Suitability QC Check Standards Duplicates and Sequence Vial Report Refer to Appendix F for information about Sequence Vial Reports Sequence Custom Report templates are created and edited using the Sequence Custom Report editor Standard Report Templates Standard templates for many report types are provided with 32 Karat Software however you may wish to create your own or edit the templates provided Standard report templates Area 96 External Standard Internal Standard and Normalization are located in the cA32Karat program directory and have the SRP extension You can create new standard report templates by saving your report template using the SRP extension Examples of the standard reports can be found in the Standard Reports section Sequence Reports Sequence reports are only created and saved using the Sequence Custom Report editor Unlike method custom reports sequence custom report templates are not saved as part of the sequence file and therefore must be saved as a template file if you want to use a sequence report template to generate a report The following standard sequence report templates are provided with 32 Karat Software Calibration Summary Duplicate Q
108. hod is saved it will contain the Quality rep as the default Method Custom Report The Custom Report saved in a method can be edited and changed at any time Saving a Report as a Default Report We can also save this report as one of the default reports in the Reports menu l Again select Report Templates Save As from the File menu 2 This time save the template with the name Quality srp 3 Click Save By saving with the Sr p extension you have created a new default template The next time an instrument window is opened the new report type will be available from the Reports menu This is a handy way to handle reports that are used frequently Report Templates Custom reports are templates for displaying and printing data and objects 32 Karat Software comes with a suite of standard report templates for a variety of report types These can be used as is or can be modified using the Method Custom Report or Sequence Custom Report editor and saved as new templates Method Custom Report Templates A method custom report template is created and saved as part of a method but it can also be saved as a template file that can be opened from within another method and used or edited Sequence Custom Report Templates User s Guide PN A33115AB Sequence custom report templates are used for reporting data generated during batch sequence operations For example certain Run Types require a report template for reporting the data W
109. ics Module with D Lamp inside 9 Capillary Cartridge 10 Detector 11 Fiber Optic Cable 12 Grounded Electrode 13 Outlet Sample Tray 14 Outlet Buffer Tray 15 Power Switch The main power switch is located on the lower right side of the front of the instrument AII connections for external system components are located on the upper left side panel of the instrument except for AC inlet and the fuse holder Three fans provide cooling airflow for internal system components air is exhausted through vents at the side and rear of the instrument Maintain proper clearances to ensure that the vents are not blocked See the P ACE MDQ Installation and Maintenance Manual for more information 32 Karat 8 0 PN A33115AB System Overview 2 Main Components The Sample Handling System The sample handling system holds four trays two sample trays inlet and outlet and two buffer trays The sample trays are primarily used for samples the buffer trays hold the other solutions required for electrophoresis e g buffer and rinse solutions The trays are arranged on two parallel tracks Under normal operating conditions the trays on the left are referred to as the inlet trays for sample and buffer the trays on the right are referred to as the outlet trays for sample and buffer Each buffer tray holds either thirty six 2 mL vials or a large reservoir tray the large reservoir tray has two 25 mL buffer reservoirs and hold
110. ies to the entire electropherogram it is not possible to use time for some peaks and mobility for others l The option to use mobility for peak identification must be selected on the Options tab of the Method Properties window under the Method menu Select Mobility based and click OK Next peaks are added to the Peaks Groups table if not already present This is most easily performed by using the Add Peaks function from the Graphical Programming menu as described earlier in the section on Integration 4 Open the Peaks Groups table and scroll to the Mobility columns The assignment of the mobility of a mobility marker has already been described To identify other peaks by mobility it is necessary to manually input their mobility value and window For the peak BETA in our example the mobility value was determined to be 0 00028134 so this value is entered into the table for the line defining BETA The default Mobility Window is zero which means that the peak would only be detected if it had exactly the same mobility value as that defined Some variation can be expected The appropriate value will be determined by trial and error A window of 5 10 is a good starting point For BETA a 596 window would be 0 00001407 the window is always an unsigned number not positive or negative At this point our Peaks Groups table looks like this Peak Group Tables PDA 214nm Eifel Named Peaks Groups Name ID Mobility M
111. ion group window which lists the configured instruments 32 Karat Software pe x Fees vj E E MDQ 1 P ACE MDQ 2 P ACE MDQ 3 Figure 3 1 32 Karat Software Enterprise Screen Main Menu User s Guide PN A33115AB 21 32 Karat Creating and Configuring an Instrument 3 2 22 Tool Bar The Tool Bar appears at the top and in some cases the bottom of the active window The active buttons within the Tool Bar allow for single click access to many common commands The following buttons are available from the opening window New e Cut Copy e Paste Delete Properties e System Administration Mode e System Administration Wizard Large Icons Small Icons List Details Help Menu Bar The Menu Bar contains all commands available in 32 Karat Software The Menu Bar can be accessed by single click or by holding down the Alt key and pressing the underlined letter Creating and Configuring an Instrument In this exercise you will create a connection to a real instrument create a virtual instrument that can be used for offline data reprocess NOTE Additional information regarding Instrument Configuration can be found in the 32 Karat Online Help IMPORTANT This section assumes that all hardware and interface boards have been installed If they have not been installed please refer to the Installation and Maintenance Manual for
112. ired for each of the other lines Sequence table run lines that have Unknown or other run types will not generate the Sequence Vial Report Confirmed Sequence Sequence TableA seq After the user has changed the Run Type of any set of sequence table run lines to be executed from Unknown to Summary the user can begin the sequence run to allow the software to collect data for the Sequence Vial Report Confirmed A Complete message in the Status column of the sequence table will confirm the end of the sequence run lii Sequence Sequence TableA seq SMB SME SMB SME SMB SME After the sequence run is completed or is terminated the user can activate the Sequence Vial Report Confirmed by selecting Sequence Custom Report under View from the Reports menu Reports Window Help Method Custom Report Ctrl Shift F5 Print nc eport Advanced Report Properties 1st Dimension Report 2nd Dimension Report Area Current Baseline Check Current System Check External Standard Internal Standard Normalization The View Sequence Reports dialog window will pop up The listings under Report Type column are the Sequence Vial Reports Confirmed for each of the sequence table run lines They are labeled as Sequence Summary reports in the dialog window The report template used are indicated in the last column The numbers under Sequence Records column are the sequence table run line numbers f
113. irmed lii Sequence Sequence TableA seq 1 Cd Unknown Unknown Unknown Alai Sample Run 5 Clear All Calibration Clear Calibration at Level Print Calibration Report JAverage Replicates Clear Replicates Begin Loop End Loop Shutdown Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability in Summary Summary Run End Summary Vial Summary QC Check Standard 3 Click on the folder icon in the Run Type Parameters region to open up a report template 4 Select Sequence Vial Summary CE brpas the report template and click Open The report template name should be in the Report Template field after opening 5 Click OK to close the Sample Run Type s dialog window and return to the sequence table 32 Karat 8 0 PN A33115AB User s Guide PN A33115AB Clear All Calibration Clear Calibration at Level Print Calibration Report J verage Replicates Clear Replicates Begin Loop End Loop Shutdown Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability Summary Run End Summary Vial Summary OAC Check Standard Unspiked Spiked Spike 1 of 2 Spike 2 of 2 Duplicate Begin Calibration End Calibra
114. it can be used to determine the amount of material in an unknown Unknowns can be added to the sequence table after calibration lines When processed the unknown peaks will be compared to the standard curve and the values calculated Open the Sequence Window 2 Inline 6 click in the Method column and select CE Calibrate met the method we have been using 3 Under Filename select Unknown1 dat Save the sequence 5 Instead of reprocessing the entire sequence click on the Range button in the Process Sequence dialog Figure 8 5 and enter 6 as the line to be run 6 Click Start To see the results of this analysis you can display the electropherogram for Unknown1 dat Or you can view a report To use a default report select Reports View External Standard from the menu bar To print this report to your system s default printer right click in the report window and select Print See Creating a Method Custom Report on page 11 102 for more information This exercise has covered only the basics of quantitative analysis For more detail and more options refer to the resources in Appendix A 32 Karat 8 0 PN A33115AB Qualitative Analysis Data Analysis and Reporting Qualitative Analysis Data Analysis and Reporting In some modes of CE separation the order of migration is determined by a molecular quality The most common applications that utilize this approach are separation of proteins by molecular weight an
115. l Instrument Options PDA s System Suitability Qualitative Analysis Caesar Integration K II L 1L Cancel Figure 3 9 P ACE MDQ Instrument Configuration Options Dialog box Analysis Options MDQ User s Guide PN A33115AB 27 32 Karat Creating and Configuring an Instrument 28 PDA Allows the analysis of multichannel data from the Photo Diode Array detector System Suitability Enables automatic review of results Results outside selected ranges can trigger defined responses Qualitative Analysis Enables the identification of peaks by migration time relative migration time or mobility Caesar Integration Used to detect peak start and stop This method is useful for peaks that have abrupt transitions from baseline to peak It is also useful when S N signal noise ratio is low Caesar Integration is the preferred method for detecting CE peaks When un checked peak start and stop will be based on slope threshold Instrument Options Use the Instrument Options function to select Standard CD default or CEC LC Configuration Options Ea General Instrument Options Standard CE Operation mode Cancel Help Figure 3 10 P ACE MDQ Instrument Configuration Options Dialog box Instrument Options Standard CE The time for a peak to reach the window is called Migration Time CEC LC The time for a peak to reach the window is called Retention Time 32 Karat
116. l peaks have dropped out of the integration results The peaks near the end of the electropherogram have been found But we now have a problem with the cluster of peaks between 24 and 26 minutes 8 Zoominto this area and observe that the baseline starts about halfway up the start of the peak This demonstrates the fact that global parameters do not always work for every peak in the data set We need to add new lines that affect only the region of the clustered peak Integrating a Peak Cluster Recall that with the original Width and Threshold values 0 2 and 50 respectively this cluster was integrated nicely Perhaps one or both of those values applied to the region around the cluster will achieve the desired results 1 While still zoomed in select Width from the Graphical Programming menu 2 As prompted click at about 24 and 25 minutes In this case we are using the graphical function here to define Start and Stop times rather than to define Width 3 In the dialog box that appears change the Value to 0 2 Click Analyze Now The cluster should appear similar to the one shown here E Channel x Time 24 6466 Minutes Amplitude 0 016032 AU o Channel Untitled Pk Area Retention Time 24 0 24 2 24 4 24 6 24 8 25 0 Minutes Figure 7 5 Well integrated peak cluster MDQ User s Guide PN A33115AB 77 Integration Optimizing Integration This cluster is well integrated Even the shoulder o
117. lder open the file CE Data Sample 3 dat and the method CE Quality met Use the copies of the data and the method that were used during the exercise on Qualitative Analysis We will be creating a report based on the analysis results generated during that activity 3 From the Method menu select Custom Report A blank window will open If a method custom report has previously been developed with the method you are using it will automatically open at this point Modifying a Report Template Rather than developing a report from nothing we will modify an existing report template to simplify this task A report template is a pre defined set of instructions for the creation of a report A number of templates are contained in the software You can add templates by creating a new one or editing an existing one and saving it under a new name We will use the Area template as the basis for our Qualitative Analysis report From the file menu select Report Template Open and select Area srp The template will open with the results from the open data file displayed The indications that we are looking at a template and not at a report are that the header information is highlighted the table has a broken border and the ruler is visible As a first step we will change the title of the report The report editor works very much like a word processor 1 Using the mouse highlight the heading Area Report 2 TypeQualitative Analysis Report
118. me This option is selected in the Initial Conditions tab of the Instrument Setup window MDQ User s Guide PN A33115AB 95 Mobility Mobility Markers 96 Instrument Setup 36 vias 28 36 vials 48 vias 8 96 positions Ed Figure 10 2 Mobility Options in the Instrument Setup dialog Mobility Plot Selecting Mobility or Apparent Mobility will cause the creation of an additional plot after data analysis This plot will show the electropherogram re scaled so that the X axis is displayed in terms of Mobility or Apparent Mobility rather than time The Plot trace after voltage ramp option should be selected unless peaks are detected during the voltage ramp up phase of the separation Deselecting this option may result in the voltage ramp interval being over emphasized in the resulting mobility plot NOTE Mobility channels cannot be generated for data files that were created with this option turned off It must be ON during data acquisition When using this option there must be at least one Mobility Marker identified in the Peaks Groups table of the method 13 After selecting the appropriate mobility channel options save the method 14 Run the sample again At the end of the run the mobility trace will be generated 32 Karat 8 0 PN A33115AB Mobility 1 Demonstration Using the Mobility Features 10 3 Demonstration Using the Mobility Features User s Guide PN A33115AB NOTE The followin
119. me the method Quality 1 met Analyze the data file again Right click on the electropherogram and select Annotations Add Quality to the right column Name should already be present Click on the word Quality to highlight it then set the number of decimals to 2 Click OK The electropherogram will now display the assigned base number as Name and the base number calculated using the standard curve as Quality The base number is an integer the small discrepancy between the assigned value and the calculated value is due to the fact that our goodness of fit was not perfect w This method can now be used to estimate the size of an oligonucleotide of unknown length To do this the unknown would be analyzed by the same method used to analyze these standards and the data analyzed with this method MDQ User s Guide PN A33115AB 91 Qualitative Analysis Data Analysis and Reporting 32 Karat 8 0 92 PN A33115AB Mobility 1 0 Functions of Mobility Mobility 10 1 Functions of Mobility Mobility is a parameter that quantifies how a charged particle migrates in an electrical field The equations for calculating mobility are defined in Appendix B Stated briefly a component with a higher mobility will move more rapidly through the separation medium than will a component with a lower mobility Because particles may be attracted to either the cathode or the anode mobility has a vector component it may be positive or ne
120. n Data path is selected in the same way as the selection of a method In this case you are selecting a directory rather than a file The data file is most easily entered by typing a unique name in the box The file name must be a name that does not already exist in the data directory if the name already exists the run will not start The remaining items on this screen are not required to run a method For information on those features see the resources in Appendix A When ready to begin press Start The system will examine the method to verify that it is appropriate for the instrument configuration The method is then downloaded to the P ACE MDQ The instrument will first perform a brief start up check and then will execute the method This is a good opportunity to become familiar with how the instrument operates Knowledge of normal operations is essential when things go wrong There is a possibility that your first run may not perform as expected MDQ User s Guide PN A33115AB 61 Running the System Single Run If error messages appear or the data does not look like the example shown below check the following Are the vials correctly filled with the proper fluid Are the vials in the positions specified by the method Was the correct method selected e Was the method written for the current instrument configuration e g PDA method cannot run on a UV or LIF system Is the method correct compare to Sectio
121. n 5 Do the tray types in the method Initial Conditions match the tray types configured for the instrument E Channel A UY L mi xj Time 4 99637 Minutes Amplitude 0 000108 Au Figure 6 2 This method will be used for additional exercises IMPORTANT Please do not continue to the next section until you have achieved a successful single run 32 Karat 8 0 62 PN 3115 Running the System 6 Programming a Sequence 6 3 Programming a Sequence A Sequence is a list of methods and data files that will be used to run a batch of samples without user intervention Sequences can be used to acquire data run the instrument or to batch reprocess existing data files In this exercise you will create a Sequence to acquire data from multiple runs of the test mix method Later in this manual you will use a sequence to perform batch reprocessing of data You must be Online for this exercise l Tocreate a new sequence select File Sequence New from the Instrument window This will open the Sequence Wizard dialog shown below The Wizard consists of 5 screens Not all of them will be used for every sequence and not every feature of each screen will be used in this exercise For information on features not covered here see the resources in Appendix A Sequence Wizard Method x CA32KaratiData SamplesiCE Quality met Figure 6 3 Sequence Wizard Method Dialog The first screen requires that you select a me
122. n easier alternative At the left of each line is a box with a red check mark Click on the red mark on line one to de select it This line will be ignored as long as it is unchecked Your Integration Events Table should look like this one ES Integration E vents UV 200nm Figure 7 4 Integration Events Table Threshold 76 1 Zoom out and click Analyze You will see a small change in the integration Obviously changing the Width alone is not sufficient to integrate this data set We are still finding too many of the very small peaks that are of no interest Right click on the electropherogram select Graphical Programming and then Threshold Following the prompts in the status bar click near the start of the electropherogram time 0 0 and at around 8 0 minutes 4 When the dialog box appears click Analyze Now The new threshold value which should be around 850 will have removed many of the small peaks in the 0 8 minute time range The new threshold value will also have been added to the Integration Events table 5 As with the Width parameter change the start and stop times of the new Threshold event to 0 and deselect the old Threshold parameter 6 Again for consistency adjust the new Threshold value to exactly 850 32 Karat 8 0 PN A33115AB Integration Optimizing Integration 7 Analyze the sample You will observe that the change in threshold has had dramatic effects Almost all of the smal
123. n place of the one shown 5 Verify that the instrument is loaded and ready to run then click Start 69 Running the System Running the Sequence Sequence Validation The software will validate that the methods in the sequence are appropriate for the current instrument configuration If any problems are detected a message will be displayed and the sequence will not be run Correct any problems noted and restart the sequence After a successful sequence validation the method in the first line will be downloaded to the P ACE MDQ and the run will begin Observe the operation of the instrument as a guide to future operations and troubleshooting You may open the Direct Control window during data acquisition to view real time information on instrument status During the run the data will be displayed in real time in the instrument windows One window will show the Absorbance or Fluorescence signal and a second window will show the Current signal as those channels were selected in the method If you are using a PDA detector a third window will display a contour plot of the absorbance signal At the end of each run the method for the next run will be downloaded afresh before the new run begins This feature allows you to make changes to a method while a sequence is processing The version of the method that is current at download last saved is the version that will run You may also edit the Sequence Table during processing Existing lines
124. n the leading edge of the cluster has been detected Use Ctrl Z to zoom out There are now two Width values in the Integration Events Table One is global the other the new one is only in effect over the time range specified in the event Integration Off This integration is nearly done but there is still a problem at the end of the electropherogram At the end of the run is a baseline shift that is being recognized as a peak l Zoom into the area from about 34 minutes to the end of the run We can see that there is really nothing of interest beyond the peak at 35 2 minutes 2 To turn off integration select Integration Off from the Graphical Programming menu while still zoomed in 3 Click at about 35 8 minutes and at the end of the electropherogram then click Analyze Now The peaks in the specified area will disappear from the integration results and the Integration Off item will be added to the table Like the Width value we used to resolve the peak cluster the Integration Off function is only active over a specific period of time of the integration functions except for Shoulder Sensitivity can be used globally or locally For example you could use another Integration Off event from Start Time 10 to Stop Time 20 to eliminate the remaining small peaks For now the integration appears to be acceptable The Integration Events table should resemble the one below The Start and Stop times may differ slightly depending on where
125. nduced Fluorescence Detector p SSSSSSS T 7 Figure 2 10 LIF Optical System Photo multiplier Laser 1 Spherical Mirror Laser 2 Capillary Ball Lens Beam Splitter 50 50 Photo Multiplier Tube 1 3 9 7 9 Laser Filters 0 Emission Filters User s Guide PN A33115AB System Overview The Laser Modules 2 9 18 The Laser Modules The following section describes the optional 488 nm and 635 nm laser modules in terms of how they interface with the MDQ instrument and the LIF Detector WARNING During normal operation of the LIF Detector laser light is not accessible to the user To prevent potentially harmful laser light from being emitted from the end of the fiber cable an interlock mechanism turns off the laser if the laser fiber cable is disconnected from the interconnect module or if the cartridge cover is opened WARNING Always turn off the laser module and P ACE instrument before removing any of the LIF system module s 488 nm Laser Module The Beckman Coulter 488 nm laser is an air cooled Argon Ion Laser Two indicator lights are located on the front of the 488 nm Laser Module The green light flashes to indicate that the interlock circuit is complete and the laser is preparing to light in approximately 40 seconds Once the laser is operating the green light will stay on continuously until the l
126. need to insert a Stop Data event l Select the new Event box and choose Stop Data as the event type 2 Entera value of 6 00 minutes to force data collection to stop at the end of the separation step Our last step in this process will be a rinse with water from the water vial EI to the empty vial BI 3 Program this rinse for 1 minute at 20 psi Select At Time and use a value of 6 00 minutes It is a good practice to Autozero the detector some time after the run starts 1 Inthe new event box select Autozero 2 Select At Time and input a value of 1 00 When this item is entered into the Time Program spreadsheet it will automatically move to the correct time sequence At this point your spreadsheet should look like that in Figure 5 14 MDQ User s Guide PN A33115AB 57 Creating and Editing a Method Time Program E Instrument Setup 1511 my Initial Conditions UV Detector Initial Conditions G3 Time Program Figure 5 14 Instrument Setup Time Program The method can still be edited at this point Items in the Time Value Duration Inlet and Outlet columns can be edited by clicking on the existing value and typing in a new value Alternatively clicking on the name of any existing event will open the drop down menu of event types Select Current Event to re open the event dialog or select a new event to change the event type Saving the Method IMPORTANT It is vital that you Save your Method l Select File
127. nt out that a loss of data may occur if information stated within the paragraph is not adhered to or if procedures are executed incorrectly NOTE Paragraphs marked by NOTE contain supplemental or explanatory information concerning the current topic or procedural step 32 Karat 8 0 X A33115AB Safety Information Safely Symbols Safety Information This section provides safety information and instructions for the hardware and accessories of the system Safety Symbols The symbols displayed below and on the instrument are reminders that all safety instructions should be read and understood before attempts at installation operation maintenance or repair to this instrument are made When symbols are displayed in this manual pay particular attention to the safety information associated with the symbol WARNING When present this symbol indicates that a potential hazard to your personal safety exists if information stated within the WARNING paragraph is not adhered to or procedures are executed incorrectly CAUTION This icon accompanies text and or other symbols dealing with potential damage to equipment When present it indicates that there is a potential danger of equipment damage software program failure or that a loss of data may occur if information stated within the CAUTION paragraph is not adhered to or procedures are executed incorrectly HIGH VOLTAGE Paragraphs marked by this symbol indicate that
128. ntegration table Width User s Guide PN A33115AB l Zoom out to full size then zoom in on the first major peak around 9 5 minutes This peak is nearly 0 5 minutes wide at the base and it is significantly wider than the surrounding smaller peaks 2 Right click on the electropherogram and select Graphical Programming A new menu of integration parameters will appear 3 Select Width A message will appear in the Status bar at the bottom of the instrument window which states Click on the start of the Peak 4 Place the mouse pointer at a point near the left edge of the major peak along the baseline and click You will be prompted to click on the End of the peak After the Start and End of the peak have been entered the following Width dialog box will appear Your values may differ slightly from those shown depending on exactly where you clicked Start Time g 439 Minutes Add to Table Stop Time 3 943 Minutes Cancel Value 0 503251 Insert into Integration E vents table C Insert into Manual Integration Fixes table Analyze Now Figure 7 3 Determining Width of Peak There are several options available in this dialog Start and Stop times refer to the points where you clicked For the Width parameter the value is the difference in these two time points the width of the peak There is an option to add this parameter to the Integration Events table or to the Manual Integration Fixes table These t
129. nterest To zoom in on the baseline to view integration results in detail you can use the mouse to rubber box the display Click and hold anywhere in the image Drag the mouse to highlight the desired area When the mouse button is released the selected area will fill the window Use Ctrl Z to zoom out Examining the area between 3 0 and 3 4 minutes will demonstrate that even negative peaks valleys are being detected MDQ User s Guide PN A33115AB 73 Integration Optimizing Integration 7 3 Optimizing Integration 1 By selecting Window Tile Horizontally the electropherogram and the Integration events table can be displayed together Resize the windows as desired by clicking and dragging a window s edge with the mouse Click in the electropherogram window then use Ctr 1 Z to zoom out if necessary Click in the Value column of the Threshold event in the Integration Events Table Enter the number 100 oO BW M Analyze the data again This time there should be about 10 peaks visible Close examination will reveal that these are most likely real peaks and not baseline artifacts although some of them are quite small Examine the area between 14 and 15 minutes for example Threshold determines how high a peak must rise above the background noise before it is recognized as a peak by the integration software Increasing the value of Threshold removes background artifacts If taken to extremes it can also remove real d
130. nts The Capillary Cartridge The CE capillary is installed in a cartridge The cartridge design protects the capillary provides a path for liquid coolant simplifies installation into the instrument and aligns the detection window in the optics The components of the cartridge are shown in Figure 8 901012LAL Figure 2 4 The Capillary Cartridge 1 Coolant tubing with capillary inside 2 Double seal inlet 3 Detector Window and Aperture LIF 4 Detector Window and Aperture UV Detector and PDA Detectors MDQ User s Guide PN A33115AB 7 System Overview Syringe Pump The detection window is an area of the capillary where the polyimide coating has been removed to reveal the transparent fused silica This area of the capillary is placed in a part of the cartridge containing a plug that connects the window to the optical system One type of plug is used for UV and PDA detectors and a second type is used for LIF detectors The procedures for installing a capillary in a cartridge are covered in the Installation and Maintenance Manual In addition to the cartridge shown here special cartridges are used with the optional External Detector Adapter see the Installation and Maintenance Manual for information on these cartridges The capillary temperature is controlled with an inert liquid that circulates through the cartridge Temperature is controlled in a range from 10 C below ambient with a minimum of 15 to 60 Cool
131. nts the separation of a mixture of poly A oligonucleotides ranging from 40 to 60 bases in length The method contains an Integration Events table that will successfully integrate these peaks and a Peaks Groups table that will name the peaks with the number of bases they represent 3 Analyze the data 4 Right click on the electropherogram and select Annotations 5 Add Name to the list of annotations to display then click Next we must enter the base numbers Y and the migration times X into the method From the Method menu select Qualitative analysis to open the dialog shown below E Qualitative Analysis X Axis Units Migration Time x Scale Linear x Minimum Value 24 5 Maximum Value 30 Y Axis Units Bases Scale Linear Goodness of Fit 0 999998 Migration Time Time 30 0785 Minutes Bases 60 50 H 50 50 1 Fit type Cubic Reference Peak Time p Reference Window 26 28 30 Migration Time Print Figure 9 2 Quantitative Analysis dialog We can select the type of data we want for the X axis In this exercise we are using migration time but we might also use another parameter such as Mobility We can select either a linear or a logarithmic scale for this axis Minimum and Maximum refer to the X axis limits over which we will allow the interpretation of qualitative data This limits how far beyond the ends
132. od Time Program 56 In the next part of this exercise we will create a time program to go with the initial conditions previously entered The example method will do the following Perform a mini regeneration on the capillary Fill the capillary with buffer Inject test mix Separate the test mix Perform a post run rinse This method will use a vial tray setup similar to that used in the section on Direct Control The vial positions are specified in the table below Solution Left inlet buffer tray Right outlet buffer tray Test Mix C1 Regenerator Solution A D1 Water E1 Run Buffer A A1 A2 A1 Empty vial B1 Click on the Time Program tab to begin The tab opens with a blank spreadsheet Click in the Event box and use the arrow From the drop down menu select Rinse Click on the Trays button Select D1 on the Inlet left side and B1 on the Outlet right side Before clicking OK right click on one of the buffer positions you just selected A dialog box will open that allows you to identify the contents of that position Mo Dur Insert the appropriate comments from the table above and repeat for the other vials 7T When done click to return to the Rinse window For this step accept the default values for all other parameters 8 Click OK to return to the Time Program spreadsheet The key parameters have been entered into the table automatically This rinse is an
133. ojects D efault T emplate S equence Vial Sumr Sequence Summary 4 4 4 C 432Karat Projects D efault T emplate Sequence Vial Sumr Cancel 32 Karat 8 0 F 12 PN A33115AB
134. on dialog box is displayed 29 32 Karat Creating and Configuring an Instrument P ACE MDQ Instrument Configuration EPBD 36 vias E 0 Tray ___ Deptt Figure 3 12 Instrument Configuration dialog For information on setting the P ACE MDQ Instrument Configuration parameters refer to the 32 Karat Online Help 4 Click OK to exit and save the module configuration Configuration Options For information on configuration options please refer to Configuration Options on page 3 27 Manual Configuration of the PDA and LIF Detectors Manual configuration of the PDA and LIF detectors is performed by selecting the appropriate icons in place of the UV detector Only one internal detector is allowed per instrument 32 Karat 8 0 30 PN A33115AB Direct Control 4 Introduction Direct Control 4 1 Introduction The Direct Control screen is essentially the front panel of the instrument It allows you to select and change operating parameters with immediate execution Direct Control is useful in preparing for Operations Maintenance and Troubleshooting The Direct Control graphical interface is used to control the instrument by clicking on hot areas of the window These areas either activate the associated dialog box allowing modification of the instrument settings or activate the task directly Direct control displays an animation of current instrument
135. on and Maintenance Manual for instructions on building a new cartridge or installing a capillary Methanol HPLC grade 0 1N HCl in water Capillary Regenerator Solution A 1M Sodium Hydroxide PN 338424 Run Buffer A PN 338426 Distilled or deionized water HPLC grade 2 mL vials and red caps 36 position buffer trays Prepare one vial each of Methanol HCl Regenerator and Water Prepare two vials of Run Buffer A Prepare one empty vial Vials should be filled to the shoulder as indicated in the Installation and Maintenance Manual All vials including the empty vial must be capped 4 Place the vials in the buffer trays as indicated in Table 4 3 Table 4 3 Vial Loading Positions Solution Left Inlet buffer tray Right outlet buffer tray Methanol B1 0 1 N HCI C1 Regenerator Solution A D1 Water E1 Run Buffer A A1 A1 Empty Vial B1 5 Check that the capillary cartridge and buffer trays are properly installed See the Installation and Maintenance Manual for more information 6 Close the doors NOTE The graphic in Direct Control indicates the presence of the cartridge and the closed door You should be able to hear the coolant circulating User s Guide PN A33115AB 35 Direct Control Exercise Conditioning the Capillary 7 Click on the Pressure hot spot on the Direct Control screen The dialog in Figure 4 2 will open Pressure Settings Ea Direction
136. privileges Follow the wizard step by step Each user can be given access to specific areas of the software and to specific instruments Selection will depend on the functions of the individual and the degree of security required in the operation of your instrument systems For example regulated quality control laboratories D 1 Appendix D System Administration generally implement tighter controls over instrument and data access than do laboratories engaged in basic research The Instrument Wizard allows the administrator to assign users to specific instruments but not to assign specific privileges on those instruments The Project Wizard allows the administrator to create projects A project is a combination of users instruments and folders Projects are most valuable when instrument systems are used by multiple operators or groups who do not need access to one another s methods and data Creating a project allows the operations on the instrument to be compartmentalized The software is shipped with one project which is called Default This is a special project in that it cannot be protected It should not be used for methods and data that are not intended to be shared For more information on System Administration see the resources in the System Administrators Guide PN A34392 32 Karat 8 0 D 2 PN A33115AB Appendix E Vial Incrementing Appendix E E 1 Vial Incrementing Introduction User s Guide PN A33115AB
137. rces in Appendix A for more information on calibrating through the Sequence Table Advance Direction Enters the advance direction for the sequence When the Advance box is enabled for either or both inlet and outlet vials the software automatically fills down vial position values in the Sample Inject Inlet Oulet columns of the sequence table This automatic fill down eliminates the need of the user to manually input one vial position at a time in the sequence table If Row Major is selected as the Advance Direction the software automatically fills down vial positions incrementing them by rows in the same tray e g Al A2 A3 A4 A5 A6 B1 B2 B3 B4 for buffer tray configuration If Column Major is selected as the Advance Direction the software automatically fills down vial positions incrementing them by columns in the same tray e g Al C1 D1 El F1 A2 B2 C2 D2 for buffer tray configuration Do not confuse Advancing vial positions with vial incrementing operation in instrument runs Advancing vial positions here refers to simplifying sequence table planning and organizing These vial position values can be changed in the sequence table by user s discretion and they are not saved with any method They are saved in the sequence For more information about vial incrementing refer to Appendix E Sequence Wizard Calibration x Calibration ID Testm Calibration path CA32Karat Projects Defaul Data Calibr
138. re The dialog should now look like the one shown above in Figure 4 2 5 Click OK The trays will move to the designated positions and the rinse will begin automatically Observe the Direct Control screen during this process Perform additional rinse steps according to Table 4 4 32 Karat 8 0 36 PN A33115AB Direct Control Exercise Conditioning the Capillary It will only be necessary to change the parameters indicated Tray Selection s es cs os Es 5 5 ess 900000000000 ses caos Ds Ea Fa Ea F2 t2 Figure 4 3 Tray Selection Dialog Table 4 4 Rinse Step Parameters Solution Inlet L Position Outlet R Position Pressure Time Methanol B1 B1 25 1 Water B1 25 0 5 0 1 N HCI C1 B1 20 2 Water B1 20 0 5 Regenerator A D1 B1 20 2 Water E1 B1 20 0 5 Buffer A A1 B1 20 2 MDQ User s Guide PN A33115AB 37 Direct Control Exercise Conditioning the Capillary At the end of these steps the capillary has been regenerated and filled with run buffer The next step is to test the electrical conductivity of the capillary 8 Click on the Voltage hot spot to open this dialog box This option is used to configure a voltage separation LX OK Voltage Settings Voltage 30 kV Voltage max 30 0 kV Duration 2 min Current max 300 0 p Cancel Ramp time 05
139. rement inlet and outlet vials after every cycle of method Injection of sample from method Increment inlet and outlet vials after every cycle of method 4 Wait Increment inlet and outlet vials after every cycle of method 5 Separate Increment inlet and outlet vials after every cycle of method After setting up Method Time Program B the user generates the following sequence table Sequence Table B1 with only 4 repetitions of the method B on a single run line Runit Run Type Reps Sample InjectInlet Sample Inject Outlet Sample Inject Duration Unknown 4 methodB met Sea 3 WI s s s User s Guide PN A33115AB E 3 Appendix E Examples of Use 4 The results of instrument sequence run of Sequence Table 1 are as follows Table E 1 Sequence Table Run Results Actual Vials Run Repetition Event Actual Vials Used Inlet Used Outlet 1 1 Rinse 1 BI A1 BO A1 1 1 Rinse 2 1 BO A1 1 1 Inject SEA1 BO A1 1 1 Wait 1 1 1 1 BI C1 BO C1 1 2 Rinse 1 BI A2 BO A2 1 2 Rinse 2 BI B2 BO A2 1 2 Inject SI A2 BO A2 1 2 Wait 2 2 1 2 BI C2 BO C2 1 3 Rinse 1 BI A3 BO A3 1 3 Rinse 2 BI B3 BO A3 1 3 Inject SI A3 BO A3 1 3 Wait BI C3 BO C3 1 3 Separate BI C3 BO C3 1 4 Rinse 1 BI A4 BO A4 1 4 Rinse 2 BI B4 BO A4 1 4 Inject SI A4 BO A4 1 4 Wait 4 4 1 4
140. ried over from the first single run to the second and third single runs In the second example of run queue that includes sequence runs of Sequence Table the first second and third sequence runs are independent of each other Vial incrementing is not carried over from the first sequence run to the second and third sequence runs 32 Karat 8 0 PN A33115AB Appendix F E Sequence Vial Report Appendix F Sequence Vial Report Overview Sequence Vial Report is a pre defined sequence summary report that shows which vials are used by the instrument system for the method time program events of rinse separate and inject The Sequence Vial Report is a description of the pre run or post run of the sequence There are two forms of the report Sequence Vial Increment Preview and Sequence Vial Report Confirmed Both forms of the report can be viewed and printed Sequence Vial Increment Preview User s Guide Sequence Vial Increment Preview report is a preview of which vials the instrument system will utilize based on what methods and other information are provided in the sequence table This Preview report can be viewed automatically before executing any sequence runs by selecting Sequence Vials Preview from the Sequence menu Sequence Analysis Control Reports Ctri Shift F7 ES Process Ctrl Shift F11 Custom Report Properties An example of the Sequence Vial Increment Preview report follo
141. rom which the Sequence Vial Reports Confirmed originate The number of repeats or iterations e g 3 3 or 4 4 4 of the sequence table run lines under Sequence Records reveals the number of repetitions that have been successfully executed by the instrument system for those run lines 32 Karat 8 0 PN A33115AB Appendix F E Sequence Vial Report Confirmed For instance run line 4 from sequence run of Sequence Table A above has successfully generated a Sequence Vial Report Confirmed for all three repetitions View Sequence Reports 1 Xi Report Type Sequence Records Report Template Sequence Summary 2 C A32Karal amp ProjectsD efaul T emplate S equence Vial Sumrr Sequence Summary 3 3 C 32Karat Projects D efaul T emplate S equence Vial Summ Sequence Summary 4 4 4 C 32Karat Projects D efaul T emplate S equence Vial Sumrr View Cancel Help emen The user can highlight any of the listed Sequence Vial Reports Confirmed and click View to open it The following example is the Sequence Vial Report Confirmed for run line 4 of Sequence Table A above E Sequence Summary Report Sequence Table C 32Karat Projects D efault Sequence Sequence TableA seq Sequence Vial Report Confirmed Data Path C 32Karat Projects D efault D ata Public Method Path C 32Karat Projects D efault Method User Systern Report Time 05 09 2003 01 05 06 PM indicates that Vial Collision will occur Cycle
142. s dialog to move a selected vial to the end of the capillary Load Button Brings all trays forward for loading Home Button Returns all trays to their home position Optics Detector Control Hot Spot Opens a dialog appropriate for the configured detector Lamp Laser Status Hot Spot Opens a dialog allowing lamp or laser s to be toggled on off Autozero Button Adjusts the detector output to zero Power Controls Voltage Button Opens a dialog to initiate voltage separation Current Button Opens a dialog to initiate current separation Power Button Opens a dialog to initiate power separation Pressure Button Opens a dialog to initiate pressure separation also used for capillary rinsing Other Controls Inject Button Opens a dialog to initiate injection of sample into the capillary Stop Button Stops all instrument operations Capillary Information Hot Spot Opens a dialog to input capillary cartridge ID information used for display only User s Guide PN A33115AB 33 4 Direct Control Direct Control Window Table 4 2 Status Display Status Display in Direct Control Status Text Indicated current instrument function and state Time Remaining Text Graphic Time remaining for current instrument process Tray position Text Graphic Indicated the vial or well at which the capillary end is located Cartridge Status Graphic Indicates if a cartridge is
143. s exercise will deal with the second question How much is present in this sample which is a question for QUANTITATIVE analysis In quantitative analysis we run a series of runs with differing concentrations of a known substance or substances For each concentration of each component we determine a detector response By generating a graph of detector response versus concentration it is possible to determine the concentration of the components in an unknown The 32 Karat Software simplifies this task It contains tools for collecting the data from calibration runs generating appropriate curve fits and using the curve fit data to analyze unknowns This exercise will also introduce you to using a sequence for post run data reprocessing We will use an External Standard calibration in this method data from standards and unknowns are acquired in separate runs MDQ User s Guide PN A33115AB 81 Calibration Creating a Calibration 8 3 Creating a Calibration Included in the Data Samples directory are five files named CE Level 1 dat CE Level 2 dat CE Level 3 dat CE Level 4 dat and CE Level 5 dat These data files are the results of runs of the same two components at five different concentrations as indicated in the table below Component units mL Filename Alpha Beta CE Level 1 dat 1 0 4 0 CE Level 2 dat 2 0 4 5 CE Level 3 dat 3 0 5 0 CE Level 4 dat 40 5 5 CE Level 5 dat 5 0 6 0 The first step in the
144. s in Appendix A for information on this feature For this exercise setup the PDA Detector Initial Conditions to match Figure 5 3 MDQ User s Guide PN A33115AB 47 Creating and Editing a Method LIF Detector Initial Conditions 5 5 LIF Detector Initial Conditions lii Instrument Setup x SS Initial Conditions LIF Detector Initial Conditions Time Program Electropherogram channel 1 4 Electropherogram channel 2 4 blec Acquisition enabled Dynamic range 100 Dynamic range 100 m Filter settings Filter settings C High sensitivity C High sensitivity Normal Normal C High resolution C High resolution Peak width pts 1625 Peak width pts 1625 Signal Signal Direct Indirect Direct Indirect r Laser filter description information only r Laser filter description information only Excitation wavelength 488 nm Excitation wavelength 635 nm Emission wavelength 520 nm Emission wavelength 675 nm m Data rate Relay 1 Relay 2 orf Both channels 4 Hz C On C On Figure 5 4 Instrument Setup dialog LIF Detector Initial Conditions tab Electropherogram Channel 1 and 2 The LIF Detector is available in one and two channel versions The programming of these channels is identical In this exercise we will use only one channel If your system is configured with non Beckman Coulter
145. s the magnitude of the pressure vacuum or voltage and how long it will be applied Higher values will inject more sample Tray Positions Functions exactly as described in the Separation event 32 Karat 8 0 PN A33115AB Creating and Editing a Method 5 Time Program Sequence Table When a method is used in a Sequence Table certain parameters in the inject event can be overridden by values input in the Sequence This parameter determines whether the method Inject event or the Sequence Table has priority Multiple injection events are allowed If multiple injections are used only one injection event may be overridden in the sequence table or incremented Autozero This event causes the detector output to be reset to zero It may be timed or untimed At Time 000 min Cancel Help Figure 5 11 Autozero Dialog Stop Data Data collection starts at time 0 00 Data collection will continue until the end of the method unless a STOP DATA event is encountered This event can be used to avoid data collection during steps such as post run capillary cleaning STOP DATA is always a timed event Time 0 01 min Cancel Help Figure 5 12 Stop Data dialog End End is an optional event The method will not continue beyond an end event It is always a timed event Time 001 min Figure 5 13 End Dialog MDQ User s Guide PN A33115AB 55 Creating and Editing a Meth
146. t D gt because date and time are unknown until the sequence is run 7 When you finished editing the table select File Sequence Save As from the menu bar Use a Sequence name that has significance to you for example TestSequence 052006 32 Karat 8 0 68 PN A33115AB Running the System Running the Sequence 6 4 Running the Sequence User s Guide PN A33115AB You will now run the sequence The instrument must be prepared for operation as described under Single Run Mode earlier in this section Verify through Direct Control that the instrument UV lamp or laser is turned on The test sample and buffer vials must be loaded and installed You must be online before proceeding l Click the double green arrow button from the Instrument window toolbar IMPORTANT This icon will not be available if you are in Offline mode The dialog shown here will be displayed Run Sequence Test Sequence 5 16 2000 seq I Figure 6 12 Run Sequence dialog 2 Clickon the Open icon and select the sequence you just created The sequence name will appear in the text box Run Range allows you to run only part of the Sequence 3 For this exercise select All Printing allows the system to automatically print reports when they are ready 4 Ifyou do not have a printer connected leave this box unchecked When complete the dialog should look like the one shown in Figure 6 12 but with your sequence name i
147. t this point we have only used one of the calibration samples to define our peaks We will use all five calibrators to generate the standard curves for Alpha and Beta 32 Karat 8 0 PN A33115AB Calibration Creating a Calibration Generating the Calibration Curve To generate the curves we will reprocess the data through a Sequence In an earlier exercise we used a Sequence to perform multiple runs Sequences can also be used to do batch reprocessing of data 1 2 Select File Sequence Sequence Wizard to open the sequence wizard Click on the File icon and select CE Calibrate met the file we have been editing For Data File Type select From existing data files Click Next The next dialog allows us to select multiple data files Click on the File icon This will open a dialog that allows the opening of multiple files at one time Navigate to the Data Samples folder Locate the file CE Level 1 dat Double click on the file name When you double click the file name is added to the list at the bottom of the dialog Repeat for CE Level 2 CE Level 3 CE Level 4 and CE Level 5 so that all five files appear in the list 9 Click Open when done 10 Verify that the five files are listed in the window then click Finish This will open a Sequence table with the method and the five data files listed 11 Scroll right to the Filename column The five files should be listed in order 12 Scroll back left to the Level
148. te 49 Relay Tant 2 cna een he ba oscar ep m dose ute Petite oss 49 POG pof decet d cct 49 Separate Dialog 51 Rins eDial g ed 0600 n ea aA voee cen dalli sands 53 54 cay Pips 55 Stop Dalat 4 22 aet ie bans 55 0 Cr CPC 59 Saving the Method RR 58 iv 32 Karat 8 0 PN A33115AB Table of Contents Section 6 Running the System Single Run and Sequence RUNS 59 Materials needed 59 DIEING UIT oan rs ode ofer nc BLA ter iof peu tus 61 Programming Sequence 63 Running the Sequence 69 Sequence Validation AE ss 804 ere kenen e hae ln le al 70 Section 7 Integration DeSCriptON Sutra d ci A EES ithe E 71 integrating Dald c ese rete ey ee sot ped 72 Optimizing Integration 74 Integration Parameters act ar e teta o e eo I GER De ERN o OLIO e eren 74 Width METRUM 75 canine mane PES 76 Integration Results an
149. tem in the sequence run This report is identical to the Sequence Vial Increment Preview report except the following 1 Filenames for the sequence run are now included in the Filename column and 2 Only the vial combinations that have been successfully used in the course of the sequence run by the instrument system are shown Sequence Table C 32Karat Projects D efault Sequence Se quence TableA seq Sequence Vial Report Confirmed Data Path C 32Karat Projects DefaulhData Method Path C 32Karat Projects D efault Method User System Report Time 05 08 2003 09 17 57 PM indicates that Vial Collision will occur Cycle Method Filename Rep Inject Inject Inject Other Other Rinse Rinse Separate Separate In Out Time Inject In Inject Out Vials In Vials Out Vials In Vials Out MDQ User s Guide PN A33115AB F 3 Appendix F Sequence Vial Report Confirmed To generate the Sequence Vial Report Confirmed the user must change the Run Type of any set of sequence table run lines to be executed from Unknown to Summary The following steps may be helpful l Begin by clicking on the blue arrow in the Run Type box of an initial sequence table run line where the sequence run will start it does not have to be run number 1 The Sample Run Type s dialog window will open 2 Select Begin Summary as the run type IMPORTANT Do not select Vial Summary as the run type This run type is not used to generate the Sequence Vial Report Conf
150. that have not been started may be edited or deleted and additional lines may be added to the table Lines that have already completed or are currently executing can not be edited NOTE When a sequence is built using a method that has auto incrementing enabled edits that change the auto generated positions are not allowed If allow override is not selected relevant sequence table entries are not editable When the sequence is complete you will have acquired data from four runs The two replicate runs from line two will have file names to which rep 1 and rep 2 have been added It will be helpful to experiment with the Sequence Table before proceeding to the next section Make changes process the sequence and observe the effects You may wish to combine these experiments with the writing of new methods A series of different methods can be entered into the Sequence Table When processed the results will demonstrate the effects of changes in parameters For example you might create a series of methods at different separation temperatures e g 20 22 24 26 28 and 30 C to examine the effect of that parameter on your separation Another series of experiments might examine the effects of changing the volume injected If your method allows override this can be done with a single method by editing the Sample Inject Duration column in the Sequence Table 32 Karat 8 0 70 PN A33115AB Integration Description Integration
151. thod 2 Click on the Open and navigate to the method file created in the last section 3 Under Data File Type select For Acquisition Amount Values will not be used in this exercise 4 Click Next User s Guide PN A33115AB 63 Running the System Programming a Sequence Sequence Wizard Unknowns Test Mix C N32Karat amp ProjectsND efault D ata ws Figure 6 4 Sequence Wizard Unknowns dialog This screen is used to create the Sample ID and Data File ID 5 Type a text string in the Sample ID field The blue arrow to the right of the text box opens the following menu Figure 6 5 Sample ID text options menu Selecting an item from this menu will cause a symbol to be inserted into the Sample ID filed In the example shown Line Number was selected This parameter will be automatically incorporated into the Sample ID when the sequence is run You may select any combination of these items Data Path specifies the directory where the data files will be stored 6 Click Open and select Data Path then navigate to the desired folder 32 Karat 8 0 64 PN A33115AB Running the System 6 Programming a Sequence 7 Typea text string into the Data File field The blue arrow to the right of the text box opens this menu Line Number Increment Number Sample ID User Name Method Name Instrument Name Date and Time Open File Figure 6 6 Data File Text options menu As described above for the Sample ID
152. tion Appendix F E Sequence Vial Report Confirmed Summary tpl Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability Summary Run End Summary Select Sequence Report Template C Template Calibration brp Duplicate brp overniteseqrep brp Vial Summary tpl Sequence Vial Summary CE Sequence Report Template brp tpl v Sequence Vial Summary CE brp rs 6 The sequence table should reveal a run type of Summary Begin for the initial sequence run line where the sequence run will start Sequence Sequence Table seq unt Status RunType Level 10 O Unknown F 5 Appendix F Sequence Vial Report Confirmed 7 Forsubsequent sequence run lines that are to be executed middle of sequence run repeat step 1 on those lines but select Summary Run as the run type There is no need to open any report template 8 Click OK to return to sequence table Sequence Sequence TableA seq Summary Begin O Unknown h 0 0 Unknown Unknown Sample Run Type s Clear All Calibration Run Type Parameters 1 Calibration at Level Print Calibration Report JAverage Replicates Clear Replicates Begin Loop End Loop Shutdown parameters necessary Print Additional Repor
153. tor or data system through an analog output The signal may be plotted graphically in the form of an electropherogram and analyzed The second method is to induce the samples to fluorescence and measure the emitted light This is done by Laser Induced Fluorescence LIF Substances in the capillary which fluoresce at the laser wavelength are detected The LIF Detector measures and records this fluorescence which appears as a peak on the computer screen or printed electropherogram The P ACE MDQ instrument may be used to separate many different kinds of samples including peptides proteins nucleic acids ions enantiomers and pharmaceuticals CE has very low sample requirements relative to other analytical techniques 5 to 30 microliters with the actual injection volume being typically between 5 and 50 nanoliters It provides a complimentary alternative to other separations techniques such as chromatography 1 3 Modes of Electrophoresis Capillary Zone Electrophoresis CZE User s Guide PN A33115AB When voltage is applied to an uncoated fused silica capillary tube filled with a uniform electrolyte solution separation of charged species occurs due to differential migration in the electrical field This process in which charged particles in solution migrate toward an electrode with opposite charge is called electrophoresis If the inner wall of the capillary is also charged the fluid in the capillary will begin to flo
154. ts Begin System Suitability System Suitability Standard End System Suitability Begin Summary Summary Run End Summary Vial Summary MAF Cheek Standard 9 Sequence table should reveal Summary Run as the run type for the subsequent sequence run lines middle of sequence run lii Sequence Sequence TableA seq Summary Begin Summary Run 10 For the last sequence run line to be executed end of sequence run repeat step 1 but select End Summary as the run type There is no need to open any report template 32 Karat 8 0 F 6 PN A33115AB Appendix F E Sequence Vial Report Confirmed 11 Click OK to return to sequence table Sample Run Type s Clear All Calibration Run Type Parameters Clear Calibration at Level Print Calibration Report Average Replicates Clear Replicates Begin Loop End Loop Shutdown No parameters necessary Print Additional Reports Begin System Suitability System Suitability Standard End System Suitability Begin Summary Summary Run End Summary Vial Summary QC Check Standard 12 Sequence table should reveal Summary End as the run type for the last sequence run line to be executed end of sequence run lii Sequence Sequence TableA seq For users who want to generate the Sequence Vial Report Confirmed
155. uld look like Figure 8 4 Sequence CE Calibrate seq Bunt Filename SampeAm ISTDAmt _ celveida s CECaiaeme __ celevel2da 1 1 o CE Calibrate met ce level dat 1 1 o CE Calibrate met __ celevl4da 1 1 CECalbrateme __ celvel5da 1 imi Sequence CE Calibrate seq Runt RunType Level Conc Override Reps _ pu Calibration 2 2 S9 Calibration 8 8 8 0 3 d Ese 4 Calibration Figure 8 4 Sequence Table 32 Karat 8 0 PN A33115AB Calibration 8 Creating a Calibration We are now ready to Process the sequence and generate the calibration Select Process from the Sequence menu The dialog in Figure 8 5 will appear Run range must be All Processing mode must be set to Reintegrate Click Start The processing will require a few seconds to complete As each data file is analyzed the status column in the Sequence table will change to Complete pe Process Sequence x Sequence information Sequence name Jrojects Default Sequences CE Calibrate seq al Cancel n Selection C Range gt Mode Tower Processing mode Rentesse Bracketing None
156. untimed step so there is no value in the Time window 1 In the blank line at the bottom of the table click in Event column again and select another rinse event This time program a rinse with water from position BI E1 to position 2 Change the rinse time to 0 5 minutes This event will be added to the table when is clicked in the dialog box 3 Click in Event column again and add a step to fill the capillary with buffer rinse from position BE A1 to 32 Karat 8 0 PN A33115AB Creating and Editing a Method Time Program 4 This time use 25 psi for 1 minute Click when done 5 Click in the Event column in the bottom row again 6 Select Inject as the event type 7T Select the Trays option and program the injection from position to 1 8 Click OK 9 Input 0 5 psi as the pressure and 4 0 seconds as the time 10 Click OK At this point in the method we are ready to separate the components in the test mix Select a new Event box and choose Separate as the event type Use the Trays button to select positions BI A2 and 1 Click OK Select a Voltage separation Set the voltage to 30 kV ramp time to 0 2 minutes and duration to 6 minutes This will be a timed step so the At time button must be checked Set the time to 0 00 minutes CO iU Mt Ioco B4 db op Click to put this event into the table We will be using a final rinse in this method so we
157. w Main Components 3 The Sample Handling System 5 The Capillary Cartridge 7 UMD iras 8 High Voltage Power Supply 8 LED Indicators 46 om opt oes deca A Ete Tu a a e a EO 9 Cartridge and Tray Cover Interlocks 10 Opening the tray cover 10 Opening the cartridge cover 10 UV Detector ODDS vost pice vm bite Nm detinent ots soror ofr 11 The Photo Diode Array PDA Detector 13 Laser Induced Fluorescence Detector 15 The klaser Modulos 2 66 tss ura chaines aca t i e eee ue ka steer cures 18 488 nm Laser 18 635 nm Laser Module 19 Using Other Lasers with the LIF 20 Section 3 32 Karat Starting the SoftWare isi NEN UU 21 TOOL Bal cr em qe eco uera d FO eoru OR C 22 MG 254 citi d ur enda dun bo temi e Scena oia utr d ac rd 22 Creating and Configuring an Instrument
158. w toward the electrode that has the 1 Introduction to CE Common Modes of Electrophoresis same charge as the capillary wall This bulk movement of fluid is termed electroosmotic flow or EOE Because EOF is usually of higher magnitude than electrophoresis analytes of both positive and negative charge will ultimately be carried in the same direction although at different rates Electrophoresis will separate like charged but otherwise distinct analytes from one another In this manner both positive and negative analyte molecules can be detected as they are swept past the detector The direction of EOF can be changed by reversing the charge on the electrodes or by changing the charge on the wall of the capillary by chemical means The amount of time required for sample molecules to migrate to the detector depends on the length of the capillary the electrophoretic mobilities of the particular sample molecules the specific electrolyte used the magnitude of the and the applied voltage Molecules having different electrophoretic mobilities will be detected at different times Other factors to be considered are the size shape and charges of the particles the electrolyte concentration the pH of the separation buffer and the dimensions of the capillary Micellar Electrokinetic Capillary Chromatography MEKC In free zone capillary electrophoresis neutral molecules travel as a single band To separate these molecules micellar additives
159. wo choices differ in one critical way Items added into the Integration Events table become part of the method they will affect 15 Integration Optimizing Integration any data file that is integrated with that method The Manual Integration Fixes table becomes part of the data file items here will affect the integration of only the single data file to which they are attached In this exercise we will work only with the Integration Events table Manual fixes are discussed in the resources in Appendix A The Add to Table option will add the event into the Integration Events table Analyze Now will add the event to the table but it will also cause the integration to be redone with the new parameter in place For now click Add to Table The new Width item will now be visible in the Integration Events table This item will only be in effect for the interval between the Start Time and Stop Time We would like this event to be in effect for the entire data set To have a parameter active over the entire run set both Start Time and Stop time 0 Change the Start and Stop times of the new width value to 0 For purposes of consistency with this manual change the Value of the new Width event to 0 5 your value should already be very close to this point Now we have a problem There are two Width statements with different values both of which are declared for the entire run We could delete the old Width statement but the software offers a
160. ws Sequence ial Increment Preview CE Irp Sequence Table C 32Karat Projects D efault Sequence Se quence TableA seq Sequence Vial Report Projected Data Path C G2Karat Projects D efaul D ata Method Path C 32Karat Projects D efault Method User System Report Time 05 08 2003 07 25 03 PM indicates that Vial Collision will occur Filename Rinse Rinse Separate Separate Vials In Vials Out Vials In Vials Out 1 methodA met 1of4 Bo A1 10 0 SEAT BO A1 BIA BO A BO C1 BEE1 BO A BI D1 BO D1 BI 51 BO B1 2 methodA met 20f4 Bo 41 10 0 5 42 1 BEAT BO A BI C2 BO C2 BIE2 BO 41 BI D1 BO D1 3 methodA met 3of4 BIAS 10 0 A2 SM BI C3 BO C3 UM BI D2 02 methodA met 4of4 Blas 10 0 SM BI C4 BO C4 E E BI D2 BO D2 1 Appendix F Sequence Vial Increment Preview F 2 At the top right hand corner or the report the report is labeled as Sequence Vial Report Projected to indicate that this report is Sequence Vial Increment Preview The Sequence Vial Increment Preview report reveals the cycles Cycle column of each method to be run as well as the repetitions Rep column from the sequence table to be executed in the run The Filename column is left blank by default as this report is only a preview of a sequence run The Inject In Out columns contain the same inlet outlet user inputted vial position
161. xecuted when the sequence is run The Run Range dialog of the Sequence Run command gives the user the choice to run some sequence table run lines x r Sequence information Sequence name Jarat Projects Default Sequence Sequence Table AB seq zm Cancel r Run range m Mode Help C All Tower N Selection Processing made Normal Range 24 1 8 Bracketing None r Printing Review Print method reports after each r Begin run 3 Immediately In the above run range of run lines 5 7 2 4 1 8 from Sequence Table the sequence run of Sequence Table AB will start on run line 5 continue on to 7 go back to 2 and continue on to 4 go back to 1 and finally end on run line 8 32 Karat 8 0 E 8 PN A33115AB Appendix E E Examples of Use The results of this instrument sequence run Range of Sequence Table AB are shown below The results are obtained sequentially in the order that are given here Again for simplicity only the separation event is considered Method Actual Vials Actual Vials Cycle Run Repetition Used Event Used Inlet Used Outlet Used 5 1 Separate BI D1 BO D1 1 6 1 B Separate BI C1 BO C1 1 7 1 Separate BI D1 BO D1 2 2 1 B Separate BI C2 BO C2 2 3 1 A Separate BI D2 BO D2 3 4 1 B Separate BI C3 BO C3 3 1 1 A Separate BI D2 BO D2 4 8 1 B Separ
162. y harmful WARNING Observe all precautionary information printed on the original solution containers e Operate the system in the appropriate environment Take all necessary precautions when using pathology or toxic materials to prevent the generation of aerosols e Observe all applicable precautionary procedures when using flammable solvents in or near the instrument Wear appropriate laboratory attire for example safety glasses gloves lab coat and breathing apparatus when working with hazardous materials e Dispose of all waste solutions in a proper manner Xii 32 Karat 8 0 PN A33115AB Safety Information Safely Symbols Electrical Safety To reduce risk of electrical shock all devices employ a three wire electrical cable and plug to connect the equipment to earth ground Ensure that the wall outlet receptacle is properly wired and earth grounded DO NOT use a three to two wire plug adapter DO NOT use a two wire extension cord or a two wire multiple outlet power strip Disconnect power to the system before performing maintenance DO NOT remove any panels Panels should be removed only by qualified service personnel WARNING A high voltage power supply is used with this instrument Safety interlocks disable high voltage output while the capillary access cover is open and remove the risk of shock while performing routine instrument functions However removal of any panel may expos
163. you clicked E Integration E vents UV 200nm iof x 78 Width Threshold Width A Threshold Width Integration Off Figure 7 6 Integration Events table 4 Delete lines one and two since they are unused Leaving them in and unchecked does no harm 5 Save the method or all previous efforts will be lost 6 Select File Method Save As T Savethe method as Integration Exercise met NOTE You may see a warning that the Separation Time of the method is 0 0 Select Yes to continue with the save This message appears because in this exercise nothing was added to the Instrument Setup window of this method and this omission has been detected by the Auto Validation function of the software In actual practice Integration Events would be added to the method used to acquire the data 32 Karat 8 0 PN A33115AB Integration Integration Results and Reports 1 4 Integration Results and Reports The results of your integration efforts go beyond some labels on the electropherogram These results may be used to generate reports and to identify and quantitate unknowns These topics are covered in later sections of this manual For now use a pre defined report to capture the current results From the menu bar select Reports View A report will appear on the screen showing the Integration results in tabular form If a hard copy of this report is desired and if a printer connect

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P/ACE™ MDQ User`s Guide