FractionLynx Software

Transcription

1 FractionLynx Software User s Guide 34 Maple Street Milford, MA , Revision A

2 NOTICE The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, the use of this document WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER. Waters and XTerra are registered trademarks, and FractionLynx, MassLynx, OpenLynx, and ZQ are trademarks of Waters Corporation. Microsoft, Windows, and Windows NT are registered trademarks of Microsoft Corporation. All other trademarks or registered trademarks are the sole property of their respective owners.

3 Table of Contents Preface Chapter 1 Introduction UV-Directed System Test Instrument Setup Required Materials Setting Up the Chromatography Test MS-Based Chromatography Test Procedure Accessing Reports Using the FractionLynx Browser Creating Report Files Accessing the FractionLynx Browser Setting Report Scheme Parameters Chapter 2 Installing FractionLynx Software as an Option Installing FractionLynx Software Chapter 3 Using the FractionLynx Editor Accessing the FractionLynx Editor Enabling Fraction Collection Resetting Communications Exiting the FractionLynx Editor Table of Contents 3

4 3.5 Selecting Fraction Collection Parameters Fraction File Editor General Page Timing Page Volume Page Positive and Negative Pages Analog Page PDA Page UV Page Timed Events Page Mixed (Boolean) Triggers Editor Fraction Collection Setup Instrument Setup Collector Configuration Gilson Comms Dialog Box Gilson Positions Bed Layout Plate Generator Manual Control Resetting Beds Chapter 4 Collection Modes Definition of Terms Overview of Each Mode Table of Contents 4

5 4.3 Fraction Collector Tube Numbering Schemes Single Plate Per Fraction Collector Multiple Plates Per Fraction Collector Multiple Fraction Collectors Method of Operation for Each Mode Sequential Collection Enable User Starts One for One Collection One for One Collection Overrides the FractionLynx Parameters Sample Plate Size Versus Fraction Collector Rack Size Sample Plate Numbering Versus Fraction Collector Rack Numbering Logging Multiple Batches Sample Running Order Multiple Fractions Per Tube Reserved Tubes OpenAccess Implementation Chapter 5 Running FractionLynx Samples Setting Up Projects Root Directory Acqudb Subdirectory Data Subdirectory Sampledb Subdirectory Table of Contents 5

6 5.2 Sample List Starting a Run from the Sample List Displaying Fractions in MassLynx Chromatograms Real-Time Display of Fractions in Chromatogram Chapter 6 FractionLynx Browser Introduction FractionLynx Browser FractionLynx Browser Toolbar Browser Files View Options Spectrum Page Chromatogram Page Default Plate Page Colors Page Copy Control Page Column Pages Other Display Options Other Menu Options File Menu View Menu Window Menu Sample Plate Pane Fraction Plate Pane Sample Description Pane Table of Contents 6

7 6.9 Fraction Collection Results Pane Fraction Trigger and Fraction Tube Export Options Results Table Pane Spectrum Pane Fraction Tube Spectra Pane Chromatogram Pane Chromatogram Fraction Display Elemental Composition / Library Search Pane Report Schemes Report Scheme Settings Print Control Page Report Column Selection Pages Sample Summary Page MS Peaks Results Summary Page Report Header Page Sample Header Page Chromatogram Report Page Fraction Tube Summary Page Fraction Trigger Summary Page Decimal Places Page Producing Tab-Delimited Files Chapter 7 FractionLynx with OpenLynx Setting Up the Walk-up Page Table of Contents 7

8 Chapter 8 Troubleshooting FractionLynx Uncollected Samples Minimum Intensity Threshold (MIT) Raw Data Files Plumbing Diagrams UV-Directed System Test Instrument Setup Delay Timing Dye Test MS-Directed System Test Measuring Flow to the Mass Spectrometer MS-Directed System Dye Test Troubleshooting Tips OpenLynx Troubleshooting Appendix A Hardware Setup A.1 Equinox Setup for Windows XP Platform Systems A.2 Equinox Setup for Windows NT Platform Systems A.2.1 Setting Up the Equinox Card A.2.2 Equinox Driver A.3 Post-Installation System Performance Enhancements A.4 Connecting the Waters Fraction Collector II or lll A.5 Connecting the Gilson 215 Collector A.6 Connecting the Gilson 204 Fraction Collector A.7 Configuring System Modules to MassLynx Table of Contents 8

9 A.8 Inlet Configuration Dialog Box A.9 Scan Command A.10 BGM and C/FO Configuration A Sample Manager Configuration Appendix B Peak Detection B.1 Analytical B.1.1 Mass Detection B.1.2 Analog Detection B.1.3 PDA Detection B.2 Preparative B.2.1 Mass Detection B.2.2 Analog Detection B.2.3 PDA Detection Index Table of Contents 9

10 List of Figures 1-1 Load Sample List Format Dialog Box Customize Field Display Dialog Box Starting Data Acquisition in MassLynx Enable Fraction Collection Dialog Boxes General and Timing Pages Volume and Analog Pages PDA and UV Pages UV-Directed Dye Test Chromatograms Starting Data Acquisition in MassLynx Enable Fraction Collection Dialog Boxes General Page Timing and Volume Pages Positive and Negative Pages MS-Directed Dye Test Chromatograms FractionLynx Browser Window Setting Print Controls in the Edit Report Scheme Settings Dialog Box MassLynx V4.0 Installation Wizard Modifying MassLynx 4.0 Program Accepting the License Agreement Selecting the FractionLynx Software Option Installing OpenLynx Options Starting the Program Installation Selecting the FractionLynx Key Disk Path Selecting the OpenLynx Key Disk Path Program Installation Complete List of Figures 10

11 3-1 FractionLynx Editor Enable Fraction Collection Dialog Box Reset Communications Command Collection Modes Dialog Box Fraction File Editor, General Page Fraction File Editor, Timing Page Fraction File Editor, Volume Page Fraction File Editor, Positive and Negative Adducts Pages Fraction File Editor, Analog Page Fraction File Editor, PDA Page Fraction File Editor, UV Page Fraction File Editor, Timed Events Page Illustrated Example of Mass A AND WaveLength B Illustrated Example of Mass A OR Mass B Illustrated Example of Mass A NOT Mass B Illustrated Example of Mass A XOR (Exclusive OR) Mass B MassLynx Boolean Trigger, Boolean Column Dialog Box FractionLynx Mixed Triggers Editor Dialog Box FractionLynx Mixed Triggers Editor Dialog Box, Expanded Selecting from the Trigger Source List Adding an Expression Selecting an Operator Selecting Wavelength A Adding the Wavelength to the Expression Saving and Closing the FractionLynx Mixed Triggers Dialog Box Sample List Page, Browse Selecting and Saving a Mixed Trigger Mixed Trigger Entered in the Boolean Logic Column Entering Components into the Boolean Expression Validation Error Message Mix Displayed on the Fraction Chromatogram List of Figures 11

12 3-32 Mix Displayed in the Fract.txt Screen Selecting a Trigger from the Sample List Delete Mixed Trigger Warning Box Expression Deleted from the Mixed Trigger Editor Viewing Trigger in the Mixed Trigger Editor and Mixed Triggers Dialog Box Boolean Logic INI File Deleting a Trigger from the Boolean Logic Section and Boolean Logic File Section Saving Changes Display of INI with Trigger Removed Triggers Removed from Mixed Triggers Editor and Mixed Triggers Dialog Boxes Collector Configuration Page (Waters Fraction Collector III) Selector Valve Page Collector Configuration (Waters 2757) Typical Gilson Fraction Collector Setups Gilson Comms Dialog Box Positions Dialog Box Bed Layout Dialog Box, Select/Create Bed Layout Page Bed Layout Dialog Box, Modify Bed Layout Tab Plate Description Dialog Box Rack Generator Window Example of Rotated Rack Scale Rack Dialog Box Manual Control Dialog Box Plate Generator Types Tube Plate Layout Tube Plate Layout Tube Plate Layout Plate Layouts for Multiple Fraction Collectors List of Figures 12

13 4-6 Plate Layout for Four Fraction Collectors Collection Modes Dialog Box Example of How Samples and Batches Collect Unable to Start Queue Warning Message Box Sample Plate Size Equals Collector Rack Size Unequal Sample and Collection Racks Sample Plates Larger than Collection Plates Mapped Fraction Collector Positions for Each Well Location Four Injection Plates Running One to One MassLynx Not Enough Vessels Error Message Box Mapping for Single Shot Samples Plate Defined with Horizontal First Priority Sequential, One to One, and MultiFraction Mapping Mini-Racks Modified Bed Layout for Mini-Racks or Fraction Blocks GeneVac Rack Sample List Sample List Example: Fewer Mass Triggers than Masses Start Sample List Run Dialog Box Fraction Display Parameters Dialog Box FractionLynx Browser Print Control Dialog Box Set Margins Dialog Box View Options Dialog Box, Spectrum Page View Options Dialog Box, Chromatogram Page View Options Dialog Box, Default Plate Page View Options Dialog Box, Colors Page View Options Dialog Box, Copy Control Page View Options Dialog Box, List Columns Page View Options Dialog Box, Fraction Display Page List of Figures 13

14 6-11 Program Not Found Dialog Box Failed Samples Options Dialog Box View Menu Window Menu Sample Plate Pane Fraction Plate Pane Sample Description Pane Fraction Collection Results Pane View by Trigger: Selecting NO For Export Trigger View by Tubes: All Fraction Tubes Set to NO for Trigger View by Tubes: Changing One Tube to YES View by Trigger: Corresponding Fraction Reverting to YES Results Table Pane Spectrum Pane Fraction Tube Spectra Pane Chromatogram Pane with Fractions Displayed on all Chromatograms Chromatogram Pane with Fractions Not Displayed on all Chromatograms Chromatogram Pane with View Fractions by Tube Edit Report Scheme Settings Dialog Box, Print Control Page Edit Report Scheme Settings Dialog Box, Sample Summary Page Edit Report Scheme Settings Dialog Box, Decimal Places Page Setting Up the Walk-up Page Adding and Deleting Input Fields OpenLynx Login Window Sample Chromatogram Using Dye Display View Normalized Data Set to Lowest Point List of Figures 14

15 8-4 Fraction File Editor MIT Example Set to 3 Million Example of MIT Set to 30 Million Absorbance Value Data Points Displayed List of Data Points Raw Data File Accessed via Explorer Typical Inject-to-Collect, Mass-Directed AutoPurification System Typical Inject-Collect Mass-Directed AutoPurification System Fraction File Editor, General Tab A-1 Found New Hardware Wizard A-2 System Setting Changes Dialog Box A-3 Installation Completed Page A-4 Start Up Screen A-5 Selecting the Driver Option A-6 Selecting the Appropriate Driver A-7 Install It Now Screen A-8 Help Screen A-9 Network Dialog Box, Adapters Tab A-10 Select Network Adapter Dialog Box A-11 Insert Disk Dialog Box A-12 Select OEM Option Dialog Box A-13 Setup Message Dialog Box A-14 Equinox SST Configuration Dialog Box A-15 EquiView Plus Datascope Path Dialog Box A-16 Network Adapters Tab Showing the Equinox Board Installed A-17 Inlet Editor, Injection Configuration Tab A-18 Inlet Editor, Dilutor Configuration Tab A-19 Inlet Editor, Wash Parameters Tab A-20 Inlet Editor, Sampler Configuration Tab A-21 Inlet Editor Window, Status Tab List of Figures 15

16 A-22 Inlet Configuration Dialog Box A-23 Contact Closure Tab A-24 Triggers Tab A-25 GPIB Communication Tab A-26 Inlet Editor, View Menu A-27 Modify Instrument Method Dialog Box A-28 Autopurification System Configuration Dialog Box A-29 Waters Instruments Dialog Box B-1 Analytical Peak Detection B-2 Analytical Peak Termination B-3 Preparative Peak Detection B-4 Preparative Peak Termination List of Figures 16

17 List of Tables 1-1 UV Test Sample Dye Compounds MS Test Sample Dye Compounds FractionLynx Browser Panes Print Reports Report Formats FractionLynx Mixed Triggers Editor Properties Bed Scheme Defined as Two Columns of Five Rows Acqu Database Control Files Sample List Properties Sample List with Fewer Mass Triggers than Masses Sample Summary MS Peak Results and Fraction Tube Summary System Pressure Readings A-1 Equinox Ports List of Tables 17

18 Preface The FractionLynx Software User s Guide is intended for a wide variety of users whose familiarity with computers and software ranges from novice to expert. This guide describes the basics of how to install, configure, and use FractionLynx software. See the MassLynx User Guide for information on using MassLynx software. Organization This guide contains the following: Chapter 1 describes how to make an injection, collect a sample, view the browser, and print basic reports. Chapter 2 describes the installation of FractionLynx software. Chapter 3 describes how to access the FractionLynx Editor, and the configuration of the screens associated with the FractionLynx parameters. Chapter 4 describes the collection modes available with FractionLynx software. Chapter 5 describes how to set up your projects for running FractionLynx samples. Chapter 6 shows how to use the FractionLynx Browser to review data. Chapter 7 briefly describes the OpenLynx portion of FractionLynx. Chapter 8 discusses troubleshooting MS- or UV-based systems using FractionLynx. Appendix A describes how to setup Equinox hardware and software, the installation of MassLynx software, how to configure system hardware, and connection of fraction collectors. Appendix B describes how a peak is detected using the analytical and preparative peak types. Related Documentation Waters Licenses, Warranties, and Support: Provides software license and warranty information, describes training and extended support, and tells how Waters handles shipments, damages, claims, and returns. Online Documentation MassLynx Help: Provides an overview of topics covering the MassLynx application. MassLynx Help is accessed either from the MassLynx top-level window or from selected program windows. 18

19 FractionLynx Help: Provides an overview of topics covering the FractionLynx application. FractionLynx Help is accessed either from the MassLynx Main window or from selected program windows. Printed Documentation for Base Products Waters 2525 Binary Gradient Module Installation and Maintenance Guide: Describes installing, operating, maintaining, and troubleshooting the Waters 2525 Binary Gradient Module. Waters 2767 Sample Manager, Injector and Collector Installation and Maintenance Guide: Describes installing, operating, maintaining, and troubleshooting the Waters 2767 Sample Manager. Waters Column/Fluidics Organizer Installation and Maintenance Guide: Describes installing, operating, maintaining, and troubleshooting the Waters Column/Fluidics Organizer. MassLynx Software Release Notes: Contains last-minute information about the product. Also provides supplementary information about specific MassLynx software releases. Printed Documentation for Options Printed documentation that supports software and hardware options includes the following: MassLynx User Guide: Describes how to use the MassLynx data system to select and display data files, process, and quantify data, and perform library searches. MassLynx Data Acquisition Guide: Describes how MassLynx is used to start, tune, and calibrate the detector and how to acquire data. MassLynx Interfacing Guide: Describes how to import and export data to and from MassLynx and other applications. MassLynx Guide to Inlet Control: Describes how MassLynx software controls mass spectrometer inlets, such as liquid or gas chromatographs, during data acquisition. OpenLynx Software User s Guide: Describes the procedures for installing, configuring, and using OpenLynx software. Waters 2487 Dual λ Absorbance Detector Operator s Guide: Describes the procedures for installing, operating, maintaining, and troubleshooting the Waters 2487 Dual λ Absorbance Detector. Waters 996 PDA Detector Operator s Guide: Describes the procedures for installing, maintaining, and troubleshooting the Waters 996 PDA Detector. 19

20 Waters 2996 PDA Detector Operator s Guide: Describes the procedures for installing, maintaining, and troubleshooting the Waters 2996 PDA Detector. Waters 515 HPLC Pump Operator s Guide: Describes the procedures for installing, maintaining, and troubleshooting the Waters 515 HPLC Pump. Waters Fraction Collector II Operator s Guide: Describes the procedures for installing, operating, maintaining, and troubleshooting the Waters Fraction Collector II. Waters Fraction Collector III Operator s Guide: Describes the procedures for installing, operating, maintaining, and troubleshooting the Waters Fraction Collector III. Waters Pump Control Module Installation Guide: Describes the procedure to install a Waters pump control module in a Waters multiple-pump gradient system. Waters Micromass ZQ Detector Operator s Guide: Describes the procedures for installing, operating, maintaining, and troubleshooting the Waters ZQ Mass Detector. Documentation on the Web Related product information and documentation can be found on the World Wide Web. The Waters Web site is Related Adobe Acrobat Reader Documentation For detailed information about using Adobe Acrobat Reader, see the Adobe Acrobat Reader Online Guide. This guide covers procedures such as viewing, navigating, and printing electronic documentation from Adobe Acrobat Reader. Printing This Electronic Document Adobe Acrobat Reader lets you easily print pages, page ranges, or the entire document by selecting File > Print. For optimum print quantity, Waters recommends that you specify a PostScript printer driver for your printer. Ideally, use a printer that supports 600 dpi print resolution. 20

21 Documentation Conventions The following conventions can be used in this guide: Notes Convention Purple Italic Courier Courier Bold Underlined Blue Usage Purple text indicates user action such as keys to press, menu selections, and commands. For example, Click Next to go to the next page. Italic indicates information that you supply such as variables. It also indicates emphasis and document titles. For example, Replace file_name with the actual name of your file. Courier indicates examples of source code and system output. For example, The SVRMGR> prompt appears. Courier bold indicates characters that you type or keys you press in examples of source code. For example, At the LSNRCTL> prompt, enter set password oracle to access Oracle. Indicates hypertext cross-references to a specific chapter, section, subsection, or sidehead. Clicking this topic using the hand symbol brings you to this topic within the document. Right-clicking and selecting Go Back from the shortcut menu returns you to the originating topic. For example, Accessing the FractionLynx Browser is described in Section 1.4, Accessing Reports Using the Fraction- Lynx Browser. Keys The word key refers to a computer key on the keypad or keyboard. Screen keys refer to the keys on the instrument located immediately below the screen. For example, The A/B screen key on the 2414 Detector displays the selected channel. Three periods indicate that more of the same type of item can optionally follow. For example, You can store filename1, filename2, in each folder. > A right arrow between menu options indicates you should choose each option in sequence. For example, Select File > Exit means you should select File from the menu bar, then select Exit from the File menu. Notes call out information that is helpful to the operator. For example: Note: Record your result before you proceed to the next step. 21

22 Attentions Attentions provide information about preventing damage to the system or equipment. For example: STOP Attention: To avoid damaging the detector flow cell, do not touch the flow cell window. Cautions Cautions provide information essential to the safety of the operator. For example: Caution: To avoid burns, turn off the lamp at least 30 minutes before removing it for replacement or adjustment. Caution: To avoid electrical shock and injury, unplug the power cord before performing maintenance procedures. Caution: To avoid chemical or electrical hazards, observe safe laboratory practices when operating the system. 22

23 Chapter 1 Introduction 1 FractionLynx is the automated purification software application that is a part of the MassLynx software suite. Using FractionLynx, molecules can be selected as they elute on the basis of the following: Molecular weight Total Ion Chromatogram (TIC) PDA Wavelength PDA TAC UV Wavelength Analog Channel This chapter is designed to assist a new user who has already had training outlining the steps required to make a successful injection and subsequent collection of the Sample Dye Kit (part number , supplied with the system). This chapter is designed to be used in conjunction with product training and should not be used as a substitute for training. The successful completion of the system level test will serve to determine that the components of the UV- or MS-based system are functioning properly. The chapter is divided into two sections: one refers to UV-based collection, and one refers to MS-based collection. Many parameters in both sections are the same, including the gradient parameters and the UV detector parameters. The first part of the UV section deals with the creation or loading of a custom FractionLynx Sample List format, a step which is a common requirement to both UV- and MS-based systems. The FractionLynx Editor parameters for both the UV- and MS-based systems are used with Waters UV detectors and the Waters ZQ Mass Detector. The threshold values may differ slightly if other manufacturers detectors are used. The resulting chromatograms at the end of each section are representative of the type of results achieved, although they can differ slightly from system to system. This chapter also describes how to: Make an injection (Section 1.2) Collect a sample (Section 1.3) 23

24 View a browser report (Section 1.4) Print basic reports (Section 1.4) If the system-level test results are not similar to the example test results presented in this chapter, see Chapter UV-Directed System Test Perform the UV-directed system test to determine that proper chromatography results are achieved. This section describes conditions originally devised for UV methods to rapidly test functionality of all component parts as a system. Actual system performance, in terms of column load per run, depends greatly on conditions such as HPLC solvent modifiers and column dimensions Instrument Setup The 2767 Sample Manager and fraction collector must be set up properly to avoid possible damage to the injectors and prevent solvent leaks. See the Waters 2767 Sample Manager, Injector and Collector Maintenance Guide and the Waters Fraction Collector II (or the Waters Fraction Collector III) Operator s Guide to set up and configure each instrument. Note: Ensure that the bed layout and reference position values are correct before turning on the pumps Required Materials The Sample Dye Kit (part number ) contains three compounds for testing the ability of the AutoPurification System to perform high flow rate targeting and fractionation of specified wavelengths. Table 1-1 lists the three compounds in the mixture. Note: The experiment can be performed for low (<10 ml/min) flows, after considering the appropriate column loading for the smaller column and the gradient to be used. Table 1-1 lists the order of elution. Table 1-1 UV Test Sample Dye Compounds Item Thionin Acetate Thioflavin T Crystal Violet UV max 590 nm 418 nm 590 nm Formula C 12 H 9 N 3 S.C 2 H 4 O 2 C 17 H 19 ClN 2 S C 25 H 30 ClN 3 Sigma part number T3387 T3516 C3886 Introduction 24

25 Retrieving or Creating Sample List Formats To display the complete Sample List view, double-click the Status icon in the toolbar. To download a previously saved Sample List format or to open the default FractionLynx formats, select Samples > Format > Load (Figure 1-1). Select a sample from the list, then click OK. 1 Figure 1-1 Load Sample List Format Dialog Box To add or remove columns, select Samples > Format > Customize. In the Customize Field Display dialog box, select the check box of the column required (Figure 1-2). Arrange the order of the columns using the arrow keys and click OK to save the selected order. UV-Directed System Test 25

26 1 Figure 1-2 Customize Field Display Dialog Box 1.2 Setting Up the Chromatography Test To perform the chromatography system test, create an inlet method using the following parameters. The column used to perform these tests is an XTerra Set up the Waters 2525 BGM Pump parameters as follows: Mobile Phase A: Water with 0.1% TFA Mobile Phase B: Acetonitrile with 0.1% TFA Flow Rate: 20 ml/min Introduction 26

27 Start Conditions: 95:5 A:B Run Time: 10 minutes Time A% B% Curve Number T = 0 minutes T = 1 minute T = 7 minutes T = 7.5 minutes T = 8.5 minutes T = 9 minutes T = 10 minutes Set up the Inlet Editor to run a 10-minute method. Label the method Dye. When using a Waters 2996 PDA Detector, set the wavelength range to be acquired in the Inlet Editor. The specific wavelength that the fraction collector will monitor for the potential fraction must be set in the Sample List as shown in Figure 1-3. This value must be within the wavelength range setup in the Inlet Editor. When using a Waters 2487 UV Detector, set up the wavelength to monitor in the Inlet Editor. In this case, the Fraction Trigger selected from the drop-down list in Figure 1-3 is UV 1, not wavelength A. The setting in the Wavelength column is for reporting purposes only and does not control the 2487 Detector. The software references the value set in the Fraction Trigger column, UV 1, UV 2, and so on, against the value set in the Inlet Editor. 3. From the MassLynx Sample List, set the Fraction Triggers. 4. Suggested Fraction File parameters are on the following pages. See Chapter 3 for Parameter page descriptions. 5. Set the Sample List to an injection volume of 100 µl. 6. The FractionLynx Editor must be open to collect fractions. Click FractionLynx in the side menu of the shortcut view. On the editor icon, minimize the FractionLynx Editor. To start data acquisitions, click (Start) in the MassLynx toolbar (Figure 1-3). Setting Up the Chromatography Test 27

28 Start Icon 1 Figure 1-3 Starting Data Acquisition in MassLynx Parameter Settings for the UV-Directed System Test Procedure Figure 1-4 to Figure 1-7 show parameter settings used in the UV-directed system test. Figure 1-4 Enable Fraction Collection Dialog Boxes Introduction 28

29 Click Collection Parameters to display the Fraction File Editor. 1 Figure 1-5 General and Timing Pages Figure 1-6 Volume and Analog Pages Setting Up the Chromatography Test 29

30 1 Figure 1-7 PDA and UV Pages UV-Chromatography Dye Test Results The chromatograms in Figure 1-8 represent the typical results of the dye test. The chromatograms show three separate color samples of blue, yellow, and violet collected into separate vials labeled 1:164, 1:165, and 1:167 to 1:168. Figure 1-8 UV-Directed Dye Test Chromatograms Introduction 30

31 This sample was collected using the Waters 996 PDA Detector. The wavelength values were put in the Sample List and the fraction triggers were set to Wavelength A and B. The extracted ion shows the correct picture of what was actually collected. Select Display > Wavelength to input the wavelengths of choice, then click OK to get the extracted wavelengths. To extract the spectrum, double-click the peak in the TIC. 1 Note: Actual results can vary if different column sizes, flow rates, and injection volumes are used. 1.3 MS-Based Chromatography Test Procedure The conditions described in this experiment were devised to produce a procedure to rapidly test that all component parts function as a system. Actual system performance, in terms of column load per run, depend greatly on conditions such as HPLC solvents or modifiers and column dimensions. The Sample Dye Kit (part number ) contains three compounds used to test the ability of the AutoPurification System to perform high flow rate targeting and fractionation of specified masses. Table 1-2 lists the three compounds in the mixture. Table 1-2 MS Test Sample Dye Compounds Item Thionin Acetate Thioflavin T Crystal Violet m/z UV max 590 nm 418 nm 590 nm Formula C 12 H 9 N 3 S.C 2 H 4 O 2 C 17 H 19 ClN 2 S C 25 H 30 ClN 3 Sigma part number T3387 T3516 C3886 Test Notes All three compounds ionize favorably in positive ion electrospray giving [M+H] ions. The following method is for a semi-prep scale purification. The experiment can be performed for low (<10 ml/min) flows after considering the appropriate column loading for the smaller column and the gradient to be used. The order of elution is as follows. Note: Report masses to one decimal place. 1. Thionin Acetate loses the Acetate salt (C 2 H 4 O 2 ) to give this [M+H] ion: Thionin ([M+H] = 228.1), Lambda Max. = 590 nm MS-Based Chromatography Test Procedure 31

32 2. Thioflavin T loses a Cl to give this [M+H] ion: Thioflavin T ([M+H] = 283.1), Lambda Max. = 418 nm 3. Crystal Violet loses HCl to give this [M+H] ion: ([M+H] = 372.2), Lambda Max. = 590 nm 1 Note: The following method is formulated using a 19 x 50 mm column. Longer columns require a longer duration gradient. To perform the chromatography system test, create an inlet method with the following parameters: 1. Set up the Waters 2525 BGM Pump parameters as follows: Mobile Phase A: Water with 0.1% TFA Mobile Phase B: Acetonitrile with 0.1% TFA Flow Rate: 20 ml/min Start Conditions: 95:5 A:B Run Time: 10 minutes Time A% B% Curve Number T = 0 minutes T = 1 minute T = 7 minutes T = 7.5 minutes T = 8.5 minutes T = 9 minutes T = 10 minutes Set up the Inlet Editor to run a 10-minute method. Label the method Dye. When using a Waters 2996 PDA Detector, set the wavelength range to be acquired in the Inlet Editor. The specific wavelength that the fraction collector monitors for the potential fraction must be set in the Sample List as shown in Figure 1-3. This value must be within the wavelength range set up in the Inlet Editor. When using a Waters 2487 UV Detector, set the wavelength to be monitored in the Inlet Editor. In this case, the Fraction Trigger selected in the drop-down list shown in Figure 1-3 is UV 1, not wavelength A. The setting in the Wavelength column is for reporting purposes only and does not control the 2487 Detector. The software references the value set in the Fraction Trigger column (UV 1, UV 2, and so on) against the value set in the Inlet Editor. Introduction 32

33 3. Set the makeup pump to run at 1 ml per minute. 4. Set up a Scan Function from 150 to 600 amu, ESP+, Centroid Data, 0.5-second scan time with a 0.1-second Inter Scan Delay, and Cone Voltage = 25 V. Set this to run for 10 minutes. 5. In the Sample List, enter the mass of the compound to be collected in the columns labeled Mass A, Mass B, Mass C, etc. The Mass and Wavelength columns are for information only unless the corresponding fraction information is inputted in the Fraction Trigger column. For example, Mass A/Wavelength A/UV1, as in Figure Suggested FractionLynx parameters are on the following pages. See the FractionLynx Installation Guide for parameter page descriptions. 7. Set the Sample List to an injection volume of 100 µl. 8. Set the MS multiplier voltage tuning to 650 V for a ZMD Detector, and to 500 V for a ZQ Detector. 9. The FractionLynx Editor must be open to collect fractions. Click FractionLynx in the side menu of the shortcut view. On the editor icon, minimize the FractionLynx Editor. To start data acquisitions, click (Start) in the MassLynx toolbar. See Figure MS-Based Chromatography Test Procedure 33

34 Start Icon 1 Figure 1-9 Starting Data Acquisition in MassLynx Parameter Settings for the MS-Directed System Test Procedure Figure 1-10 to Figure 1-13 show parameter settings used in the MS-directed system test. Note: The Split/Collector Delay value (Timing page) may need to be adjusted depending on the flow rate. To determine the proper value, see Chapter 8. Introduction 34

35 1 Figure 1-10 Enable Fraction Collection Dialog Boxes Figure 1-11 General Page MS-Based Chromatography Test Procedure 35

36 1 Figure 1-12 Timing and Volume Pages Figure 1-13 Positive and Negative Pages MS-Chromatography Dye Test Results The chromatograms in Figure 1-14 represent typical results of the dye test. The three components of the dye mix have been separated and collected into separate vials labeled 1:1, 1:2 to 1:3, and 1:4 to 1:5. The extracted ion shows the correct picture of what was actually collected into each vial. Double-click the peak in the TIC to extract the spectrum, and double-click the spectrum of Introduction 36

37 the mass of interest to extract the mass chromatograms. Always look at the extracted ion or wavelength when troubleshooting as it will give the correct representation of what was actually collected. 1 Figure 1-14 MS-Directed Dye Test Chromatograms MS-Based Chromatography Test Procedure 37

38 1.4 Accessing Reports Using the FractionLynx Browser The FractionLynx Browser allows you to view, print, or export your results to other applications (such as a LIMS system or Excel) using the Clipboard or tab-delimited text files Creating Report Files OpenLynx creates a Browser report file (*.rpt) each time the Login PC submits a job. For compound targeting, you must enter a Mass to search for, whether you are using a Login PC or the Sample List. OpenLynx also creates a Browser report file when the Sample List is used and the Process_Params column has the.olp method entered into it Accessing the FractionLynx Browser To access the FractionLynx Browser, select FractionLynx > FractionLynx Browser from the MassLynx shortcut bar. The FractionLynx Browser window appears (Figure 1-15). Figure 1-15 FractionLynx Browser Window Introduction 38

39 The window has seven panes (described in Table 1-3). Some panes may not appear, depending on the processing you selected in the OpenLynx method. Table 1-3 FractionLynx Browser Panes Plate Pane Sample Description Fraction Collection Results List Results Table Spectrum Elemental Composition Results List and Library Search Results List Chromatogram Description Displays the plate layout and compounds found. Displays sample information and indicates if a search designated the compound as found, found tentative, or not found. Displays fraction collection information. This pane appears only if you collected fractions. Lists detected compounds. Displays the spectrum for the highlighted sample well and the selected retention time. Displays the results of any elemental composition and library searching for the peak selected in the Results Table pane. This pane appears only if you perform elemental composition calculations or library search. Displays the integrated chromatogram for the highlighted sample well Setting Report Scheme Parameters Report schemes (*.ors files) specify the information contained in printed reports. To define the information on the printed report, select File > Report Scheme Settings. The Edit Report Scheme Settings dialog box appears (Figure 1-16). Click the Print Control tab if it does not appear to set the printing parameters. Accessing Reports Using the FractionLynx Browser 39

40 1 Figure 1-16 Setting Print Controls in the Edit Report Scheme Settings Dialog Box Select the appropriate check boxes in the Print Reports and Format Sample Report areas, then click OK. For information on other areas in this screen, see Chapter 6, FractionLynx Browser. Introduction 40

41 Table 1-4 Print Reports Plate summary Sample summary Sample Report Sample on a New Page Produces a plate report. This is a picture of the plate showing: 1 for found compounds? for found tentative compounds 0 for compounds not found + if no search was performed on the compound for unused wells Produces a summary report for a sample. Define the number of samples printed in the report on the Print Control dialog box. Produces a more detailed sample report. Starts each sample in the sample report on a new page. 1 Table 1-5 Report Formats Spectra height (mm) Chromatogram height (mm) All Chromatograms On One Axis Mass Chromatogram/Axis Side by Side Chromatograms Side by Side Spectra Prints the spectra associated with the sample. Select the check box, then enter the required height. Prints the chromatogram associated with the sample. Select the check box, then enter the required height Uses one axis for all chromatograms displayed. Select, then enter the number of chromatograms to display on each axis. Use this option to make overlaid mass chromatograms easier to view. The default of 1 displays each chromatogram on a different axis. Prints two chromatograms per line. Note: The Peak Information table is not displayed if you select this option. Prints two spectra per line. Note: Library search results and elemental calculations are not displayed if you select this option. Accessing Reports Using the FractionLynx Browser 41

42 Chapter 2 Installing FractionLynx Software as an Option This chapter describes installing the FractionLynx software as an option after MassLynx has been installed. See the MassLynx installation instructions provided with the CD disk. Note: You must be logged on to a user account that has administrative privileges. Install MassLynx only to the C:\MassLynx directory, and do not move the MassLynx folder after installation. FractionLynx results can also be processed by using the OpenLynx program to produce Browser Report files Installing FractionLynx Software Many of the installation screens in this document show installation on a system running Windows XP Professional. These screens appear slightly different on a system running Windows NT or Windows 2000 platforms, but the procedure is identical. To install FractionLynx version 4.0: 1. Close all running programs, including existing versions of MassLynx. 2. Insert the MassLynx for Windows Installation CD disk into the CD-ROM drive.the installation should start automatically. 3. If installation does not start automatically, select Start > Run. In the Run dialog box, enter the following command using the letter that represents your CD-ROM drive. For example: C:\SETUP 4. Click OK to continue. Installing FractionLynx Software 42

43 The Welcome to the Wizard for MassLynx v4.0 page appears (Figure 2-1). 2 Figure 2-1 MassLynx V4.0 Installation Wizard 5. Click Next to go to the Program Maintenance page (Figure 2-2). Figure 2-2 Modifying MassLynx 4.0 Program 6. Click Modify, then click Next to go to the License Agreement page (Figure 2-3). Installing FractionLynx Software as an Option 43

44 7. Read the license agreement, click I accept the terms in the license agreement, then click Next. If you do not accept the terms, the installation will terminate. 2 Figure 2-3 Accepting the License Agreement 8. The Application Manager Options page appears (Figure 2-4). Select OpenLynx and FractionLynx, and click Next. The Application Manager Options page appears (Figure 2-5) with additional OpenLynx options. Note: The OpenLynx option must be installed with FractionLynx. FractionLynx requires some of the OpenLynx processing and reporting capabilities. Figure 2-4 Selecting the FractionLynx Software Option Installing FractionLynx Software 44

45 9. Select OpenLynx Global Server if this option is to be installed. If not, leave the check box empty and click Next. 2 Figure 2-5 Installing OpenLynx Options 10. Click Install to start the program installation (Figure 2-6). Figure 2-6 Starting the Program Installation Installing FractionLynx Software as an Option 45

46 11. The KeyDisk pages appear (Figure 2-7 and Figure 2-8). Insert the FractionLynx and OpenLynx key disks according to the prompts. Figure 2-7 Selecting the FractionLynx Key Disk Path 2 Figure 2-8 Selecting the OpenLynx Key Disk Path 12. Click Continue. When the installation is finished, the Wizard Completed page appears (Figure 2-9). Installing FractionLynx Software 46

47 13. Click Finish to close the wizard. 2 Figure 2-9 Program Installation Complete Installing FractionLynx Software as an Option 47

48 Chapter 3 Using the FractionLynx Editor Use the FractionLynx Editor to configure the system and to develop FractionLynx methods for use by MassLynx. You also use it to define bed and plate layouts and to start and stop fraction collection. FractionLynx methods describe how a fraction will be detected. These methods have *.frc file extensions and are in the Acqudb directory of the current project. 3.1 Accessing the FractionLynx Editor To access the FractionLynx Editor, select FractionLynx > FractionLynx Editor from the MassLynx shortcut bar. The FractionLynx Editor appears (Figure 3-1). 3 Fraction Kernel State Area Figure 3-1 FractionLynx Editor Communication Collection Enabled If the PC and the fraction collector are communicating normally, then this light is green, otherwise it is red. Click the red light for an explanation of the problem. If fraction collection is enabled, then this light is green. If fraction collection is not enabled, then it is red. See Section 3.2, Enabling Fraction Collection, for details on how to enable and disable fraction collection. Accessing the FractionLynx Editor 48

49 Fraction Collection Area State Location Next Collection Site Current state of the fraction collector. Location of the vessel currently used for collection in the format Fraction Collector Number:Vessel Number. Location of the vessel used for collecting the next fraction using the format Fraction Collector Number:Vessel Number. Peak Detection Area State Peak Start Time Trigger Additional Information Collection Mode Current state of the peak detection: Idle No FractionLynx methods are running. Solvent Delay A sample is being acquired but the system is in the solvent delay period. The system will prevent any triggers during this period. (defined in the FractionLynx Parameters). Peak Detected A peak was detected that conforms to the peak detection criteria in the parameters file. It has yet to pass the minimum peak width parameter and is therefore not classed as a fraction. Fraction Detected A peak was detected and passed the minimum peak width criteria. It is now classed as a fraction and will be collected if there are any collection vessels available. Start time of the peak being collected. This is the time since the injection, i.e., real retention time. All times referenced in the FractionLynx system relate to the time that the peak was detected in the mass spectrometer, UV detector, or analog device. Mass or wavelength of the fraction trigger, TIC, or analog. Ionization mode. 3 The currently selected collection mode appears, which can be Sequential, One to One, or Reserve tubes Per Injection. See Chapter 4 for further details. Using the FractionLynx Editor 49

50 3.2 Enabling Fraction Collection Select Fraction System > Enable Fraction Collection to enable fraction collection (Figure 3-2). Figure 3-2 Enable Fraction Collection Dialog Box Select the Enable Fraction Collection check box, then click OK. Note: If the check box is not selected, then fractions will not be collected. 3.3 Resetting Communications Select Fraction System > Reset Communications from the FractionLynx Editor to reset communications between the PC and the fraction collectors (Figure 3-3). 3 Figure 3-3 Reset Communications Command Note: Communications cannot be reset when the instrument is acquiring data. Enabling Fraction Collection 50

51 3.4 Exiting the FractionLynx Editor Select Fraction System > Exit to close the FractionLynx Editor. Note: The FractionLynx Editor must be open, but can be minimized, for fractions to be collected. If the FractionLynx Editor is closed, then fraction collection will not be performed. 3.5 Selecting Fraction Collection Parameters There are several modes available for fraction collection, which are described in Chapter 4, Collection Modes. To enable collection modes, select Collection Parameters > Collection Modes from the FractionLynx Editor. The Collection Modes dialog box opens (Figure 3-4). Note: The default collection parameter is sequential collection. 3 Figure 3-4 Collection Modes Dialog Box For further details about this dialog box, see Section on page Fraction File Editor 1. Select Collection Parameters > Fraction File Editor from the FractionLynx Editor to access the fraction collection properties pages. The Fraction File Editor opens (Figure 3-5). The FractionLynx Editor allows you to do one of the following: Create a new file by selecting File > New. Open an existing file by selecting File > Open and selecting the required file from the browser displayed. Using the FractionLynx Editor 51

52 2. Define the parameters as needed (see Section to Section 3.5.9). 3. Select File > Save or File > Save As. Existing files are labeled as *.frc files and the default location is the Acqudb directory of the current project. When creating a Sample List, double-clicking the Fraction File column and selecting the required file from the displayed list will include these files. Parameters on the General, Timing, and Volume pages are common to many types of purification runs. Parameters on the Positive, Negative, PDA, and Analog pages are used only when required by the current injection. MassLynx determines which parameters to use, based on the type of data acquisition method and fraction triggers requested. This allows you to use the same method for different purification triggers General Page Use the General page to enable the general parameters of Fraction Detection, Multiple Fractions, Rinse Between Fractions, and Span (Figure 3-5). 3 Figure 3-5 Fraction File Editor, General Page The following topics describe the General page parameters. Selecting Fraction Collection Parameters 52

53 Fraction Detection Area Fraction Collection Peak Type Select On to perform fraction collection for the current sample, otherwise select Off. Note: If Off is selected, then no fraction collection takes place using that fraction file. Select Preparative or Analytical to define the sliding window size to use when monitoring data to identify peak status. Analytical = 3-point sliding window. Use this for sharp peaks usually produced under analytical HPLC flow rates, e.g., <= 2 ml/min. with peaks <30 secs wide. Preparative = 5-point sliding window. Necessary for peaks eluting more slowly, usually as a result of high column loading in semi-prep flow rates, e.g., > 5 ml/min. See Appendix B for an explanation of the 3- and 5-point sliding windows used. Multiple Fractions Area Max. Fractions per Injection Max. Tubes per Injection Enter the maximum number of fractions to be collected for a single sample in the Sample List. Range: 1 to (A single fraction can be collected over several tubes depending on the parameters described later. The Max. Fractions per Injection value is the number of triggers, not the number of tubes). Enter the maximum number of tubes to be collected for a single sample in the Sample List. Range 1 to Rinse Between Fractions Area Rinse Time (secs) Enter the time in seconds to rinse the needle between fractions. Range: 0 to 10 seconds. Be careful when using the rinse time feature for closely eluting peaks, i.e., fractions that collect immediately after each other. The fraction collector will collect the first fraction, perform the rinse procedure, then move to the next tube for collection; and so the start of the second fraction may be missed. Using the FractionLynx Editor 53

54 Span Area Span (+/ amu) The size of the window to search the spectrum for the mass entered in the Sample List (fraction trigger 1-4), or adducts entered in the Positive or Negative pages. Note: The value is before and after the mass of interest. Therefore, the total window size is double the actual value entered. Experience has shown that a value of 0.5 (i.e., 0.5 amu either side of the peak of interest) yields the best results. Valves larger than 0.5 can result in overlapping windows Timing Page Use the Timing page to set the parameters for Trace Monitoring and Peak Timing (Figure 3-6). 3 Figure 3-6 Fraction File Editor, Timing Page Trace Monitoring Area Solvent Front Delay (secs) With many large-scale injections, some sample (especially when diluted in DMSO) can blow by the column, i.e., it is not retained and appears as a solvent front peak. It may not be desirable to collect this peak as it may contain other impurities. Set this parameter to ignore peaks eluting during the specified time. Range: 0 to 300 seconds. Selecting Fraction Collection Parameters 54

55 Peak Timing Area Split/Collector Delay (secs) MS/Aux. Delay (secs) Volume Page Specify the time difference for a peak to travel from the splitter to the mass spectrometer and from the splitter to the fraction collector. When a peak is detected, the collection valve will not be triggered until this time period passes, i.e., the peak has actually reached the head of the collection valve. Range: 0 to 180 seconds. Recommended minimum delay is 8 seconds. For more information on the delay time, see Section 8.5.2, Delay Timing Dye Test. Specify the time difference between a peak being seen at the auxiliary detector (PDA, UV, etc.) and being detected at the mass spectrometer. Range: 60 to 60 seconds. Peaks detected by the auxiliary detector after the mass spectrometer will use a negative value, i.e., align the trace backward to match the mass spectrometer trace. Peaks detected by the auxiliary detector before the mass spectrometer will use a positive value, i.e., align the trace forward to match the mass spectrometer trace. Note: In UV-only mode, this option is not available. 3 Use the Volume page to define fraction width and collection capacity (Figure 3-7). Figure 3-7 Fraction File Editor, Volume Page Using the FractionLynx Editor 55

56 Fraction Sizes Area Minimum Fraction Width Maximum Fraction Width Select Time (secs) or Volume (ml) from the Units list and enter the time or volume in the Value field. Any fractions narrower than the entered value are not collected. Range: 0 to 60 seconds or 0 to 100 ml. The peak volume can be calculated from the flow rate defined in the Inlet Editor. (Assume that flow rate remains constant during acquisition and can only change before the injection and after data is acquired, i.e., column wash/reequilibration.) Select Time (secs), Volume (ml), or Volume (tubes) from the Units list and enter the time or volume in the Value field. Range: 0 to 300 seconds, 0 to 1000 ml, or 0 to 100 tubes. For peaks terminated using the Below Gradient option on the Positive, Negative, PDA, and Analog pages, the gradient may not be met. This option is an extra safeguard to terminate fraction collection for peaks with long trailing edges. At this point only a small quantity of sample is being collected compared to the high volume of solvent, and the drying process becomes expensive compared to the level of sample recovery. If the gradient is not met within the time or volume specified here, then fraction collection is terminated for this peak. The peak volume can be calculated from the flow rate defined in the Inlet Editor. (Assume that flow rate remains constant during acquisition and can only change before the injection and after data has been acquired, i.e., column wash/reequilibration.) 3 Collection Capacity Area Maximum Tube Fill (%) Enter the maximum percentage of the tube volume to fill a tube. If a peak causes the volume to be exceeded, the fraction collector head moves to the next tube and continues collection, providing the subsequent collection does not exceed the specified Max Peak Width. Range: 0 to 100 (%). Tube volume is defined in the Fraction Collection Setup page (Bed Layouts tab - see page 80). A peak collected over multiple tubes counts as one fraction when comparing to Max Fractions per Injection. Note: If using an injector/collector such as a 2767, the tube volume is set in the bed layout editor of the inlet method, not in the fraction bed layout. Selecting Fraction Collection Parameters 56

57 3.5.5 Positive and Negative Pages The parameters for negative ion-based fraction collection are the same as those for positive ion. Note: In UV-only mode, these pages do not appear on the Fraction File Editor. FractionLynx evaluates positive and negative traces independently, therefore separate parameters are available for each ionization mode. The layouts of the pages for positive and negative parameters are identical (Figure 3-8). 3 Figure 3-8 Fraction File Editor, Positive and Negative Adducts Pages Positive/Negative Ion Adducts Enter the list of adducts separated by a comma: positive = +1, +23 negative = 1 This allows simultaneous targeting of the mass and adduct losses or gains for each ion mode. Each adduct is added to, or subtracted from, the target mass and the Span applied to it. This generates multiple, independent traces any of which can trigger fraction collection. If the true molecular weight is used in the Sample List, an adduct of +1 needs to be used to detect the (M + H) produced during positive ionization scan; an adduct of 1 needs to be used to detect the (M H) produced during a negative ionization scan. Use M + 23 for sodium adducts produced in + ion mode. Using the FractionLynx Editor 57

58 Min. Intensity Threshold (MIT) Peak Start Enter the value above which peak intensities must be before they will be considered for peak detection. It is used to avoid false triggers from the baseline and is applied to all target masses and adducts in the relevant ionization mode. Values can be input using absolute or exponential format, e.g., or 2.5e5. Range: 0 to or 1.34e8. Select the method of detecting the start of the peak from the drop-down list: Leading Edge Gradient (%) Enter the minimum gradient required to trigger fraction collection in the Value field. The leading edge of a peak is normally steeper/more defined than the trailing edge. Use a high value for a steep gradient and a low value for a shallow gradient. It is applied to all target masses and adducts. Range: 0 to % End point must be more intense than the start point 50% 1.5 times 100% 2.0 times 200% 3.0 times Use MIT Only If this option is selected, then the Value field is disabled and the Min. Intensity Threshold defined above is used. 3 Terminate Peak Terminate peaks in one of three ways: Below Gradient (%) Terminates the peak when the gradient of the peak falls below the value defined. Usually the trailing edge is less well defined than the peak front and results in a tail. To detect a shallow/flat gradient, use a high value. Steeper gradients can be detected using low values. See peak detection algorithms in Appendix B for clarification. Use Max Peak Width Terminates the peak when the Maximum Peak Width defined on the Volume page is met. This is useful for collecting a predefined amount of sample/solvent irrespective of gradient/min. intensity threshold. Use MIT Only If this option is selected then the Value field is disabled and the Min. Intensity Threshold defined above is used. Selecting Fraction Collection Parameters 58

59 3.5.6 Analog Page Figure 3-9 Fraction File Editor, Analog Page Min. Intensity Threshold (MIT) Set the value according to the parameters in Section Peak Start Set the value according to the parameters in Section Terminate Peak Set the value according to the parameters in Section PDA Page Figure 3-10 Fraction File Editor, PDA Page Using the FractionLynx Editor 59

60 Span (+ / nm) Min. Intensity Threshold (MIT) Specify the window size when searching the spectrum for the wavelength entered in the Sample List (Fraction Trigger 1 to 10). Range: 0 to 50 nm. Typical value = 3. Set the value according to the parameters in Section Peak Start Set the value according to the parameters in Section Terminate Peak Set the value according to the parameters in Section UV Page 3 Figure 3-11 Fraction File Editor, UV Page Min. Intensity Threshold (MIT) Set the value according to the parameters listed in Section Peak Start Set the value according to the parameters listed in Section Terminate Peak Set the value according to the parameters listed in Section Selecting Fraction Collection Parameters 60

61 3.5.9 Timed Events Page Adding Events 1. Enter a time in the Time field and choose an event from the Event list (Figure 3-12). If Disable Collect is selected, the Parameter field is disabled. If the Parameter field is not disabled, enter a parameter. 2. Click (Line Insert) to place the event in the Event table. Note: In the table, timed events are allowed between 0 and minutes. Deleting Events To remove a single event, highlight it in the Time column of the Event table and click (Line Remove). Clearing the Event Table To clear the entire Event table, click (Table Remove). 3 Figure 3-12 Fraction File Editor, Timed Events Page Using the FractionLynx Editor 61

62 Enable/Disable Collect Start/Stop Collect MIT Settings Turns on and off the Fraction Collection functionality of the software during a run. These events prevent the software from collecting peaks that are not required at a given time, such as the solvent front. However, if a fraction is being collected at the same time the event is programmed to occur, the fraction collection will override the timed event and the timed event will not occur. This functionality is for timed collection and is not based on any of the criteria in the FractionLynx Editor method. Start and Stop will override all threshold criteria in the method, and will override the Enable/Disable event as well. These events allow users to change the MIT Threshold values for Fraction Collection during the run. However, if a fraction is being collected at the same time the event is programmed to occur, the fraction collection will override the timed event and the timed event will not occur Mixed (Boolean) Triggers Editor Note: The terms Boolean expressions, Boolean triggers, and mixed triggers are used interchangeably in this document. Boolean expressions created within FractionLynx allow you to customize fraction triggering, based on fraction triggering parameters used in conjunction with one another. Four Boolean operators are available within the Mixed Triggers Editor (AND, OR, NOT, and XOR) for creating fraction triggering expressions. Figure 3-13, Figure 3-14, Figure 3-15, and Figure 3-16 provide an illustrated example of each of these operators. 3 AND Operator Example Mass A AND Wavelength B Collects Mass A Wavelength B Figure 3-13 Illustrated Example of Mass A AND WaveLength B Selecting Fraction Collection Parameters 62

63 OR Operator Example Collects A Collects A and B Collects B Figure 3-14 Illustrated Example of Mass A OR Mass B Note: Peak A is collected in its entirety, then Peak B is collected due to the OR function. NOT Operator Example Collects A Only Mass A NOT Mass B 3 Mass A Mass B Figure 3-15 Illustrated Example of Mass A NOT Mass B Using the FractionLynx Editor 63

64 XOR Operator Example Mass A XOR Mass B Collects A Collects B Mass A Mass B Figure 3-16 Illustrated Example of Mass A XOR (Exclusive OR) Mass B Using Boolean Triggers 1. Add the Fraction Boolean Logic column to the Sample List format. Right-click the Sample List page and select Customize Display. Click in the box preceding Fraction Boolean Logic and click OK. 2. Create the Boolean expression as follows: a. Highlight and right-click in a cell of the Fraction Boolean Logic column. Select Edit (Figure 3-17). 3 Figure 3-17 MassLynx Boolean Trigger, Boolean Column Dialog Box Selecting Fraction Collection Parameters 64

65 The FractionLynx Mixed Triggers Editor dialog box is used to create the Boolean logic expression that is applied to existing fraction triggers to create a mixed trigger. It has two views: a reduced view (Figure 3-18) and an expanded view (Figure 3-19). The More>> / Less<< buttons are used to switch between views. Figure 3-18 FractionLynx Mixed Triggers Editor Dialog Box b. Click More to expand the FractionLynx Mixed Triggers Editor dialog box. Table 3-1 lists the properties of the expanded Fraction Mixed Trigger Editor dialog box. 3 Figure 3-19 FractionLynx Mixed Triggers Editor Dialog Box, Expanded Using the FractionLynx Editor 65

66 Table 3-1 FractionLynx Mixed Triggers Editor Properties Property Description OK Delete Cancel More Less Trigger Source Trigger Expression Add AND OR NOT XOR Open Brace Close Brace Backspace Clear Description Lists Boolean trigger expression descriptions. It is this description that is entered into the Fraction Boolean Logic column in the MassLynx Sample List. Exits the dialog box and saves any changes to the Boolean expressions. Removes a description from the list. Exits the dialog box without saving any changes to the Boolean expression. Switches to the expanded view. Switches to the reduced view. Lists all the fraction trigger types that can be used to create a mixed trigger. The trigger types should match those in the Sample List. Displays the Boolean trigger expression. Adds the Trigger Source to the Trigger Expression field. Adds the Boolean AND operator to the Trigger Expression field. Adds the Boolean OR operator to the Trigger Expression field. Adds the Boolean NOT operator to the Trigger Expression field. Adds the Boolean XOR operator to the Trigger Expression field. Adds an open brace to the Trigger Expression field. Adds a close brace to the Trigger Expression field. Removes the last Trigger Source or operand from the Trigger Expression field. Clears the Trigger Expression field. 3 c. Enter a trigger description in the Description field (Figure 3-19). d. Select Mass A from the Trigger Source list (Figure 3-20). Selecting Fraction Collection Parameters 66

67 Figure 3-20 Selecting from the Trigger Source List e. Click Add. The message Warning: Invalid Syntax appears in the FractionLynx Mixed Triggers Editor dialog box until the expression is created. If the warning message still appears after the expression is created, check the syntax (Figure 3-21). 3 Syntax Warning Figure 3-21 Adding an Expression f. Select the operator (Figure 3-22). Using the FractionLynx Editor 67

68 Figure 3-22 Selecting an Operator g. Select WaveLength A (Figure 3-23). 3 Figure 3-23 Selecting Wavelength A h. Click Add to include the chosen wavelength to the editor file (Figure 3-24). Selecting Fraction Collection Parameters 68

69 Figure 3-24 Adding the Wavelength to the Expression i. Click OK to close the FractionLynx Mixed Triggers Editor dialog box (Figure 3-25). Note that the syntax is valid because the expression is complete. 3 Figure 3-25 Saving and Closing the FractionLynx Mixed Triggers Dialog Box 3. On the Sample List page, highlight the cell in the Fraction Boolean Logic column, right-click, then select Browse (Figure 3-26). Using the FractionLynx Editor 69

70 Figure 3-26 Sample List Page, Browse 4. Select the mixed trigger and click OK (Figure 3-27). 3 Figure 3-27 Selecting and Saving a Mixed Trigger Selecting Fraction Collection Parameters 70

71 The mixed trigger is entered in the Fraction Boolean Logic column (Figure 3-28). Figure 3-28 Mixed Trigger Entered in the Boolean Logic Column 5. Enter each component of the Boolean expression into a Fraction Trigger column in the Sample List (Figure 3-29). Parameters such as minimum intensity threshold, leading edge gradient, minimum fraction width, maximum fraction width, etc., which all describe how a trigger is defined in the fraction method, are applied to each term in the mixed trigger. When all trigger conditions are satisfied, the mixed trigger activates, and a fraction is collected. 3 Using the FractionLynx Editor 71

72 Figure 3-29 Entering Components into the Boolean Expression If individual triggers are not entered into the Sample List fraction trigger columns when the Sample List is starting an acquisition, an error message is immediately displayed. The error message indicates that the FractionLynx validation failed and that the FractionLynx Boolean logic is incorrect (Figure 3-30). 3 Selecting Fraction Collection Parameters 72

73 Figure 3-30 Validation Error Message 3 6. Save the Sample List and begin acquisition. 7. Fractions collected from Boolean expressions are labeled mixed in the fraction display of the Chromatogram window (Figure 3-31). The expression is also defined as mixed in the _fract.txt file located in the raw data file (Figure 3-32). Using the FractionLynx Editor 73

74 Figure 3-31 Mix Displayed on the Fraction Chromatogram 3 Figure 3-32 Mix Displayed in the Fract.txt Screen Selecting Fraction Collection Parameters 74

75 Deleting Boolean Triggers Boolean triggers can be deleted from two sources: Sample List Project acquisition database (Acqudb) To delete Boolean triggers from the Sample List: 1. Open the FractionLynx Mixed Triggers Editor (Figure 3-33). Select a trigger description from the list, then click Delete. Figure 3-33 Selecting a Trigger from the Sample List 2. Click Yes at the warning message asking if you are sure that you want to delete the mixed trigger expression (Figure 3-34). 3 Figure 3-34 Delete Mixed Trigger Warning Box The expression is deleted from the description list in the FractionLynx Mixed Triggers Editor (Figure 3-35). Using the FractionLynx Editor 75

76 Figure 3-35 Expression Deleted from the Mixed Trigger Editor To delete Boolean triggers from the Project Acquisition Database (Acqudb): 1. Note the description of the Boolean trigger in the FractionLynx Mixed Triggers Editor and in the Mixed Triggers selection box. To access this description, highlight the Fraction Boolean Logic column, right-click the Sample List, then select Browse. In this example, the mixed trigger MT AND WLG will be deleted (Figure 3-36). 3 Figure 3-36 Viewing Trigger in the Mixed Trigger Editor and Mixed Triggers Dialog Box 2. Using Notepad, open the Boolean Logic.ini file (Figure 3-37). Access the file by selecting Windows Explorer > MassLynx > Project > Acqudb > Boolean Logic.ini >. Note: The mixed trigger MT and WLG is present in the BOOLEAN LOGIC section and in the BOOLEAN LOGIC file section of the Boolean Logic.ini file. Selecting Fraction Collection Parameters 76

77 Figure 3-37 Boolean Logic INI File 3. Delete the trigger from the BOOLEAN LOGIC section and the BOOLEAN LOGIC file section of the Boolean Logic.ini file (Figure 3-38). 3 Using the FractionLynx Editor 77

78 3 Figure 3-38 Deleting a Trigger from the Boolean Logic Section and Boolean Logic File Section 4. Click Save to save the changes (Figure 3-39). Selecting Fraction Collection Parameters 78

79 Figure 3-39 Saving Changes The trigger is now removed from the.ini file (Figure 3-40). 3 Figure 3-40 Display of INI with Trigger Removed The trigger is also removed from the menus in the FractionLynx Mixed Triggers Editor and in the Mixed Triggers dialog boxex (Figure 3-41). Using the FractionLynx Editor 79

80 Figure 3-41 Triggers Removed from Mixed Triggers Editor and Mixed Triggers Dialog Boxes 3.6 Fraction Collection Setup Instrument Setup To access the Setup pages, select Collector Setup > Instrument Setup from the FractionLynx Editor. The Fraction Collector Setup dialog box appears (Figure 3-42). 3 Figure 3-42 Collector Configuration Page (Waters Fraction Collector III) Fraction Collection Setup 80

81 3.6.2 Collector Configuration Number Of Collectors Collector Model Collection Type Comm Port Enter the number of fraction collectors that are being used. Range 1 to 9. Select the fraction collector model that is being used. If you select WFC II/III, the configuration is as shown in Figure Variations for the Waters 2757 and the Gilson 215 and Gilson 204 are shown in Figure For the selected fraction collector, click Collect Fractions to collect fractions or Collect Waste to collect the sample using a waste valve. For the Gilson 215, click Inject/Collect. If incorrect parameters are defined or a problem occurs which results in no fractions being collected, the sample can be lost to waste. To solve this when a sample is being scanned but a fraction is not being collected, the solvent flow can be directed to another fraction collector known as a waste collector. The waste for each sample is then deposited in a separate vessel so that it can be reused if required. Enter the name of the comm port that the fraction collector is connected to (COM1, COM2, etc.). 3 Baud rate Select the baud rate of the current instrument. This value (1200, 2400, or 4800 bps) should be the same as that defined in the fraction collector hardware manual. Bed Layout for Fraction Collector n Select the bed layout for the selected fraction collector. n updates to the number of the fraction collector selected. Selector Valve Setup The selector valve can be used when there are multiple collections in the system. The delay time in the FractionLynx Editor is the delay to the first collector. To ensure uniformity, the tubing from the valve to each collector must be the same length. To enable the fraction collector selector valve, select the Use Selector Valve check box. Select the comm port from the Com Port list (Figure 3-43). In this configuration, waste collection is not supported. Using the FractionLynx Editor 81

82 Figure 3-43 Selector Valve Page Communications (Waters 2757) 3 Figure 3-44 Collector Configuration (Waters 2757) Fraction Collection Setup 82

83 Gilson Setup Gilson 215 Gilson Using the FractionLynx Editor 83

84 Gilson 215 Inject/Collect Figure 3-45 Typical Gilson Fraction Collector Setups ID Gilson Comms Enter the ID number displayed at the back of the instrument. For more information, see Section A.5 on page 211. Click this button to open the Gilson Comms dialog box (Figure 3-46). 3 Positions Click this button to open the Positions dialog box (Figure 3-47). Fraction Collection Setup 84

85 3.6.3 Gilson Comms Dialog Box Use this dialog box to define the serial line communication between the Gilson and the PC (Figure 3-46). Figure 3-46 Gilson Comms Dialog Box Note: These settings are defined by your Waters Service engineer during installation and should not need changing. For details, consult the instrument manual Gilson Positions Use this dialog box to define the integer position (Figure 3-47). Figure 3-47 Positions Dialog Box Using the FractionLynx Editor 85

86 Rinse Station for Collector 1 Travel Height Dispensing Height Specifies the coordinates of the rinse station from the home position of the needle (i.e., the position of the needle when not in use). Defines the travel height above the plate that the rack will sit on. Defines the dispense height above the plate that the rack will sit on Bed Layout Use this dialog box to create, delete, or modify bed layouts (Figure 3-48). To display the Bed Layout dialog box, select Collector Setup > Bed Layout Editor. Note: References to rows and columns in the Bed Layout dialog box relate to the number of physical plates on the bed and not the number of tubes. 3 Figure 3-48 Bed Layout Dialog Box, Select/Create Bed Layout Page Creating a New Bed Layout 1. Highlight a bed layout similar to the one you want to create, then click the button to create a new layout. The layout appears in the Bed Layouts list as the same name with a 1 at the end, e.g., Waters x100 Rack. 2. To change the name of the layout, enter the new name in the Bed Layouts field and click the button. The name is updated in the Bed Layouts list. New bed layouts are saved to the MassLynx Racks directory and have a *.bwf extension. Fraction Collection Setup 86

87 Deleting a Bed Layout 1. Highlight the bed layout you want to delete, then click the button. A dialog box asks you to confirm the deletion. 2. Click OK to delete the bed layout. Other Bed Layout Options To change the number of rows in the current column, enter the new number in the Rows field and click the button. To append a new column, click the button. To delete the current column, click the button. To insert a column, click the column before which you want to insert and click the button. Note: The column inserted will have the same number of rows as the column highlighted. Modifying a Bed Layout 1. Click the Modify Bed Layout tab in the Bed Layout dialog box to change the plate position or type (Figure 3-49). 3 Figure 3-49 Bed Layout Dialog Box, Modify Bed Layout Tab Using the FractionLynx Editor 87

88 2. Click one of the plates to open the Plate Description dialog box (Figure 3-50). Use this dialog box to define the type of plate to use in this location. Figure 3-50 Plate Description Dialog Box 3. Select a new plate from the Plates list. 4. Change the plate position on the bed by entering a value for each measurement as follows: X value: Enter the measurement from the currently selected plate to the plate immediately to the left. Y value: Enter the measurement from the currently selected plate to the plate immediately above. If there is no plate (to the left or above), then measurements are taken from the Home position, which is where the needle sits when not in use. Volume of collection vessels: Enter the volume each tube will hold in milliliters. Note: Measurements for plate positions are always taken from the top left corner of each plate. Distances are measured in 1/10 th of millimeters, e.g., 1 mm = 10 units and 1 cm = 100 units. 5. Click OK to accept the new values. 6. Click OK to exit the Bed Layout dialog box Plate Generator Select Collector Setup > Plate Generator from the FractionLynx Editor to open the custom Rack Generator window (Figure 3-51). Fraction Collection Setup 88

89 Rack Name Rows Columns Vial Offsets Origin Grid Reference Figure 3-51 Rack Generator Window Enter the name of the plate to edit. Specify the number of vials in a horizontal row and the distance between the center of one tube and the next tube in the same row. In the example plates, there are four rows. Specify the number of vials in a vertical column and the distance between the center of one tube and the next tube in the same column. In the example plates, there are five columns. (These parameters do not affect fraction collection.) Specify alternate vial rows or columns to be offset. Specify the corner of the rack from which the vial grid referencing starts. Indicate how to reference the vial rows and columns, e.g. whether the rows are alphabetical or numerical. 3 Using the FractionLynx Editor 89

90 Referencing Select one of three options: XY, which references the vials A1, B1, etc. Sequential Discontinuous, which numbers the vials 1, 2, 3, etc. across a row, left to right, and then starts the next row from the left again. Sequential Continuous which numbers the vials 1, 2, 3, etc. across a row, left to right, then continues numbering the next row, right to left, etc. XY Referencing Sequential Continuous Sequential Discontinuous Priority Plate Size Top Left Vial Offset Select the Horizontal first check box if samples are to be acquired horizontally across the plate: If Referencing = X, Y, Horizontal = Letter, Vertical = Number, and Horizontal first is selected, samples are acquired in the order A1, A2, A3, etc. If the Horizontal first check box is not selected, samples are acquired in the order 1A, 1B, 1C, etc. If Referencing = Sequential Continuous or Sequential Discontinuous and Horizontal first is selected, samples are acquired from row 1, then row 2. If the Horizontal first check box is not selected, samples are acquired from column 1, then column 2, and so on. Define the size of the plate to its outside edges. Define the measurement to the center of the first vial from the top-left corner of the plate. 3 Creating and Deleting Plates To create a custom rack: 1. Click (New Rack) or select Rack > New Rack. This creates a new rack based on the Default Settings for New Rack described below. 2. Enter the appropriate values and click (Save) or select Rack > Save Current Rack. Fraction Collection Setup 90

91 To create a new rack based on an existing rack: 1. Display the existing rack. 2. Change the Rack Name. 3. Enter the appropriate values and click (Save) or select Rack > Save Current Rack. Use the (Previous Rack) and (Next Rack) toolbar buttons to page through the list of saved custom racks. The Previous Rack and Next Rack options on the Rack menu perform the same operation. New racks are saved to the MassLynx Plates directory. To delete a custom rack: 1. Select the rack to delete by typing the name in the Rack Name box or by paging through as above. 2. Click (Delete) or select Rack > Delete Current Rack. Note: All spacings and the vial section are stored in 0.1-mm units. Rotating Plates Select View > Rotate Rack to rotate a rack by 90 (Figure 3-52). 3 Scaling Plates Figure 3-52 Example of Rotated Rack 1. Select View > Scale Rack to open the Scale Rack dialog box (Figure 3-53). Using the FractionLynx Editor 91

92 Figure 3-53 Scale Rack Dialog Box 2. Move the slider or enter a new value to change the size of the rack as displayed in the Rack Generator window. 3. Click Close. Defining Default Settings for New Plates Select Tools > Default Settings for New Rack to open the Default Settings dialog box. This dialog box defines the default settings to use when creating a new rack. Field descriptions are the same as those for Figure Manual Control 3 When creating new plates, the Manual Control option allows plate positions to be checked by moving the needle to the specified coordinates. To display the Manual Control dialog box (Figure 3-54), select Collector Setup > Manual Control from the FractionLynx Editor. Figure 3-54 Manual Control Dialog Box Fraction Collection Setup 92

93 Moving to a Vessel on a Plate 1. From the Collector Number list, select the number of the fraction collector for which you want to verify parameters. 2. From the Plate Number list, select the number of the plate for which you want to verify parameters. 3. Enter the number of the vessel to move to in the field below Plate Number, then click Move. The needle moves to the required position. For WFCII units, the valve switches to the Open/Collect position. For Gilson units, the Divert to Vessel (collect) and Divert to Drain (waste) options can be used (see Diverting Sample on page 94). Moving to a Vessel 1. From the Collector Number list, select the number of the fraction collector for which you want to verify parameters. 2. Enter the number of the vessel to move to in the field below Move To Vessel, then click Move. The needle moves to the required position. Note: If the collector has more than one plate, then the numbers of the vessels on the second, third, etc. plate will continue from the previous plate. For example, enter 97 to move to vessel one on the second plate, 193 to move to vessel one on the third plate, etc. For WFC II units, the valve switches to the Open/Collect position. For Gilson units, the Divert To Vessel (collect) and Divert to Drain (waste) options can be used (see Diverting Sample on page 94). 3 Moving to an XY Coordinate STOP Attention: Do not move in the XY direction before changing Z. This will damage the needle. 1. From the Collector Number list, select the number of the fraction collector for which you want to verify parameters. 2. Enter values in the X and Y fields and click Move XY. The needle moves to the required position. Using the FractionLynx Editor 93

94 Moving to a Z Coordinate Note: This is a Gilson 215 only command. STOP Attention: Do not move in the XY direction before changing Z. This will damage the needle. 1. From the Collector Number list, select the number of the fraction collector for which you want to verify parameters. 2. Enter a value in the Z field and click Move Z. The needle moves to the required position. Moving to the Home Position 1. From the Collector Number list, select the number of the fraction collector for which you want to verify parameters. 2. Click Move Home. The needle moves to the Home position. Moving to the Rinse Station Position 1. From the Collector Number list, select the number of the fraction collector for which you want to verify parameters. 2. Click Rinse Station. The needle moves to the Rinse Station position. 3 Diverting Sample Note: This does not apply to Waters Fraction Collector II or III. Click Divert to Vessel or Divert to Drain to divert the sample to the required location. If Divert to Vessel is selected, then the sample is diverted to the current vessel. 3.7 Resetting Beds The reset option allows the status of a full bed to be reset to empty so that it can be reused for collection. The beds for all or selected collectors can be reset. Unless a reset is performed, when the next analysis starts, the first fraction will be collected in the next available vessel. Select this option from the FractionLynx Editor as follows: Select Reset Beds > Reset All Beds to set all collector beds to empty (the next available collection vessel will be set to vessel 1 at fraction collector 1). Note: This option cannot be selected if the mass spectrometer is acquiring and fraction collection is enabled. Resetting Beds 94

95 Select Reset Beds > Reset Collector n (where n is the number of a collector) to set the bed for the selected collector to empty. The next available collection vessel remains unchanged. Use this option when more than one collector is configured, so that a full bed can be changed for an empty bed and then reset while fraction collection is continuing in the other detector. Note: If the mass spectrometer is acquiring, the bed containing the next available collection vessel cannot be reset. 3 Using the FractionLynx Editor 95

96 Chapter 4 Collection Modes This chapter describes the various collection modes available in FractionLynx. Select these modes from the Collection Modes dialog box. 4.1 Definition of Terms Vessel / Well / Tube / Location Rack / Plate Bed / Bed Layout Collection tube or bottle. Physical holder or tray for fraction vessels. Configuration of a fraction collector in terms of the number and types of racks present. 4.2 Overview of Each Mode You can collect fractions into vessels using the following modes: Sequential Collection Each fraction (up to the maximum defined in the Fraction Collection Parameters) is collected into a separate tube. Subsequent fractions within a single run will be collected into sequential tubes. Fractions from sequential sample injections will be collected immediately following the last location from the previous run. One for One Collection Reserves a single tube for each run. The tube will be the same location as the injection position. In this mode only a single fraction will be collected. Any peak volume that does not fit into the collection tube or subsequent instances of a peak with the same mass will be ignored. Multiple Fractions Per Tube This mode is an extension to the One for One collection. FractionLynx allows for multiple instances of the same mass, or different masses, to be triggered and collected into the same tube up to its volume. Reserved Tubes A fixed number of tubes are allocated per run. 4 Definition of Terms 96

97 At the start of the next sample and tubes that were not used from the previous run are skipped. In this way the user will always know where on the bed their sample will collect. Once the reserved tubes have been used for a sample subsequent peak volume or new peaks will be ignored. Note: The collection mode is specified as a system-wide parameter rather than specified on a sample-to-sample basis. All runs will be collected using the same collection type, but can be triggered using different triggers and parameter files (*.frc). 4.3 Fraction Collector Tube Numbering Schemes Single Plate Per Fraction Collector All tubes are labeled numerically irrespective of the injector configuration (numerical or alphanumerical) or fraction collector rack type. The Plate Generator (select Collector Setup > Plate Generator) for FractionLynx will label each location starting from 1. See Figure Collection Modes 97

98 WFCII/III, 120 Tube Rack with Sequential Continuous using Horizontal first priority Gilson Code 21 Rack with Sequential Discontinuous Gilson Code 212 Plate with Sequential Discontinuous Microtitre plate with Sequential Discontinuous using Horizontal First Priority Microtitre plate with Sequential Discontinuous Figure 4-1 Plate Generator Types Multiple Plates Per Fraction Collector When there are multiple plates on a single fraction collector, the tube numbering will be sequential (see the examples in Figure 4-2, Figure 4-3, and Figure 4-4). Fraction Collector Tube Numbering Schemes 98

99 Each plate holds 30 locations/tubes. In FractionLynx, the tubes are reported as: Actual plate/reported as Figure Tube Plate Layout Each plate holds 60 locations/tubes. In FractionLynx, the tubes are reported as: Actual plate/reported as Figure Tube Plate Layout Collection Modes 99

100 Each plate holds 96 locations/tubes. In FractionLynx, the tubes are reported as: Actual plate/reported as Multiple Fraction Collectors Figure Tube Plate Layout Where multiple fraction collectors are used in a single system, the location of each vessel is indicated by FractionCollector:VesselPositionOnCollector a b c Figure 4-5 Plate Layouts for Multiple Fraction Collectors Fraction Collector Tube Numbering Schemes 100

101 TP01606A TP01606A TP01606A TP01606A In Figure 4-5: Fraction Collector 1 (WFCII/III with 2x code 21 racks) is being used to collect triggered fractions. Fraction Collector 2 (Fraction Funnel Bed) is being used to collect waste per sample. Any triggered fractions would be in positions 1:1 1:80. The waste collections would be in positions 2:1 2:90. In Figure 4-6: Four fraction collectors are being used to collect fractions. Each collector is configured with the same 120-location plate. Collected fractions would be labeled as: 1. 1:1 1: :1 2: :1 3: :1 4: Figure 4-6 Plate Layout for Four Fraction Collectors 4.4 Method of Operation for Each Mode This section describes how the fraction collector beds are used for each collection mode. For a more detailed description of how to use peak detection parameters to trigger actual fractions, see Appendix B. Set up collection modes from the FractionLynx Editor by selecting Collection Parameters > Collection Modes. This opens the Collection Modes dialog box. Collection Modes 101

102 4.4.1 Sequential Collection This is the basic method of collecting fractions and the most efficient in terms of maximizing the number of tubes that can be used. It is the default method. Make sure Enable sequential collection is selected, then enter a value in the Minimum tubes per run field (Figure 4-7). Figure 4-7 Collection Modes Dialog Box Key Features Successive fractions (whether from the same sample or different injections) will be collected into sequential tubes until the fraction collector is full. If there are multiple fraction collectors, then the next fraction will be collected from position 1 on the next fraction collector. After the last location is collected, MassLynx does the following: Finishes the current sample, but does not collect any more fractions Pauses the sample queue and indicates that the collector(s) are full Does not run any more samples until the user has indicated that new tubes have been provided Prevents OpenLynx from logging in any more samples until new tubes have been provided 4 Figure 4-8 shows how a series of samples and batches will collect into the fraction collector bed. Method of Operation for Each Mode 102

103 Figure 4-8 Example of How Samples and Batches Collect Batch Sample Num Fractions Samples continue to be run and the fraction collector bed will fill until it becomes full n 1 3 Once the fraction collector is full, it generates the following message (assuming that it is the last collector in the system configuration): The MassLynx Queue remains Waiting until the FractionLynx Editor has been used to Reset Beds. The MassLynx Sample List goes into Pause mode. If you try to remove the Pause, the Unable to Start Queue warning message box appears (Figure 4-9). Collection Modes 103

104 Figure 4-9 Unable to Start Queue Warning Message Box MassLynx will not run another sample until you indicate to the FractionLynx Editor that new tubes have been supplied, i.e., a Reset Collector. Select Reset Beds from the FractionLynx Editor menu. There are various options depending on the number of collectors configured in the system. For example: Reset All Beds Reset Collector 1 Reset Collector N Ctrl+A Ctrl+1 Ctrl+N (where N is the number of collectors in the system) Reset All Beds Reset Collector N Resets the FractionLynx counter to the first position of the first collector and assumes that all collectors are now free for use, i.e., have clean tubes. After this command executes and the Pause is removed from the MassLynx Sample List, the system continues to inject any outstanding samples until the end of the batch or the collector(s) fill up again. Resets individual collectors. For example: The system is configured with four collectors and they become full. You know that the fractions on collector 2 belong to you. The fractions on collectors 1, 3 and 4 belong to somebody else. You remove your samples from Collector 2, replace the tubes, and issue a Reset Collector 2 command. When the Pause is removed from the MassLynx Sample List, the system knows that a collector is now free to use. 4 Method of Operation for Each Mode 104

105 4.4.2 Enable User Starts When you select Enable User Start Locations, you can specify the collection tube for the first fraction of each injection. This is done by entering the collection tube number in the Fraction Start column on the MassLynx Sample List. Tubes are filled sequentially unless you specify a Fraction Start. In the following table, the first 9 tubes are skipped, as are tubes 26 to 29 and 42 to 49. Injection Fraction Start Number of Fractions Tubes Filled a a a a. If a Fraction Start is not specified, collection continues into the next sequential tube. STOP Attention: Fraction Start locations must be consistent with sequential collection. It is not allowed to start fractions out of numerical sequence (i.e., if the user start tube has been previously filled, the collection will go into the next available tube). User start locations could be used to force specified samples to start collecting on a new plate. 4.5 One for One Collection 4 Click Enable one for one collection in the Collection Modes dialog box. One for One Collection (or plate mapping) is designed to maintain integrity of the sample plate to allow for easy reformatting of samples from an application, such as library cleanup. Key Features Fraction collection location is determined by the injection location. Although there is one tube per run, waste can still be collected on a second collector. Collection Modes 105

106 4.5.1 One for One Collection Overrides the FractionLynx Parameters One for One Collection is designed to collect into one tube only. For this reason the Max. Fraction per Injection parameter (in the General tab of the Fraction File Editor) is ignored during operation. This feature means that a single method (*.frc file) could be used for both sequential and one to one mapping. This can be advantageous if the same trigger parameters are required for each Sample Plate Size Versus Fraction Collector Rack Size FractionLynx compares the size (in terms of number of locations/wells/tubes) of the injector plate to the size of the fraction collector racks and determines how the mapping will take place. In Figure 4-10, the sample plate (left) size is the same as the fraction collector rack size (right). Plate mapping is one sample plate to one collector rack. 4 Inject Collect Figure 4-10 Sample Plate Size Equals Collector Rack Size In Figure 4-11, the injector plate is smaller than the collector rack size. The extra locations on the collector rack will not be used for this batch or subsequent batches. One for One Collection 106

107 Wasted Inject Collect Figure 4-11 Unequal Sample and Collection Racks Figure 4-12 shows sample plates (#1 and #2 on the left) that are larger than the fraction collector racks. FractionLynx assigns enough racks to facilitate the collection of one plate into unique racks so that any two sample plates do not share a common collection rack. #1 4 #2 #1 #2 Figure 4-12 Sample Plates Larger than Collection Plates Collection Modes 107

108 4.5.3 Sample Plate Numbering Versus Fraction Collector Rack Numbering Create fraction collector racks using a numerical numbering scheme. It does not matter what the orientation is sequential continuous, sequential discontinuous, or with horizontal priority. Sample racks can, however, have a variety of numeric or alphanumeric schemes. In the case of a numeric sample plate, the mapping is straightforward. For alphanumeric plates, a translation must take place. Figure 4-13 shows a Microtitre Plate together with the different priorities that can affect the sampling and numbering order. The contents of the table represent the mapped fraction collector positions for each well location. 4 One for One Collection 108

109 4 Figure 4-13 Mapped Fraction Collector Positions for Each Well Location Collection Modes 109

110 4.5.4 Logging Multiple Batches Figure 4-14 shows four injection plates that could be submitted to run in a One to One Mapping mode for purification. (It is assumed that the plates will be submitted as four separate batches.) Injector Plates Collector Plates #1 #2 #3 #4 #1 #2 #3 #4 Figure 4-14 Four Injection Plates Running One to One If a user attempts to run samples from a fifth plate, the MassLynx Not Enough Vessels error message box appears (Figure 4-15). 4 Figure 4-15 MassLynx Not Enough Vessels Error Message Box OpenLynx The login procedure reports back that the fraction collector is full and that the plate could not be accepted. SampleList The user presses the Start button and the MassLynx Not Enough Vessels error message box appears, indicating that there was no more room. This same message appears if an attempt was made to run a batch (e.g. 96-well plate) and less than that number of tubes were available in free racks or plates. One for One Collection 110

111 In either case, you must select Reset Beds to replace used tubes. Figure 4-16 shows the unsuitability of this type of mapping for single shot samples. Single shot means small batches (e.g., one or two samples). Each time a batch is logged through OpenLynx or via the Sample List, then a fraction collection rack is assigned to that batch. In the example below: Batch 1 is logged into the system and rack 1 is assigned for it. Batch 2 is logged into the system and rack 2 is assigned for it. Batch 3 is logged into the system and rack 3 is assigned for it. Batch 4 is logged into the system and rack 4 is assigned for it. Collect Inject #1 #2 #3 #4 Figure 4-16 Mapping for Single Shot Samples 4 The fraction collector is now full despite only six actual samples being run Sample Running Order The order in which samples are run within a batch does not matter, i.e., they do not have to be in ascending or descending sample location order. Figure 4-17 (the plate was defined with Horizontal first priority) shows that the samples would be normally be run in the order: 1,A 3,G 6,E 6,G 9,C Collection Modes 111

112 However, running them as: 1,A 9,C 6,E 3,G 6,G or any other order does not matter. Multiple injections from the same location within a single batch will work; however, collection happens in the same tube until it is full. When the tube is full, the error message described in Section 4.5.4, Logging Multiple Batches, is generated. Figure 4-17 Plate Defined with Horizontal First Priority 4.6 Multiple Fractions Per Tube Select Collection Parameters > Collection Modes from the FractionLynx Editor. Click Enable one for one collection and Enable multiple fractions per tube in the Collection Modes dialog box (Figure 4-7). MultiFraction is an extension to the One to One Mapping feature. It uses the same rules with regards to rack allocations and tube numbering. 4 Multiple Fractions Per Tube 112

113 Consider a sample where the target mass elutes more than once from the column (Figure 4-18). Where the target equals The Sequential Mapping Technique (left) collects both the peaks, but loses the integrity of the plate format. The One to One mapping (right) retains the mapping integrity, but fails to collect the major peak. Enabling the MultiFraction option allows FractionLynx to collect multiple instances of the same mass into the same tube (up to the limit of the tube volume). 4 Figure 4-18 Sequential, One to One, and MultiFraction Mapping Collection Modes 113

114 4.7 Reserved Tubes The Reserve tubes per injection collection mode reserves a specified number of tubes to be used per injection. The range is 1 to 240. To use this mode: 1. Select Collection Parameters > Collection Modes from the FractionLynx Editor. 2. Click Reserve tubes per injection in the Collection Modes dialog box (see Figure 4-7). At the end of the run, any unused tubes are skipped before the next sample is injected (irrespective of whether samples are run individually or in a batch mode). Before the sample is injected (i.e., when the Sample List is started), FractionLynx checks the status of the fraction collector bed against the number of samples required for the whole batch. If there are not enough free tubes for collection, then the Sample List will not be run until enough tubes are available. This is achieved by several methods: Using Reset Bed type commands Reducing the number of samples in the list Adding extra capacity with another fraction collector(s) If there are not enough tubes available, then the error message in Section 4.4.1, Sequential Collection, appears. This mode of collecting is particularly attractive for labs that perform large batch runs (e.g., overnight/weekend) and need to perform manual or robotic reformatting of the samples at the end OpenAccess Implementation Many labs performing OpenAccess allow a chemist to log in a single sample. After processing, the chemist receives electronic results and fractions in a rack that contains nobody else s fractions. This implementation requires the minimum of handling, i.e., removing tubes from a large rack to place into a smaller one to take away. To keep separate results and fractions, labs can develop mini-racks or fraction blocks (Figure 4-19). 4 Reserved Tubes 114

115 Figure 4-19 Mini-Racks Each block contains 6 18 mm diameter tubes. These mini-racks or blocks can be placed into the fraction collector, and the chemist will have this as part of his or her results. The individual blocks can then be placed into a holder in the fraction collectors. The MassLynx/FractionLynx representation is shown in Figure 4-20 and Table 4-1. A tray has been designed to hold five of these blocks. Two trays can then be placed onto the WFC II or WFC III. In the bed layout, each block has been defined as a separate rack/plate. 4 Figure 4-20 Modified Bed Layout for Mini-Racks or Fraction Blocks Collection Modes 115

116 In Table 4-1 the bed is defined as two columns of five rows each, to provide the numbering scheme as shown below. Where: #n = physical block number a to b = reported tube location in FractionLynx Table 4-1 Bed Scheme Defined as Two Columns of Five Rows Row Column 1 Column 2 1 #1: 1 6 #6: #2: 7 12 #7: #3: #8: #4: #9: #5: #10: The same rules apply for this collection mode as for the other modes: Fractions will be collected into separate sequential tubes. Once the Max Tubes per Injection value is reached, no more fractions are collected for that run. Available from Genevac is a new style rack, compatible with the 2767 and 2757 (Figure 4-21). It is able to fit four special holder blocks that are also available from Genevac. The rack breaks up into four sample holders which do not require swings for loading directly into the evaporator. They are compatible with the EZ-2, HT-4X, HT-8, and HT-12. The rack is currently located in the Genevac Accessories Brochure, which can be downloaded from 4 Figure 4-21 GeneVac Rack Reserved Tubes 116

117 Chapter 5 Running FractionLynx Samples Use this chapter to set up your projects for running FractionLynx samples. 5.1 Setting Up Projects MassLynx uses a project structure that is designed to assist laboratories in GLP compliance. All acquisition settings files, raw data files, Sample Lists, etc., can be stored within a project. It is strongly recommended that you create a master project for each laboratory containing the basic information for the control of the instrumentation and data acquisition. The master project can then be used as a template to create individual projects. MassLynx comes with some predefined projects. Default.pro is the default project where all data are stored until a new project has been selected or created. For more information on projects, see the MassLynx User s Guide Root Directory The project root directory (<PROJECT_NAME>.PRO) contains seven subdirectories of which Acqudb, Data, and Sampledb are the most important Acqudb Subdirectory The Acqudb directory contains the instrument control files used in data acquisition. Table 5-1 lists typical file extensions found in the Acqudb directory. 5 Setting Up Projects 117

118 Table 5-1 Acqu Database Control Files Mass spectrometer tune Mass spectrometer acquisition Fraction collection parameters HPLC pump control: Waters 515 Waters 600 Waters 1525 Waters 2525 Waters 2690/5 Waters 2700 Waters CapLC Waters 2790/5 HP1100 Gilson Shimadzu Jasco 900 Jasco 1500 Ultimate ZMD*.dbf, ZQ*.ipr *.mdb *.frc *.w51 *.w60 *.w25 *.bgm *.wat *.w27 *.clc *.w29 *.h11 *.gil *.szu *.jas *.j15 *.ult Data Subdirectory The Data directory contains the raw data directories associated with a particular project. Each raw directory contains the raw mass spectral information, processed mass spectral information, fraction collection data, and any reports generated Sampledb Subdirectory The Sampledb directory contains the Sample List files (*.spl) and corresponding OpenLynx report files (*.rpt). 5.2 Sample List The Sample List (Figure 5-1) shows the key fields for a FractionLynx acquisition that will: Run samples, acquire data, and collect fractions. Perform OpenLynx processing and create an OpenLynx Browser report that displays FractionLynx target masses, other masses of interest, and the sites used for collection on the fraction collector. (These parameters are defined in the FractionLynx.olp file). Running FractionLynx Samples 118 5

119 A customized summary report can also be produced from the OpenLynx Browser. MassLynx is supplied with a default fraction collection Sample List format called FractionLynx. For details on Sample List formats, see the Sample List section of the MassLynx User s Guide. Define the following columns. If any of the required columns are not present, right-click in the Sample List, select Customize Display, then select the boxes for the required fields. Save this format by selecting Samples > Save > Format from the Sample List menu. Once the Sample List has been prepared, save it by selecting File > Save or Save As before continuing. Figure 5-1 Sample List 5 Sample List 119

120 Table 5-2 Sample List Properties Property File Name MS File Inlet File Bottle Inject Volume Process Parameter File Fraction File Mass A to Mass T Wavelength A to Wavelength J Description Raw data file name under which the data will be stored. Method containing the parameters that will control the mass spectrometer during data acquisition. Inlet method file that will control the HPLC gradient and injection parameters. Location of the sample in the autosampler tray. Volume of sample to be injected. Type of processing that will be performed on the data. This does not need to be defined if an OpenLynx Browser Report file is not required. If a Browser report file is required then it should be defined as OpenLynx (selected from the drop-down menu). Note: To produce the Browser report, the Auto Process Samples check box must be selected on the Start Sample List Run dialog box (Figure 5-3). Contains the name of the OpenLynx parameter file (*.olp), which defines the OpenLynx processing to be performed. Name of the Fraction Collection parameter file described in Chapter 3, Using the FractionLynx Editor. Different Fraction Collection parameter files can be selected for each sample, allowing a high degree of specificity for samples on the same Sample List requiring different detection techniques. If OpenLynx processing is to be performed, then define the masses to search for in these columns. Defined masses can also be used as fraction triggers. Masses can be supplied as the molecular weight (e.g., 357.2) or as the molecular formula (e.g., C22H31NO3). If the molecular formula is entered, the software translates it into the Monoisotopic Mass. Ionization is not accounted for, so the detection parameters in the Fraction Collection parameters file should include suitable references to +H and H on the Positive and Negative pages. (Not shown in example Sample List) If OpenLynx processing is to be performed, then define the wavelengths to search for in these columns. Defined wavelengths can also be used as fraction triggers. 5 Running FractionLynx Samples 120

121 Table 5-2 Sample List Properties (Continued) Property UV1 to UV4 Fraction Trigger 1 to Fraction Trigger 10 Mass A to Mass T Description When using a Waters 2487 UV Detector, set up the wavelength to be monitored in the Inlet Editor. In this case, the Fraction Trigger selected in the drop-down list shown in Figure 1-3 is UV1, not wavelengtha. The setting in the Wavelength column is for reporting purposes only and does not control the 2487 Detector. The software references the value set in the Fraction Trigger column (UV 1, UV 2, and so on) against the value set in the Inlet Editor. Specify which trigger type (up to 10) to use as the trace to determine when a fraction is to be collected. The triggers can be chosen from: Mass A to Mass T Wavelength A to Wavelength J Analog 1 to Analog 4 Mass TIC PDA TAC UV1 to UV4 No Trigger / Blank (If the field is left blank or No Trigger is selected, then no fraction is collected.) Mass-derived fraction collection will use the relevant masses from the Sample List together with the mass detection parameters from the Fraction Collection parameters file to perform fraction collection. It is possible to specify fewer mass triggers than masses displayed on the Sample List. An example of this is Sample 2 on Figure 5-2. In this acquisition Mass B will be used as the single Fraction Trigger. Mass A and Mass C will be ignored by FractionLynx for this sample. They will, however, be processed when OpenLynx is run to produce the Browser report. This is useful when you want to collect one specific mass, but are also interested in the presence of others. 5 Sample List 121

122 Figure 5-2 Sample List Example: Fewer Mass Triggers than Masses Table 5-3 Sample List with Fewer Mass Triggers than Masses Property Wavelength A to Wavelength J Analog Mass TIC PDA TAC Description Wavelength-derived fraction collection will use the relevant wavelengths from the Sample List, together with the detection parameters from the fraction file. If a PDA detector is being used, then PDA detection parameters are used. If an IEEE-controlled UV detector (Waters 2487) is being used, then UV detection parameters are used. Note: If the UV detector is not controlled via IEEE-488, the data must be acquired via the analog channels of the mass spectrometer (see Analog below). Takes the Analog Detection parameters from the Fraction parameter file together with the relevant analog input to determine when fractions should be collected. Enter Mass TIC in the Fraction Trigger 1 field. Mass TIC collection uses the same detection parameters (positive or negative depending on data acquisition mode), in terms of MIT, slopes and peak widths, as the Mass collection. The adducts are not used in Mass TIC detection. Enter PDA TAC in the Fraction Trigger field. PDA TAC collection uses the PDA detection parameters. 5 Running FractionLynx Samples 122

123 Table 5-3 Sample List with Fewer Mass Triggers than Masses (Continued) Property Fraction Start Fraction Boolean Logic Description Enter the tube number at which to start collection. The tube numbering begins at the top-left position of the collector rack and continues constructively until the last tube of the last rack. For example, in fraction rack 1, the last tube is number 100. The first tube on the next collection rack is number 101. Browse for a named Boolean expression by right-clicking in this column and selecting Browse. For details, see Section on page 62. Expressions can also be created by selecting Edit, which opens the FractionLynx Mixed Triggers Editor (see Figure 3-19 on page 65). 5.3 Starting a Run from the Sample List Select Start from the Run menu or click (Figure 5-3).. The Start Sample List Run dialog box opens Project Acquire Sample Data Auto Process Samples Auto Quantify Samples Run From Sample n To Sample m Shows where the acquired data will be stored, or from which directory the previously acquired data will be retrieved for processing. If this is incorrect, click Cancel and select the correct project before starting the Sample List run again (see MassLynx User Guide for further details). Allows the user to specify whether or not data is to be acquired. Do not check this box if you are post-processing only previously acquired data. Allows the user to specify whether or not data is to be processed. Selecting both this and the Acquire Sample Data check box acquires and processes the data automatically. Do not select this check box if you are only acquiring data. In order to generate an OpenLynx report (.rpt), specify the Process and Process Parameters fields (.olp) in the Sample List. Automatically quantifies the results and is not applicable to the FractionLynx process. Automatically defaults to the first and last samples in the current Sample List. You can alter these values to select a smaller range by typing new values into the relevant boxes. 5 Starting a Run from the Sample List 123

124 Priority Night Time Process Flags the Sample List as a priority process and put it above all nonpriority lists in the queue. Runs the Sample List at night. Figure 5-3 Start Sample List Run Dialog Box Process Allows for routines to be executed before, during, and after the Sample List has been run. For the FractionLynx application, there are no pre- or post-run processes. Click OK to add the Sample List to the Sample List queue on the mass spectrometer. The queue appears at the bottom of the MassLynx screen and allows the user to delete, prioritize, and pause Sample Lists. See the MassLynx User Guide for more details. 5 Running FractionLynx Samples 124

125 5.4 Displaying Fractions in MassLynx Chromatograms In addition to the options described in the Chromatogram chapter of the MassLynx User Guide, fractions can be displayed on a chromatogram. Select Fraction Display from the Chromatogram Display menu to open the Fraction Display Parameters dialog box (Figure 5-4). Display Fractions Fill Fractions Show On All Chromatograms Lines Fill Fraction Display Displays the Lines and Fill Colors on the chromatogram. Displays the region of the chromatogram from which the fraction was collected, using the fill colors described below. Shows the fraction collection regions on all chromatograms. If this box is not checked, only the fraction collection regions relating to the chromatogram display. For example, mass triggered fractions are displayed only on mass chromatograms. There are two sets of lines that can appear on the chromatogram. Start Line at the start of each fraction End Line at the end of each fraction For each line required: Select a Line type. Select a Colour from the list. Select a Width from the list. Select Solid, Dotted, or Dashed to display the line. Select None to hide the line. Displays the region of the chromatogram where the fraction was collected, using the fill colors described below. 5 Displaying Fractions in MassLynx Chromatograms 125

126 Figure 5-4 Fraction Display Parameters Dialog Box First Colour Second Colour Start Vessel Select the required colour from the list. The area of the chromatogram where the first fraction was collected will be displayed in this colour. If there are more than two fractions collected from a chromatogram, this colour will also be used to show the 1 st, 3 rd, 5 th, 7 th, etc. fractions. Select the required colour from the list. The area of the chromatogram where the second fraction was collected will be displayed in this colour. If there are more than three fractions collected from a chromatogram, this colour will also be used to show the 2 nd, 4 th, 6 th, 8 th, etc. fractions. Displays the location of the vessel where the collection of the fraction started. The value displayed on the chromatogram will be collector number:vessel number. If the collector has multiple plates, then the numbers of the vessels on the second, third, etc. plate will continue from the previous plate. For example, if there are 96 vessels to a plate, then the numbering will be: 5 Running FractionLynx Samples 126

127 Last vessel on first plate 1:96 First vessel on second plate 1:97 First vessel on third plate 1:193 Number Of Vessels Fraction Trigger Displays the number of vessels used to collect the fraction. Displays the mass that triggered the fraction. Chromatogram Type TIC Diode Array Mass Displayed TIC wavelength nm m/z = mass 5.5 Real-Time Display of Fractions in Chromatogram Select Real Time Update from the Chromatogram Display menu (or click the button) to display the chromatogram as the data is being acquired. If Display Fractions is selected on the Fraction Display Parameters dialog box, then when a fraction has been collected it will appear in the Chromatogram window. If Fill Fractions was selected, then the fraction will be filled with the selected fill colour. Note: Fractions are not displayed on the chromatogram until they have finished collection. This display lag time does not affect the performance of collection. 5 Real-Time Display of Fractions in Chromatogram 127

128 Chapter 6 FractionLynx Browser Introduction The FractionLynx Results Browser (Figure 6-1) simplifies the data review process for fraction collection. It is based on the OpenLynx Diversity Results Browser but differs in these important respects: Two plate panes can be viewed: one containing the sample plate and the other the fraction plate. A Fraction Tube Spectra pane is displayed. Note: As for the Diversity Browser, collected fractions can be viewed on the Chromatogram pane. It uses the same report file format (*.rpt) as the OpenLynx Browser and you can view both types of file in the other s browser. However, fraction plate data cannot be viewed within the Diversity Browser. Introduction 128

129 6.2 FractionLynx Browser 6 Figure 6-1 FractionLynx Browser There are eight panes displayed within the FractionLynx Browser: Sample Plate pane Fraction Plate pane Sample Description pane Fraction Collection Results pane Results Table pane Spectrum ane Fraction Tube Spectra pane Chromatogram pane FractionLynx Browser 129

130 6.2.1 FractionLynx Browser Toolbar 6 Button Menu Equivalent Purpose File > Open Open an existing FractionLynx Browser report. Edit > Copy File > Print Copy the selection and put it on the Clipboard. Print a FractionLynx Browser file. Go to first Well. Applies to sample plate only. Go to previous Well. Applies to sample plate only. Go to next Well. Applies to sample plate only. Go to last Well. Applies to sample plate only. Display previous plate. Applies to sample plate only. Display next plate. Applies to sample plate only. Display default range. View > Fractions by Tube Swap Plate View View > Swap List View Help > About FractionLynx Results Browser Swap between Fractions by Tube and Fractions by Trigger. Swap between normal plate view and multiple injection plate view. Swap between the Elemental Composition and the Library Search Results views. Display program information, version number, and copyright. FractionLynx Browser 130

131 6.3 Browser Files Opening an Existing FractionLynx Browser File 1. Click (Open), or select File > Open. 2. Select the required FractionLynx Browser results file (*.rpt) and click Open. 6 Copying a FractionLynx Browser File You can copy Plate, Sample Summary, and Spectrum List information to the Clipboard and paste it into other Windows applications. See Section on page 140 for more details. To copy a FractionLynx Browser file, click (Copy), or select Edit > Copy. Printing a FractionLynx Browser File 1. Click (Print), or select File > Print. The Print Control dialog box appears (Figure 6-2). Figure 6-2 Print Control Dialog Box 1. Click Print Current Sample to print information for the currently selected well. 2. Click Print All to print information for all wells on all plates. 3. Click Print Selection and select a From and To well number from the lists. 4. Click Print All Colours Black to print all colored data in black. 5. Change the margin settings for the printed report by clicking Margins. The Set Margins dialog box appears (Figure 6-3). FractionLynx Browser 131

132 6 Figure 6-3 Set Margins Dialog Box 6. Enter the required width in centimeters for the Top, Bottom, Left, and Right margins and click OK. 7. From the Print Control dialog box, click OK to print the report. Reports can also be printed from Windows Explorer. Select the required files, then select File > Print. Note: This method does not allow the user to select which samples are printed. Browser Files 132

133 6.4 View Options A number of options are available to change the appearance of the Browser screen. To access them, select View > Options Spectrum Page Figure 6-4 View Options Dialog Box, Spectrum Page FractionLynx Browser 133

134 Peak Annotation Area Decimal Places Accurate Mass Error Reporting Horizontal Axis Area Decimal Places WaveLength Label Daltons Label Peak/Tube Number Retention Time Process Description Set DAD Axis Label Select all Peak Spectra Default range to data Changes the number of decimal places displayed on peak annotation. Click the arrows to change this value. Range: 0 to 4 decimal places. Click Threshold (abs) or Threshold (%) and enter a value above which peaks must be, to be annotated on the spectrum. Displays the difference between the observed mass and the expected mass. Select the PPM or mda check box to display the results in parts per million or millidaltons. Changes the number of decimal places displayed on the horizontal axis. Click the arrows to change this value. Range: 0 to 4 decimal places. Enter the text to appear as the label on the horizontal axis for diode array data. Enter the text to appear as the label on the horizontal axis for mass spectral data. Displays the Peak number before the retention time in the Spectrum header. The Tube number is placed similarly for Tube Spectra over all tubes for the current sample. Displays the Retention Time in the Spectrum header. Displays the Process Description in the Spectrum Header. Displays a label on the DAD Axis. Enter the label to display in the adjacent text box. Displays all spectra for the selected well. If selected, displays the default mass display range of a spectrum (based on the peaks the spectrum contains). Displays only the mass range that actually contains data. If unselected, the default display range is the actual mass range the spectrum was originally acquired over. 6 Most of the options on the Spectrum page can be applied to the Tube Spectrum Display as well as the peak spectra. The only exceptions are the Accurate Mass Error Reporting group and Select All Peak Spectra. View Options 134

135 6.4.2 Chromatogram Page 6 Figure 6-5 View Options Dialog Box, Chromatogram Page Peak Integration Chromatogram Purity Peak Numbers Found Mass Displays the integrated baselines on the chromatogram trace. Displays the chromatogram peak purity on the chromatogram peak tops. Displays the peak number above a peak on the chromatogram peak tops. Displays the mass of a found peak. If selected, the Spectrum Purity and Decimal Places options become available. Click (arrows) or enter a value for the number of decimal places to display for the found peak. FractionLynx Browser 135

136 Spectrum Purity Base Peak Mass Retention Time Threshold (%) Horizontal Axis Decimal Places Label Fill Peaks Fill Background Process Description Replace DAD Set DAD Axis Label Displays the spectrum purity on the chromatogram peak top of a found peak. Displays the base peak mass values on the chromatogram peak tops. If selected, the Decimal Places option becomes available. Click (arrows) or enter a value for the number of decimal places to display. Displays the retention time values on the chromatogram peak tops. If selected, the Decimal Places option becomes available. Click (arrows) or enter a value for the number of decimal places to display. Value that peaks must be above in order to be annotated on the chromatogram. Change the number of decimal places displayed on the horizontal axis. Click the arrows to change this value. Enter the text to appear as the label on the horizontal axis. Select this check box to fill integrated peaks in green if found (or found tentative) and red if not found. This option uses colours from the Colors page. Select this check box to fill the area under integrated peaks in the Unused color, specified on the Colors page. Displays the process description on the chromatogram. Select this check box and enter the text to use in the process description displayed on the chromatogram. This replaces the text DAD which would normally be displayed. Select this check box to display a label on the DAD Axis and enter the label to display in the adjacent text box Default Plate Page For autosamplers controlled by the MassLynx software, a plate layout has already been defined (see the MassLynx Guide to Data Acquisition). This cannot be changed. For autosamplers not controlled by the MassLynx software, use the Default Plate page to organize the data acquired so that it can be displayed in plate format (Figure 6-6). View Options 136

137 6 Figure 6-6 View Options Dialog Box, Default Plate Page Click the Samples or Fractions option button to set up the default plate for either the sample or fraction plates. Rows Columns Origin Enter the number of rows on the plate to be used. Enter the number of columns on the plate to be used. This is the corner of the rack that the vial grid referencing starts from. Select either Top Right, Top Left, Bottom Right, or Bottom Left from the list. FractionLynx Browser 137

138 Method Method of numbering the wells on a plate. There are three options: XY which references the vials A1, B1, etc. Sequential Discontinuous which numbers the vials 1, 2, 3 across a row, left to right if origin is top left, and then starts the next row from the left again. Sequential Continuous which numbers the vials 1, 2, 3 across a row, left to right if origin is top left, then continues to number the next row, right to left, etc. 6 STOP Attention: If a Gilson autosampler or Waters 2700, 2790/2795 autosamplers are used with FractionLynx, then the vial referencing must be set to either sequential continuous or sequential discontinuous. Horizontal Vertical Horizontal priority If the method chosen is X,Y, then this check box becomes enabled. It allows horizontal referencing of the plate to be a number or a letter. If the method chosen is X,Y, then this check box becomes enabled. It allows vertical referencing of the plate to be a number or a letter. Select this check box if samples are to be acquired horizontally across the plate. If Referencing = X, Y, Horizontal = Letter, Vertical = Number and Horizontal First priority is selected, samples are acquired in the order A1, A2, A3. If Horizontal First priority is not selected, samples are acquired in the order 1A, 1B, 1C, etc. If Referencing = sequential continuous or discontinuous and Horizontal First priority is selected, samples are acquired from row 1, then row 2. If Horizontal First priority is not selected, samples are acquired from column 1, then column 2, etc Colors Page The Colors page (Figure 6-7) allows the user to define the colors used for graphic display in the FractionLynx Browser. 1. Select a color from the list. 2. Enter the mass to search for in the MassLynx Sample List or the OpenLynx Login PC. Threshold values appear on the Spectrum Test page of the OpenLynx Setup. View Options 138

139 6 Sample Plate Area Figure 6-7 View Options Dialog Box, Colors Page Found Found (Tentative) Not Found Not Searched Unused Multiple Injection The intensity of the peaks is above all threshold values. The intensity of the peaks is above the primary threshold but below the Confirmation threshold. For more details, see the Spectrum Test page in the OpenLynx Setup. There were no peaks for the mass entered. No mass was entered. There was no sample defined for this position on the plate. Multiple injections were made from the same well or vial. FractionLynx Browser 139

140 Fraction Plate Area Current Sample Fraction Collected No Fraction Select the color to be displayed for fractions belonging to the currently selected sample. Select the color to be displayed for the vials where fractions were collected. Select the color to be displayed if no fraction was collected Copy Control Page The Copy Control page (Figure 6-8) allows the user to define which information is copied to the Clipboard, for use in other Windows applications. Figure 6-8 View Options Dialog Box, Copy Control Page View Options 140

141 Sample Plate Fraction Plate Sample Summary Spectrum List Select this check box to copy Sample Plate information for all plates in the run. This copies a series of rows containing 1 for found compounds,? for found tentative compounds, 0 for compounds not found, + if no compound was searched for, for unused wells. Select this check box to copy Fraction Plate information for all plates in the run. This copies a series of rows containing 1 for fractions collected and otherwise. Select this check box to copy summary information for each sample. The Sample Summary information is written to the Clipboard using the settings for the Sample Summary and Results Summary in the current Report Scheme. Select this check box to copy details of each peak for each sample Column Pages The Column pages have similar formats and allow users to define which information is displayed in the Results Table, Fraction Results List, Elemental Composition Results List, and Library Search Results List panes. The Column pages are: List Columns page Elemental Columns page Fraction Columns page Library Columns page. Tube Columns To define information in each Column page: 1. Click the boxes next to the fields required. 2. To remove a column from the Results Table pane, right-click a column and select Remove Column. Columns can only be restored by selecting them again on these pages. List Columns Page The List Columns page (Figure 6-9) shows the number of decimal places to display for the relevant fields. These can be changed by right-clicking the field and selecting the number of decimal places from the pop-up menu. FractionLynx Browser 141

142 6 Fraction Display Page Figure 6-9 View Options Dialog Box, List Columns Page The Fraction Display page (Figure 6-10) allows the fraction collection regions on the chromatogram to be highlighted. For some examples of how this might affect the display, see Section 6.4.2, Chromatogram Page. View Options 142

143 6 Figure 6-10 View Options Dialog Box, Fraction Display Page Show Fractions Show on all Chromatograms Select this check box to display the Lines and Fill Colours on the Chromatogram pane. If this check box is selected, the fraction collection regions are shown on all chromatograms. If this check box is not selected, only the chromatograms relating to the fraction display fraction regions. FractionLynx Browser 143

144 Displaying Lines There are three sets of lines that can be displayed on the chromatogram. For each line required: 1. Select Line Type. 2. Choose a color from the drop-down list. 3. Select a width from the drop-down list. 4. Click Solid, Dotted, or Dashed to display the line. Click None to hide the line. 6 Start Line End Line Current Fill Fraction Display First Colour Second Colour Current Colour Shown at the start of each fraction. Shown at the end of each fraction. A set of lines displayed at each end of the fraction selected in the Fraction Results List pane. Select this check box to display the region of the chromatogram that the fraction was collected from, using the fill colours described below. Select the required colour from the drop-down list. The area of the chromatogram that the first fraction was collected will be displayed in this colour. If there are more than two fractions collected from a chromatogram, this colour will also be used to show the 1 st, 3 rd, 5 th, 7 th, etc. fractions. Select the required colour from the drop-down list. The area of the chromatogram that the second fraction was collected will be displayed in this colour. If there are more than three fractions collected from a chromatogram, this colour will also be used to show the 2 nd, 4 th, 6 th, 8 th, etc. fractions. Select the required colour from the drop-down list. The area of the chromatogram for the fraction selected in the Fraction Results List pane will be displayed in this colour. Double-clicking, with the right mouse button, on a fraction area of the chromatogram will make the fraction the current fraction. The fraction will be selected in the Fraction Results List pane and the Current colours and lines will be used in the Chromatogram pane Other Display Options Displaying Two Documents Simultaneously 1. Select File > Open. This displays a new window over the top of the previous one. View Options 144

145 2. Select Tile or Cascade from the Window menu. This displays the two child windows in the style chosen. 3. Repeat steps 1 and 2 as often as required. Displaying Two Different Views of the Same Document 1. Select Window > New Window to create a copy of the current window, named filename:2. 2. Select Window > Cascade > Tile Horizontally or Window > Cascade > Tile Vertically to display the new window along side the existing document to compare results of similar searches. 6 Changing the Size of a Pane To change the size of the panes on display, position the mouse pointer on the line between the two panes until the symbol appears. Alternatively, select Window > Split, hold down the left mouse button, and drag until the pane is the required size. 6.5 Other Menu Options File Menu Load on startup Send To If this option is selected, the next time the Browser is opened, the last Browser file viewed is automatically loaded. A tick mark appears next to the item when selected. Selecting this option again will turn it off. If this option is selected, an message is created with the currently selected report attached to it. Enter the address to send it to and click Send. STOP Attention: To view the entire report the recipient of the must have the MassLynx (with the FractionLynx option) or FractionLynx Browser software installed. The attachment can then be saved as normal and opened in MassLynx or the Browser. Double-clicking the report in the message can also open it. If the Browser software is not installed as a stand-alone program, the Program Not Found dialog box appears (Figure 6-11): FractionLynx Browser 145

146 6 Figure 6-11 Program Not Found Dialog Box Click Locate and select the MassLynx directory, or enter the location of the MassLynx directory and then click OK. Failed Samples Options Select File > Failed Samples Options. The Failed Samples Options dialog box (Figure 6-12) allows parameters to be defined when the Create List of Failed Samples option is selected. Figure 6-12 Failed Samples Options Dialog Box Enable ing of Report File Create File In Select this check box to the *.rpt file (formed when the *.olb file is processed) to the recipient specified in the Address field. The *.olb file is processed when the user submits it to the MassLynx queue. Enter the location to which you want the failed samples *.olb file to be saved. Other Menu Options 146

147 Create List of Failed Samples Select this option to create a *.olb file containing all of the samples that were marked as Not Found. The *.olb file can then be submitted to the MassLynx queue and analyzed, using a different ion mode or ionization method. This procedure must be carried out in the following order: 1. Analyze the original Sample List in electrospray positive mode. 2. Select File > Create List of Failed Samples to collect the Not Found samples into a *.olb file. 3. Analyze the samples in the *.olb file in electrospray negative mode. If necessary, repeat this procedure using APCI positive mode and APCI negative mode View Menu Figure 6-13 View Menu Refresh Swap List View Fractions by Tube Multiple Injections Per Vial This option rereads the current Browser Report and updates the display information. This command should be used if the content of the Browser Report has changed as a result of processing further samples. This option allows the user to swap between the Elemental Composition Results List view and the Library Search Results List view, if both lists have been generated. Select this option to view fractions by Tube. Unselected fractions will be viewed by Trigger. When this option is selected the user can swap between normal plate view and multiple injection plate view. FractionLynx Browser 147

148 Toolbar Status Bar If this option is selected from the View menu, then the toolbar will be visible. A tick mark appears next to the item when selected, selecting the option again will turn it off. If this option is selected from the View menu, then the status bar will be visible. A tick mark appears next to the item when selected, selecting the option again will turn it off Window Menu The Window menu (Figure 6-14) provides window options and allows you to view other open Browser files. The currently viewed file is checked. Figure 6-14 Window Menu New Window Cascade Tile Arrange Icons Split Select this item to create a copy of the current window. This is useful for displaying different views of the same document. The second document will have a :2 after the name. To change between documents, select the document required from the documents listed at the bottom of the Window menu. A tick will appear next to the document that is currently active Select this option to arrange document windows so that the title bar of each window is visible Select this option to arrange open windows side by side on the screen, dividing the available space equally between the open windows so that they are all visible. Select this option to arrange all iconized windows into rows. Selects the splitter bar on the selected pane. Other Menu Options 148

149 6.6 Sample Plate Pane The Sample Plate pane (Figure 6-15) displays information about all the samples from the currently selected plate. The vials are color-coded to indicate whether they contained a sample in which compounds were: Found Light green Found (Tentative) Olive green Multiple Sample Pink Not found Red Not Searched Blue Contained no sample Grey. 6 Figure 6-15 Sample Plate Pane The default colors can be changed (see Figure 6-7). The currently selected vial is shown as recessed. Change the current vial by clicking a new vial, using the vial selection toolbar buttons (,,, and ) or the arrow keys. Information about a highlighted well will be displayed in the other panes. Use the previous plate and next plate toolbar buttons to change the current plate number. FractionLynx Browser 149

150 If multiple samples were injected from a well, it will be pink and, if highlighted, the rest of the panes will be blank. The following message appears in the Sample Description pane (Figure 6-17): Multiple samples in this batch were injected from this position. Please use the Multiple Injection Plate View. View the samples by selecting View > Multiple Injections Per Vial, or by clicking (Swap Plate View). Use the View Options command to modify the appearance of a plate. This enables the number of rows and columns, reference labels, and vial colors to be set. See Section 6.4 on page 133 for more details Fraction Plate Pane The Fraction Plate pane (Figure 6-16) gives a graphical representation of the fractions collected from the samples injected from the sample plate. The currently selected sample in the Sample Plate pane displays the fractions collected from it in the Fraction Plate pane. By default,the colors are as follows: Current samples Blue Collected Fraction Green No Fraction Grey The default colors can be changed (see Section on page 138). Figure 6-16 Fraction Plate Pane Fraction Plate Pane 150

151 6.8 Sample Description Pane This pane shows a list and description of the masses found in the selected well (Figure 6-17). The pane can be expanded using the arrow controls to disclose source information. The width of the pane can be changed by positioning the mouse pointer on the heading between two columns until the symbol appears, and then clicking and dragging the column separators in the list header. 6 Figure 6-17 Sample Description Pane Submitter Sample Vial ID Description The username entered on the first page of the OpenLynx Login program. The file name of the sample, as entered in the Login dialog box or Sample List. The plate number followed by the well column and row references. The sample ID as entered in the Login dialog box or Sample List. The file description, as entered in the Login dialog box or Sample List. 6.9 Fraction Collection Results Pane The Fraction Collection Results pane (Figure 6-18) lists fractions collected and will not appear if fraction collection was not performed. The width of the columns can be changed by clicking and dragging the column separators in the list editor. Figure 6-18 Fraction Collection Results Pane FractionLynx Browser 151

152 The two available views are View by Trigger and View by Tube. These views can be toggled by selecting View > Fractions by Tube from the Browser menu (see Section 6.5.2, View Menu). View by Trigger When you select View > Fractions by Tube, the following columns are displayed within the Fraction Collection Results pane: 6 Fraction Trigger Original Target Ion Mode Start Time End Time Collection Site No. Of Tubes Export Fraction Pool Tubes The mass on which the fraction collection was triggered. Choosing a fraction trigger from the list will select the first tube collected from that fraction on the Fraction Plate pane and highlights the corresponding peak on the Chromatography pane. The mass minus any adducts. The ion mode of the collected fraction. The retention time at the beginning of the fraction collection. The retention time at the end of the fraction collection. The vial numbers of the fraction collected. The number of tubes the fraction collection required. Indicates whether to export the fraction to the text file. Default = YES. Double-clicking this column opens a combo box, which allows the default value to be changed, see Section 6.9.1, Fraction Trigger and Fraction Tube Export Options. Indicates whether all tubes for the fraction should be pooled or not. This information is exported to the text file. Default = NO. Double-clicking this column opens a combo box, which allows the default value to be changed. View by Tube If you do not select View > Fractions by Tube, the following columns are displayed within the Fraction Collection Results pane: Tube Trigger Original Target The mass on which the fraction collection was triggered. Choosing a tube from the list displays the corresponding tube on the Fraction Plate pane. The time period over which the selected tube was collected is highlighted on the Chromatogram pane. The mass minus any adducts. Fraction Collection Results Pane 152

153 Ion Mode Start Time End Time Collection Site Export Tube The ion mode of the collected fraction. The retention time at the beginning of the tube collection. The retention time at the end of the tube collection. The vial number of the tube collected. Indicates whether to export the tube to the text file. Default = YES. Double-clicking this column opens a combo box, which allows the default value to be changed (see Section 6.9.1, Fraction Trigger and Fraction Tube Export Options) Fraction Trigger and Fraction Tube Export Options Individual Fraction Tubes and Triggers can be included or excluded from the exported file so as to create a file with the required information for further processing of the collected fractions. In View by Trigger, the export and pool columns can be edited by double-clicking the entry in the appropriate column of the Fraction List pane. Figure 6-19 View by Trigger: Selecting NO For Export Trigger Selecting NO for a Fraction Trigger (see Figure 6-19) causes all Fraction Tubes for this trigger to be set to NO (as shown in Figure 6-20). Figure 6-20 View by Tubes: All Fraction Tubes Set to NO for Trigger Changing the export field of one tube back to YES (see Figure 6-21) causes the corresponding fraction to be exported (as shown in Figure 6-22). FractionLynx Browser 153

154 6 Figure 6-21 View by Tubes: Changing One Tube to YES Figure 6-22 View by Trigger: Corresponding Fraction Reverting to YES The default behavior is for all fraction trigger and tube information to be written to the exported text file. If the option Include All Fraction Triggers in Delimited File is cleared (described in Section ), then only fraction triggers with YES in their export field will be included in the exported file. The same is true for fraction tubes Results Table Pane Figure 6-23 Results Table Pane For the analysis of complex libraries where online chromatography is used, the YES/NO answer is supplemented with a results table. This table is split into the following columns: Results Table Pane 154

155 Peak Number Compound Mass Function Time Trace This lists the number of compounds that have been detected using the parameters specified in the OpenLynx Setup. Each detected compound has its own peak number. If an entered mass is detected in a particular Peak Number then this column will display 'Found'. If an entered mass is detected in a particular spectrum then this column will display the mass followed by the % Purity. If no compounds were found this will be blank. This column states the MS function and ionization technique in which the chromatographic peak was detected. The retention time of the spectrum in decimal minutes. Description of the chromatogram used to locate this spectrum. This may be blank if a specific retention time was used to locate spectrum. The information listed here can be, among others, TIC, DAD, or a mass chromatogram. 6 % Total Area The area of a particular peak represented as a percentage of the total area of all the peaks detected using a particular trace. Height RT Index RT Log P Concentration Amount State The height of a particular peak. The retention index of a particular peak. The measure of the hydrophobicity of a particular peak. If quantification was selected, this column will display the concentration of the detected compound. '.' will be displayed if the concentration was not calculated for this peak. This column will display the Concentration, multiplied by a User Factor defined in the Sample List, or the Factor specified on the Quantify page of the OpenLynx Setup. The Factor can be a user-specified number, or the number of nitrogens in the chemical formula, depending on the option selected. '.' will be displayed if the amount was not calculated for this peak. If a good quality spectrum was found, the State field contains the message OK. If the State was not found to be OK, then the State field may contain one or more of the following messages: Overloaded, Noisy, or Too few peaks. FractionLynx Browser 155

156 Changing the Width of a Column The width of the columns can be changed, by positioning the mouse pointer on the heading between two columns until the symbol appears, and then clicking and dragging the column separators in the list header. Removing a Column Columns can be removed from the display. Right-click a column heading, then select Remove Column. 6 Note: Columns can only be restored from the List Columns dialog box. Selecting a Result The result highlighted will be displayed in the Spectrum, Chromatogram, and Sample Description panes. To select another result to view, click any part of the row of the result required or use the arrow keys to page up and down the list of results Spectrum Pane This pane displays the Mass Spectra of the index entry highlighted in the Results Table pane (Figure 6-24). Figure 6-24 Spectrum Pane When a new sample is selected, the first spectrum in each of the acquisition functions is displayed. Individual spectra can be displayed by selecting the corresponding Peak Number field in the Results Table pane. Multiple entries can be displayed by holding down the Ctrl key and clicking the required entries or holding down the Shift key and clicking the last entry in a block. The Spectrum pane will appear blank if no sample is currently selected or if the current size of the pane is not large enough to show all the spectra currently required. The text in red on the left side of the pane is the mass spectrum retention time. Spectrum Pane 156

157 The text on the right side of the pane shows the acquisition function type and ionization mode followed by the absolute intensity of the largest peak in the spectrum, on the next line. Altering the Range of the Horizontal Axis (Zoom) with the Mouse Press at one end of the region of interest, and without releasing the button, drag the mouse horizontally to the other end. As you drag the mouse you will see a rubber band stretched out to indicate the range you have selected; do not go beyond the bounds of the axis. When you release the mouse button, the selected range will be redisplayed to fill the current window. This operation can be repeated as often as required. Clicking the toolbar button will restore both the Spectrum and Chromatogram display to the default range Fraction Tube Spectra Pane The Fraction Tube Spectra pane (Figure 6-25) displays the spectra for the tubes of collected fractions. The display range defaults to take into account the peaks being shown. Figure 6-25 Fraction Tube Spectra Pane The text in red on the left side of the pane is the mass spectrum retention time. The text on the right side of the pane shows the acquisition function type and ionization mode followed by the absolute intensity of the largest peak in the spectrum, on the next line. The range of the horizontal axis can be changed as described in the pervious section. The spectra displayed depend on whether view by fractions/tube is selected (see Section 6.5.2, View Menu). FractionLynx Browser 157

158 View by Trigger When a new fraction vial location is selected on the Fraction Plate pane, the spectra for all acquisition functions for that tube will be displayed. When a new fraction is selected from the Fraction Collection Results pane, all spectra for all functions and all tubes belonging to that fraction will be displayed. When a new sample is selected, all spectra for the first fraction will be displayed. A vertical scroll bar is available to scroll through all spectra for the currently selected fraction. 6 View by Tube When a new fraction vial location is selected from the Fraction Plate pane, the spectra for acquisition functions for that tube will be displayed Chromatogram Pane This pane displays the processed chromatogram of the Peak Number entry highlighted in the Results Table pane. The text in red on the left side of the pane is the chromatogram description. The text on the right side of the pane is an indication of the maximum intensity of the chromatogram. For further details see the OpenLynx Software User's Guide, Chapter 3, OpenLynx Browser. See Altering the Range of the Horizontal Axis (Zoom) with the Mouse on page 157 for details on how to change the display. Viewing Other Chromatograms Each entry in the results table can have one or more chromatograms associated with it. To view another chromatogram, page down using either the scroll bar or the arrow keys Chromatogram Fraction Display The Chromatogram display can be annotated to show the region of collection for the current tube/trigger. Other tubes/triggers are shown in a different color. Colors are selected from the Fraction Display page (see page 142) by selecting View > Options. Examples of the various viewing options are given below. Figure 6-26 shows the Chromatogram pane with the options Show Fractions and Show on all Chromatograms selected (see Fraction Display Page on page 142). In addition, dashed line was selected for the currently selected fraction and solid line for other fractions, both at the start and end of the fraction. Fractions are being displayed by trigger. Chromatogram Pane 158

159 6 Figure 6-26 Chromatogram Pane with Fractions Displayed on all Chromatograms Figure 6-27 Chromatogram Pane with Fractions Not Displayed on all Chromatograms Figure 6-28 Chromatogram Pane with View Fractions by Tube In Figure 6-28, the same display as Figure 6-26 is shown but with Show on all Chromatograms not selected. Figure 6-28 shows the same display as Figure 6-27 but with View > Fractions by Tube cleared, thus each tube is displayed. FractionLynx Browser 159

160 6.14 Elemental Composition / Library Search Pane Under some circumstances additional panes can be present. See Chapter 3, OpenLynx Browser, in the OpenLynx Software User s Guide Report Schemes Opening a Report Scheme 1. Select File > Report Scheme Open. 2. Select the required *.frs file from the dialog box. 3. Click Open. Saving a Report Scheme 1. Select File > Report Scheme Save As. 2. Select the required location and enter a *.frs filename in the dialog box. 3. Click Save Report Scheme Settings The information on printed reports can be defined using the Edit Report Scheme Settings dialog box (Figure 6-29). To open this dialog box, select File > Report Scheme Settings. Elemental Composition / Library Search Pane 160

161 Print Control Page 6 Figure 6-29 Edit Report Scheme Settings Dialog Box, Print Control Page FractionLynx Browser 161

162 Print Reports Area Sample Plate summary Fraction Plate summary Sample summary Delimit Sections New Line Per Peak New Line Per Trigger New Line Per Tube Sample Report Sample on New Page Select this check box to produce a Sample Plate report. This is a picture of the plate showing 1 for found compounds,? for found tentative compounds, 0 for compounds not found, + if no compound was searched for, and for unused wells. Select this check box to produce a Fraction Plate report. This is a picture of the plate showing 1 for a fraction vial containing a collected fraction and a for unused wells. Select this check box to produce a summary report for a sample. Note: The number of samples printed is controlled from the Print Control dialog box. See Printing a FractionLynx Browser File on page 131. When selected allows the MS Peak Results, Fraction Tube Results, and Fraction Trigger Results to be printed in separate sections. In each section, the sample summary fields will be appended to the beginning of each line. It also enables the New Line Per Peak/Trigger/Tube options (below). These options together allow control over the formatting of files exported to Microsoft Excel (see Section , Producing Tab-Delimited Files). Select this check box to start each peak on a new line. Select this check box to start each trigger on a new line. Select this check box to start each tube on a new line. Select this check box to produce a more detailed report for a sample. Select this check box to start each sample of the Sample Report on a new page. 6 Peak Information Tables Area To display peak information tables below a chromatogram, select the check box relevant to the type of information required. Report Schemes 162

163 Format Sample Report Area Spectra height (mm) Chromatogram height (mm) All Chromatograms On One Axis Mass Chromatograms/Axis Maximum Spectra Displayed Side by Side Chromatograms Side by Side Spectra Page Orientation Select this check box and enter the required height, to print the spectra associated with a sample. Select this check box and enter the required height, to print the chromatograms associated with a sample. Select this check box to use one axis for all chromatograms. Select this check box and enter the number of chromatograms to display on each axis. This makes overlaid mass chromatograms easier to view. The default of 1 will display each chromatogram on a different axis. To limit the number of spectra printed on a page, select this check box and specify the maximum number of spectra to print. Select this check box to print two chromatograms per line. Note: The Peak information table is not displayed if this option is chosen. Select this check box to print two spectra per line. Note: Library search results and elemental calculations are not displayed if this option is chosen. Click Portrait or Landscape to print the report with the required page orientation. 6 Chromatogram/Spectrum Print Order Area Define the order in which chromatograms and spectra are printed by clicking MS First, Analog First, or DAD First for chromatograms, and Time Order or Chromatogram Order for spectra. FractionLynx Browser 163

164 6.16 Report Column Selection Pages The basic format of the Report Column Selection pages (Sample Summary Report page) is the same; each lists fields that can appear as column headings on the type of report selected. Click the corresponding tab for the type of report required. The Report Column Selection pages are as follows: Fraction Tube Summary page Fraction Trigger Summary page Spectrum Report page Spectrum Search Report page Sample Header page Chromatogram Report page Sample Summary page MS Peaks Results Summary page Report Header page Elemental Report page. Decimal Places page. 6 Adding a Field 1. Select a field from the Available Fields list. 2. Click the button. The new field is added to the bottom of the Reported Fields list. Removing a Field 1. Select the field to remove from the Available Fields list. 2. Click the button. The field is removed from the Reported Fields list. Changing the Order of Fields 1. Select the field to be moved in the Available Fields list. 2. Click the or button until the field moves to the required position. Changing the Field Alias If the field name in the Available Fields list does not correspond to a description the user will recognize, enter a different name in the Alias field, e.g., State could be displayed on the report as Pass/Fail. This field can also be used to display field names in another language. Report Column Selection Pages 164

165 Changing the Field Width 1. Enter a new value in the Width field. Note: This option is not available on the Sample Header and Report Header pages. 2. Click OK to accept the new value. 6 Figure 6-30 Edit Report Scheme Settings Dialog Box, Sample Summary Page Sample Summary Page Select Include Sample Summary Fields in Summary Text File to select or clear all Summary fields at once. FractionLynx Browser 165

166 MS Peaks Results Summary Page 1. Select Nominated Results Trace, Largest Peak Only, or All Found Peaks from the Nominated Results Trace list to define the type of trace used for calculating the Area % Total. 2. If a chromatogram has more than one found peak, you can include the Largest Peak Only, All Found Peaks, or All Peaks in the report. 3. Select Include MS Peaks in Summary Text File to select or clear all highlighted MS Peak fields at once Report Header Page 1. Enter text to appear at the top of a report in the Report Header Text field. This text appears between the Report name and Submitter name, if selected (see below). 2. Select Display report name to print OpenLynx Report at the top of the report. If this text is not required, clear the check box and enter your own text in the Report Header Text field. 3. Select Display Submitter name to print the submitter s name as defined in the OpenLynx Login program (User name). 4. Select Display date and time to print the date and time of printing Sample Header Page 1. Select Print Sample Report Title to print Sample Report at the top of each page of the report. 2. Select Print Sample Header to print the sample header for each sample Chromatogram Report Page The Chromatogram Report page has one extra field, Current Trace. Each type of trace can have a different set of fields associated with it. Select each option from the list and select the fields you want to report for this type of chromatogram Fraction Tube Summary Page Select Include Fraction Tube Spectra in Sample Report File to print spectra for fraction tubes. Select Include all Fraction Tube Fields in Summary Text File to select or clear all highlighted fields at once. Select Include all Tubes in Delimited File to display information for all tubes in a delimited text file (see Section 6.9.1, Fraction Trigger and Fraction Tube Export Options). Report Column Selection Pages 166

167 Fraction Trigger Summary Page Select Include all Fraction Trigger Fields in Summary Text File to select or clear all highlighted fields at once. Select Include all Triggers in Delimited File to display information for all triggers in a delimited text file (see Section 6.9.1, Fraction Trigger and Fraction Tube Export Options) Decimal Places Page Use this page to choose the number of decimal places to display (0 to 4) for the criteria shown (Figure 6-31). Figure 6-31 Edit Report Scheme Settings Dialog Box, Decimal Places Page FractionLynx Browser 167

168 Producing Tab-Delimited Files From the Print Control page, select Sample Summary (see page 162). The four check boxes below sample summary produce the behavior described in the following section. Delimit Sections Output will be in sections with the following headings: PEAKSUMMARYRESULTS FRACTIONTRIGGERRESULTS FRACTIONTUBERESULTS Each section can be excluded by clearing the Include <selected> Fields in Summary Text File check box on the corresponding tab on the Edit Report Scheme Settings dialog box. For example, clearing the Include MS Peaks Results Fields in Summary Text File on the MS Peaks Results page omits the PEAKSUMMARYRESULTS section from the Sample Summary. Thus, none of the fields selected on the MS Peaks Results page would appear in the exported information. 6 Within each section, the results for each sample will be on one line unless any of the following options are selected: New Line Per Peak New Line Per Trigger New Line Per Tube Output to the Sample Summary page will be formatted in delimited sections with the information for each MS Peak/Fraction Trigger/Fraction Tube on a new line as depicted in Table 6-1. The Sample Summary information is repeated on each line. Table 6-1 Sample Summary PEAKSUMMARYRESULTS SampleSummary[sample 1] MSPeaksSummary[sample 1, peak 1] SampleSummary[sample 1] MSPeaksSummary[sample 1, peak 2]... SampleSummary[sample 1] MSPeaksSummary[sample 1, peak n] SampleSummary[sample 1] MSPeaksSummary[sample 2, peak 1].... SampleSummary[sample n] MSPeaksSummary[sample n, peak n] Report Column Selection Pages 168

169 Table 6-1 Sample Summary (Continued) FRACTION TRIGGER RESULTS SampleSummary[sample 1] FractionTriggerSummary[sample 1, trigger 1] SampleSummary[sample 1] FractionTriggerSummary[sample 1, trigger 2]... SampleSummary[sample 1] FractionTriggerSummary[sample 1, trigger n] SampleSummary[sample 1] FractionTriggerSummary[sample 2, trigger 1].... SampleSummary[sample n] FractionTriggerSummary[sample n, trigger n] FRACTIONTUBERESULTS SampleSummary[sample 1] FractionTubeSummary[sample 1, tube 1] SampleSummary[sample 1] FractionTubeSummary[sample 1, tube 2]... SampleSummary[sample 1] FractionTubeSummary[sample 1, tube n] SampleSummary[sample 1] FractionTubeSummary[sample 2, tube 1].... SampleSummary[sample n] FractionTubeSummary[sample n, tube n] 6 In Table 6-1 above example: SampleSummary refers to the fields chosen on the Sample Summary page. FractionTriggerSummary refers to the fields chosen on the Fraction Trigger Summary page, similarly for FractionTubeSummary and MSPeaksSummary. If Include Fraction Triggers in Summary Text File is cleared but the corresponding check boxes on the Sample Summary, MS Peaks Results Summary, and Fraction Tube Summary pages are selected, the output in Table 6-2 would be produced. Table 6-2 MS Peak Results and Fraction Tube Summary PEAKSUMMARYRESULTS SampleSummary[sample 1] MSPeaksSummary[sample 1, peak 1] SampleSummary[sample 1] MSPeaksSummary[sample 1, peak 2]... SampleSummary[sample 1] MSPeaksSummary[sample 1, peak n] SampleSummary[sample 1] MSPeaksSummary[sample 2, peak 1].... SampleSummary[sample n] MSPeaksSummary[sample n, peak n] FractionLynx Browser 169

170 Table 6-2 MS Peak Results and Fraction Tube Summary (Continued) FRACTIONTUBERESULTS SampleSummary[sample 1] FractionTubeSummary[sample 1, tube 1] SampleSummary[sample 1] FractionTubeSummary[sample 1, tube 2]... SampleSummary[sample 1] FractionTubeSummary[sample 1, tube n] SampleSummary[sample 1] FractionTubeSummary[sample 2, tube 1].... SampleSummary[sample n] FractionTubeSummary[sample n, tube n] 6 Report Column Selection Pages 170

171 Chapter 7 FractionLynx with OpenLynx You should read the OpenLynx Software User s Guide before attempting to work through this chapter. Before attempting to fill in the required fields, it is important that the methods be created first. The only extra parameter required to use FractionLynx with OpenLynx is the Fraction Method. The Fraction Method is the fraction collection parameters file. Select the required file from the drop-down list. All fraction collection parameters files (*.frc) in the Acqudb directory of the current project are available for selection. 7 Note: Ensure that the Loop Mode option remains unselected when performing chromatographic processing. For additional information, see the Waters OpenLynx Software User s Guide. 7.1 Setting Up the Walk-up Page Access the Walk-up page by selecting OpenLynx > Setup. This page (Figure 7-1) is used to define parameters for each run. Setting Up the Walk-up Page 171

172 7 Figure 7-1 Setting Up the Walk-up Page Parameter Description Input Fields Maximum Samples Priority Process Night Time Process Description Displays a description of the method. Lists the fields that are displayed on the Login PC for user input. See Adding or Deleting Input Fields on page 173 for information on adding or deleting input fields. Enter the maximum number of samples to be processed in one OpenLynx login session. Defines the job submitted from the OpenLynx Login program as a priority process. Note: All samples processed using this method become priority processes. For more information, see MassLynx User Guide. Defines the job submitted from the OpenLynx Login program as a night-time process. FractionLynx with OpenLynx 172

173 HPLC File Parameter Time of Analysis (mins) Create Failed Samples Files MS Tune MS Method Inlet Method Injection Volume (µl) Pre-Run Method Post-Run Method Switch Mode Fraction Method Loop Mode Loop Time (mins) Description Note: All samples processed using this method become nighttime processes. For more information, see the MassLynx User Guide. Allows you to define an LC method when logging in a sample. Enter the time, in minutes, to analyze one sample. Saves failed sample data as an *.olb file. Select the tune parameters file from the list. Select the scanning parameters file from the list. Select the LC parameters file from the list. File extensions vary depending on the type of inlet you selected. Enter the volume of sample to be injected. Select the pre-run parameters file from the list to condition the column. File extensions vary depending on the type of inlet you selected. Select the post-run parameters file from the list to return the column to a known state. File extensions vary depending on the type of inlet you selected. Select the method from the list to run when a column is switched. File extensions vary depending on the type of inlet you selected. The switch method runs if the inlet method differs from the previous one. Select the fraction collection parameters file from the list. Performs a loop injection, which you should only use for samples requiring no chromatography. Enter the retention time at which to acquire spectral data during processing. 7 Adding or Deleting Input Fields Input Fields are displayed on the Login PC for you to enter information. To add or delete fields, click Edit Fields or double-click any entry in the Input Fields or None if no fields are displayed to access the Field Mapper dialog box (Figure 7-2). Setting Up the Walk-up Page 173

174 Input Fields Available Fields Order of Fields to Use Append Insert Remove Clear Field Alias Figure 7-2 Adding and Deleting Input Fields Description Lists available fields. Lists the fields displayed on the Login PC for users. Adds a highlighted field to the end of the list. Adds a field immediately before that highlighted in the Order of Fields to Use list. Deletes a highlighted field. Deletes all fields. Enters a description of the field. 7 Collecting Fractions in OpenAccess Mode MS TRIGGERS To collect fractions, the Mass and Fraction information must be entered. Mass Specifies the Target Mass that is to be reported. Fraction Specifies the Target Mass Fraction to be collected. The information for Mass and Fraction must be present in order to collect the fraction. PDA FRACTIONS PDA Fraction Specify the Wavelength of the fraction to be collected. In OpenLynx Login only, the only information required for the PDA to trigger collection is the PDA Fraction. No PDA wavelength is required, and no wavelength 1 and 2 selections are required. These are for reporting purposes only. FractionLynx with OpenLynx 174

175 2487 IEEE UV Fraction 1 and 2 Specifies the fraction to be collected using the 2487 Dual Wavelength Detector. The wavelength chosen must correspond to that in the Inlet method; there is no control of the detector through OpenAccess. As an example, Channel A in the Inlet method = UV Fraction 1 Trigger. The software looks at the order of the wavelengths in the inlet method and will collect the first or second wavelength as specified. Enter a 1 or a 2 in the UVFraction 1 field. For example, in the inlet method, A=418nm, B=590nm. If you enter UVFraction1=2, then it will collect the 590-nm wavelength.it works the same way for the reverse order of the wavelengths, i.e., A=590nm, B=418nm in the inlet method. 7 Figure 7-3 OpenLynx Login Window Setting Up the Walk-up Page 175

176 Chapter 8 Troubleshooting FractionLynx The most common problem encountered is the absence of the desired fraction in the collector tube. There are several reasons why this might occur. Before determining that hardware or plumbing is the cause of a problem, it is important to verify that the problem has not been caused by programming. It is important to first verify, through the chromagram window, that the mass or wavelength in question is visible. If the extracted mass or wavelength is not visible, then the collector could not have collected it. In this case, it is important to recheck the masses and wavelength entered as the triggers. See Section 8.1, Uncollected Samples. If the extracted mass or wavelength is visible, but was not collected, then check the following: The FractionLynx Editor must be open. If not, fractions cannot be collected. The MIT value must be met (Section 8.2, Minimum Intensity Threshold (MIT). The fraction collector must have enough tubes available for collection. The Sample List must contain the fraction trigger information. The hardware configuration. The information contained within the raw data file (Section 8.3, Raw Data Files). If it can be determined that this software is not the cause of the problem, then a check of the system hardware and plumbing configurations must be performed. See Section 8.5, UV-Directed System Test, and Section 8.6, MS-Directed System Test Uncollected Samples If the fraction collector did not collect the sample, it is important to determine if the sample was even seen by the MS. Is the mass in question visible in the chromatogram? Is the mass present in the extracted ion chromatogram? Does the peak meet the Minimum Intensity Threshold criteria? When looking at the mass in question, refer to the extracted mass or wavelength. Never troubleshoot with the TIC as it is not the same as the extracted chromatogram. Uncollected Samples 176

177 Figure 8-1 Sample Chromatogram Using Dye 01 8 If the mass that is being sought is not visible here, then the MS did not detect it and therefore could not collect it. Ensure that the view screen is set so that all of the chromatogram is visible by selecting Display > View from the Chromatogram window. Troubleshooting FractionLynx 177

178 Figure 8-2 Display View Under Normalize Data To, click Lowest Point. 8 Figure 8-3 Normalized Data Set to Lowest Point Uncollected Samples 178

179 8.2 Minimum Intensity Threshold (MIT) If the Minimum Intensity Threshold has not been met, then no sample can be collected. The easiest way to determine this is to review the settings from the FractionLynx Editor (Figure 8-4). Figure 8-4 Fraction File Editor If the MIT was accidentally set to 30 Million, instead of 3 Million (Figure 8-5), the volume of sample collected would have changed greatly. See the chromatograms examples shown on the following pages. 8 Figure 8-5 MIT Example Set to 3 Million Troubleshooting FractionLynx 179

180 Here are two example chromatograms. Figure 8-5 has the Baseline abs set to 3 million, and Figure 8-6 has the Baseline set to 30 million. The value of 30 million can be obtained by following this example. Select Display > View and set the Baseline abs to the MIT value, in this case 30 million gives the following effect. The only part of the sample visible is the tip, therefore that is all that would have been collected. If the MIT is too high, then no sample will be collected. 8 Figure 8-6 Example of MIT Set to 30 Million It is also possible to determine the absorbance value of the data points from the Chromatogram window (Figure 8-7). Figure 8-7 Absorbance Value Data Points Minimum Intensity Threshold (MIT) 180

181 Go to the Chromatogram view, then extract the wavelength/mass required. Click to copy the list of data points to the Clipboard. Click to display the list of the data points on the screen (Figure 8-8). It can be determined from this list what the approximate threshold value would be to not include the noise in the chromatogram. Figure 8-8 Displayed List of Data Points This list now gives an idea of what threshold values that can to be set in FractionLynx Editor, as above. 8.3 Raw Data Files 8 The raw data file contains lots of useful information that can be accessed through Explorer. The data file will contain some or all of the following components (Figure 8-9). Troubleshooting FractionLynx 181

182 Figure 8-9 Raw Data File Accessed via Explorer ChroInfo.txt File The ChroInfo.txt file contains the trigger values, start and end times, and tube information. The following list is an example of the contents: 8 Collected Fractions Number 2 Trigger OrgTrigger IonMode1 +ve ion StartTime EndTime CollectionSite1 1:5 to 1:6 NumOfTubes 2 Trigger OrgTrigger IonMode2 +ve ion StartTime EndTime CollectionSite2 1:7 to 1:8 NumOfTubes 2 Raw Data Files 182

183 DiverseFractions.txt File The DiverseFractions.txt file contains much of the same information, with some additions. The following list is an example of some of the information available. TargetFractions DetectorMethod Analog Triggers Intern Not Used trigger1 trigger2 trigger3 trigger ;PosAdd1 PosAdd2 PosAdd3 PosAdd4 PosAdd NegAdd1 NegAdd2 NegAdd3 NegAdd4 NegAdd CollectedFractions 8 Trigger OrgTarget IonMode StartTime EndTime CollectedTubes Collection Site ve ion :5 to 1: ve ion :7 to 1:8 2 NumTubes Fraction Number Fract.txt File The Fract.txt file contains all the information associated with the collection methods, as well as the fraction information. The important values here are the threshold settings for triggers, minimum and maximum fraction times and tube values, and adduct information. Span = 0.50 Tube Number Collector Number Well StartTime StopTime Plate , 1: Troubleshooting FractionLynx 183

184 RinseTime = 0.0 MassSpecToFracCollectorDelay = AuxToMSDelay = SolventFrontDelay = 0.00 MinFractionValue = 3.00 MaxFractionValue = (If the sample is greater than 120 seconds wide, the collector would stop collecting.) MinFractionType = Time MaxFractionType = Time MaxTubeFill = NegAdducts = -1 NegThreshold = 0.00 NegStartType = Leading Edge NegRise = 0.00 NegEndType = Below Gradient NegEndUnits = 0.00 PosAdducts = 1,23 (Example of possible issue: If no adducts were in the method, then no adducts would be collected.) PosThreshold = (If the threshold was set too high, then the mass would not be identified by the computer.) PosStartType = MIT Only PosRise = 0.00 PosEndType = MIT Only PosEndUnits = PDASpan = 3.00 PDAThreshold = (If the threshold was set too high, then the wavelength would not be identified by the computer.) Plumbing Diagrams Figure 8-10 and Figure 8-11 representat typical system configurations for all systems to follow as a basic template. The pressure restrictions and requirements are the same for all systems, regardless of which vendor supplied the components. Plumbing Diagrams 184

185 Autosampler Makeup Pump Splitter Gradient Pump Column 19 x 50 mm 1000:1 Splitter ZQ MS Excess Waste Waste Collection Purified Fraction UV-Vis Figure 8-10 Typical Inject-to-Collect, Mass-Directed AutoPurification System 8 Troubleshooting FractionLynx 185

186 T T WASTE T T106 T112 T104 6 T117 5 LFT INJ RGT INJ P-1 T107 T114 P WASTE T T T T111 8 T115 T Fraction Flow Splitter ZQ T122 T121 T116 Notes: 1. Rheodyne pin plugs ( ) are required and supplied in the UV-directed Startup Kit ( ). 2. Replace T105, T106, T107, T108, and T111 with HF equivalents for flow rates greater than 100 ml / minute. 3. This tubing and fitting are provided with the 2767 product. 4. Detector flow splitter is included in the MS-directed Startup Kit. Port closest to the adjustment dial connects to the ZQ Detector. 5. T117 is needed for 2996 PDA, 2487 Inlet connects directly into the detector flow splitter. 6. T120 connects to the outlet port of 2996 PDA, or to the outlet tube of the 2487 with plastic union from the 2487 Startup Kit. 7. T121 is required in some high-flow applications. 8. T122 is required to guarantee the split ratio. Figure 8-11 Typical Inject-Collect Mass-Directed AutoPurification System Plumbing Diagrams 186

187 8.5 UV-Directed System Test The UV-directed system test is performed to determine that the delay time is set correctly for fraction collection and that proper chromatography results are achieved. After making plumbing and electrical connections of the components for the UV-directed system, it is necessary to measure the delay time between the detector and the Waters Fraction Collector II or III. It is essential that the peak reach the detector before the fraction collector, to allow the computer ample time to send an event signal to the fraction collector that a peak was detected. This delay must be at least 8 seconds, with typical time delays being about 15 seconds Instrument Setup The 2767 Sample Manager and fraction collector must be set up properly to prevent possible damage to the injectors or solvent leaks. See the Waters 2767 Sample Manager, Injector and Collector Maintenance Guide and the Waters Fraction Collector II / III Operator s Guide on how to set up and configure each instrument. Note: Ensure that the bed layout and reference position values are correct before to turning on the pumps Delay Timing Dye Test 8 A quick and reliable method of determining the delay time at different flow rates is to perform a sample injection made up from the Sample Dye Kit. The dye is a mixture of three compounds (Table 1-1). The following test was conducted using a Waters 19 mm 50 mm XTerra column. Note: When a different size and type of column is being used, you must adjust flow and injection volumes accordingly. Required Materials Sample Dye Kit, part number (in System Startup Kit) Stopwatch Mobile phases: A Water with 0.1% V:V TFA B Acetonitrile with 0.1% V:V TFA Troubleshooting FractionLynx 187

188 Setting Up the Delay Time Test Note: When using the column isocratically, the retention time is about 90 to 120 seconds, based on the column size and flow rate. 1. Check that the MassLynx software is configured for the correct inlet system. 2. Set the split collector delay for 30 seconds. See Figure In MassLynx, set up an HPLC method to run at the maximum user flow rate for 3 minutes with a 10:90 A:B. 4. Set the detectors to the following parameters: Waters 2996 PDA Detector to scan from 200 to 600 nm Water 2487 UV Detector to monitor 220 nm Ensure that the flow rate corresponds to the inlet and outlet on the UV Fraction Manager. 5. Set up the Sample List (see the example list in Figure 8-7): Injection Volume = 100 µl Wavelength A = 220 nm Fraction Trigger 1 = Wavelength A for the 2996 Detector, or UV 1 for the 2487 Detector 6. Once the injection occurs, monitor the waste line from the fraction collector valve. 7. As soon as the dye appears in the waste line of the fraction collector valve, start the stopwatch. 8. When you hear the fraction collector valve click to the dispense position, stop the stopwatch. The dye should have eluted before the collection valve opened. 9. The actual Splitter Collector Delay can be calculated as follows: Actual Delay = Original Fraction Parameters Delay (e.g., 30 seconds) time recorded on the stopwatch. For example, if the dye is seen in the waste line 15 seconds before the valve clicks to the collect position: Actual Splitter Collector Delay = seconds Actual Splitter Collector Delay = 15 seconds 10. Change the Splitter Collector Delay on the Fraction Collection - Timing page to the value just calculated and rerun the experiment to ensure that the delay time is correct. 11. Repeat steps 4 to 10 for all flow rates that might be used, e.g., 10, 15, 20, or 25 ml/min. Note: When running the timing test, if the dye elutes and the fraction collector valve do not open, recheck the pressures and splits. 12. Perform a chromatography test to test the complete system, as described in Section 1.2. UV-Directed System Test 188 8

189 8.6 MS-Directed System Test Note: The system pressure balancing must be carried out before the timing test can be properly performed. The test must be performed using 50% organic mobile phase because 50% is the midway point in most gradients. The pressure must be constant to obtain accurate readings. Using degassed solvents and an inline prep column will help stabilize the pressure. 1. Set both the prep pump and the makeup pump to the desired flow rates. Record the system pressures from the front panels of the pumps, or from the Inlet Editor (whichever is the most convenient) in the chart provided in Table To measure the pressure on the prep side of the pump, disconnect the fittings at the collection port of the LC Packing Splitter and record the pressure drop of the prep pump. Reconnect the fittings when finished. 3. The low flow to the MS exits the splitter opposite the makeup flow side. The exit is denoted by the 1/1000 or 1/10,000 value, depending on the style of splitter. Disconnect the fitting from the LC Packing Splitter. Wait 30 seconds to allow the pressure to stabilize before taking a reading. Reconnect the fittings when finished. 4. Once the chart has been filled out, the value that is required is at least 100 psi in the box marked Delta of two sides. The LC Packing Splitter must have 100 psi more backpressure on the prep side of the fraction collector system if the splitter is to function correctly. If the pressure difference is <100 psi, then the actual split will be less than the label claim, i.e., 1/1000 or 1/10,000. The pressure difference can be adjusted by adding or remove restriction from the flow path. Usually a short piece of small diameter tubing, typically inch is introduced on the prep side of the flow path to increase the backpressure. Note: Any addition of restrictions after the LC Packing Splitter can affect the split ratio and will require the system pressures to be balanced again. 8 Troubleshooting FractionLynx 189

190 Table 8-1 System Pressure Readings System Pressure Readings Prep side before disconnection: psi Delta: psi Prep side after disconnection: psi Delta of Prep and Make-up Sides: ps i Make-up flow side before disconnection: psi Make-up flow side after disconnection: psi Delta: psi 8 Pressure test performed by: Title/Dept.: Date: Comments and notes: Mobile phases: Flows: Note: Waters recommends that a copy of this page be retained as a template for future pressure tests. MS-Directed System Test 190

191 8.6.1 Measuring Flow to the Mass Spectrometer To measure the flow to the mass spectrometer (MS): 1. Set the makeup pump to deliver 1 ml/min of solvent, preferably methanol. Measure the flow from the makeup pump for 1 minute. Collect the solvent into an Eppendorf tube and use a syringe to draw out the solvent collected. This value should be 1 ml. The MS should be receiving 20% of the 1 ml/min flow coming from the makeup pump. This can be measured, as follows. 2. Place an Eppendorf tube at the outlet of the waste line from the UV detector. Measure the flow output for 1 minute. Use a syringe to draw out the solvent collected. This should not be less than 750 µl. If the collected solvent is less, then too much solvent is going to the MS, and the split needs to be adjusted accordingly. For example, if 1000 µl/min is going to the split before the MS and UV, and 750 µl/min is coming out of the UV, then 250 µl of solvent must be going to the MS. The optimum value for ESI is 150 to 250 µl/min to the MS. The split can be adjusted using the variable splitter, by turning the micrometer thimble. When a split has been created using a tee and tubing of different diameters, the split can be adjusted by adding or removing tubing. Replacing a piece of tubing with a longer piece of the same diameter will create more pressure on that part of the split, thus not allowing as much solvent to pass as before. If not enough solvent is flowing to the MS, either add more tubing to the UV side of the split, or remove some tubing from the MS side MS-Directed System Dye Test A quick and reliable method of determining the delay time at different flow rates is to perform a sample injection made up from the Sample Dye Kit. The dye is a mixture of three compounds (Table 1-2). The following test was conducted using a Waters 19 mm 50 mm XTerra column. Note: When a different size and type of column is being used, you must adjust flow and injection volumes accordingly. Required Materials Sample Dye Kit, part number (in the MS-directed system Startup Kit) Stopwatch Mobile phases: A - Water with 0.1% V:V TFA B - Acetonitrile with 0.1% V:V TFA Troubleshooting FractionLynx 191

192 Delay Time Test Note: When using the column isocratically, the retention time is about 90 to 180 seconds, depending on the column size. 1. Check that MassLynx software is configured for the correct inlet system. 2. In MassLynx, set up an HPLC method to run at the maximum user flow rate for 3 minutes with 90:10 organic:water composition. 3. Set up a 3-minute scanning acquisition for ESP + ve. For example: Centroid = 150 to 550 amu scan for 0.5 seconds Inter-Scan Delay = 0.1 seconds Cone Voltage = 20 V 4. Set up the fraction collector parameters to have a Split/Collector Delay of 30 seconds. 5. Set up the Sample List as follows: Injection volume = 100 µl Mass A = 227 Da Fraction Trigger 1 = Mass A 6. Start the MassLynx acquisition. 7. Once the injection occurs, monitor the waste line from the fraction collector valve. 8. As soon as the dye appears in the waste line of the Fraction Collector valve, start the stopwatch. 9. When you hear the fraction collector valve click to the dispense position, stop the stopwatch. The dye should have eluted before the collection valve opened. 10. The actual Splitter Collector Delay can be calculated as follows: Actual Delay = Original Fraction Parameters Delay (e.g., 30 seconds) time recorded on the stopwatch. For example, if the dye is seen in the waste line 15 seconds before the valve clicks to the collect position: Actual Splitter Collector Delay = seconds Actual Splitter Collector Delay = 15 seconds 11. Change the Splitter Collector Delay on the Fraction Collection - Timing page to the value just calculated and rerun the experiment to ensure that the delay time is correct. 12. Repeat steps 6 to 11 for all flow rates that might be used, e.g., 10, 15, 20, or 25 ml/min. 13. Repeat the complete procedure for APCI and ESP if both probes are to be used. 8 MS-Directed System Test 192

193 14. Upon completion of the delay time test, perform the MS-based chromatography test in Section 1.3, to rapidly test that all the component parts function as a system. Note: When running the timing test, if the dye elutes and the fraction collector valve do not open, recheck the pressures and splits. 8.7 Troubleshooting Tips Ensure that the ZQ Photomultiplier is set to 500 V; this will prevent the detector from being saturated. ZMD Photomultipliers can be reduced from 650 to 550 V when detector saturation is a problem. If the makeup pump is not turned on, the flow to the MS and UV is greatly diminished, it is still possible to see the sample in the MS, but the peaks will be later than expected, and may be wider than expected. This is because without the makeup pump, the flow rate is now 1:1000 or 1:10,000 of the prep flow, depending on the splitter type, thus not reaching the detectors quickly. Consequently, any collection will not be the correct material, since the real sample will have passed through the fraction collector. If the UV peak is normal, but the MS is small, late or nonexistent, check the split and the flow to the MS. It is not uncommon for the split to deteriorate and send all of the sample to the UV. It may be necessary to back flush the splitter. Disconnect the splitter from the system and remove the inlet frits before back flushing. Keep the cone voltage low to prevent too much fragmentation of the sample ion. Keep the span setting for the Fraction File Editor at 0.5 amu (Figure 8-12). This will keep the window of detection to a minimum; enough to compensate for small mass calculation errors that occur when using average and monoisotopic weights. It is recommended that masses to one decimal place are used. If the span is too large, too much zero intensity data is averaged in the Fraction determination, thus reducing the overall intensity of the chromatographic peak. 8 Troubleshooting FractionLynx 193

194 Figure 8-12 Fraction File Editor, General Tab 8.8 OpenLynx Troubleshooting OpenLynx Manager must be open for the OpenLynx Login to be able to operate. If the OpenLynx Login has the error Please contact your administrator, it is possible that the status.ols file cannot be found. This file is in the OpenLynx/Batch database along with the Processed folder. Locate status.ols and double-click it. The user may delete it as it will automatically be recreated the next time Login is opened. 8 OpenLynx Troubleshooting 194

195 Error Message Field 8 If the login fails to allow the user to complete the login procedure, the method path may need to be set. Double-click the method, within the MassLynx directory. Troubleshooting FractionLynx 195

196 By highlighting the xxx.olp method, the path for the software to locate the methods of use is being located, and therefore this should no longer be an issue. 8 Note: For more information on OpenLynx troubleshooting, see the OpenLynx Software User s Guide. OpenLynx Troubleshooting 196

197 Appendix A Hardware Setup A This appendix describes how to set up the hardware and software applications that interface with the FractionLynx application. Note: If more than one Waters Fraction Collector ll or lll is to be used in the system, then a Waters Equinox card is required (part number WAT280125). This will allow up to eight additional WFC units to be configured into the system. A.1 Equinox Setup for Windows XP Platform Systems After installing the Equinox PC card in a workstation, the Equinox application must be downloaded to support the newly installed hardware. On Windows and Windows XP-based workstations, the installation of the Equinox application and run startup is automatically wizard driven. The following figures exemplify the automatic run sequence. Figure A-1 Found New Hardware Wizard Hardware Setup 197

198 A Figure A-2 System Setting Changes Dialog Box Figure A-3 Installation Completed Page Hardware Setup 198

199 A.2 Equinox Setup for Windows NT Platform Systems A.2.1 Setting Up the Equinox Card Figure A-4 shows the opening Start Up Screen when the Equinox CD is loaded. If your CD does not have AutoRun, enter d:/setup.exe, where d: is your CD drive. A Figure A-4 Start Up Screen A.2.2 Equinox Driver The Equinox card requires a device driver that can be installed from the supplied CD. 1. Select Windows NT from the Drivers & Software area of the Start Up screen. 2. Select Windows NT 4.0 Driver (Figure A-5). Hardware Setup 199

200 A Figure A-5 Selecting the Driver Option 3. Select SST Driver (Figure A-6). Figure A-6 Selecting the Appropriate Driver Hardware Setup 200

201 4. The Install it Now screen (Figure A-7), then the Help screen (Figure A-8) appear. Click Windows NT 4.0 SST Driver under Install it Now, and continue. A Figure A-7 Install It Now Screen Figure A-8 Help Screen Hardware Setup 201

202 5. The Network dialog box appears (Figure A-9). The first pane describes the process of installing the card (as a network device). The second pane is the Windows NT Network screen from Settings > Control Panel. The type of network card listed will vary from the following example, according to the specifics of your system. A Figure A-9 Network Dialog Box, Adapters Tab 6. To install the Equinox card, follow step a and onward from the Help screen. 7. Click Add. Hardware Setup 202

203 A Figure A-10 Select Network Adapter Dialog Box 8. Click Have Disk and specify the path d:\drivers\ras\ as specified in the Equinox Help screen, where d: is the CD drive. Figure A-11 Insert Disk Dialog Box Hardware Setup 203

204 A Figure A-12 Select OEM Option Dialog Box 9. Click OK. Figure A-13 Setup Message Dialog Box 10. Make sure that COM3 is the First COM Port selected, then accept the rest of the defaults offered (Figure A-14). Figure A-14 Equinox SST Configuration Dialog Box Hardware Setup 204

205 The Equinox ports are labeled 1 to 8. Because there are already two communication (COM) ports on the computer, the connectors will actually be at COM (label + 2). Table A-1 Equinox Ports Labeled Port Number Software (MassLynx) Port for Configuration A 11. When multiple WFC II units are connected into the system, they can be connected to any of the extension ports supplied by the Equinox unit. The actual order/functionality that they will be used in/assigned is determined by the configuration process used in MassLynx. 12. Click Next. Figure A-15 EquiView Plus Datascope Path Dialog Box 13. The Network Adapters tab now shows the Equinox card installed (Figure A-16). Clicking Close will result in the system configuration being updated and a prompt to shut down and restart the system. Upon system reboot, the Equinox card will be available for use. Hardware Setup 205

206 A Figure A-16 Network Adapters Tab Showing the Equinox Board Installed A.3 Post-Installation System Performance Enhancements Once MassLynx, FractionLynx, and any updates have been loaded, several key configurations must be taken into consideration to ensure correct operation of the software and hardware components in the system. FractionLynx is predominantly a semi-prep/prep scale application with longer cycle times than those used by analytical methods. The sample handling is also different. In analytical applications, only a small aliquot is taken, whereas in purification, the entire sample can be committed to the system. Aspirate and Dispense Speeds This section describes the Waters 2700 Sample Manager. Similar parameters are also configurable for Gilson, CTC, and other Waters 27xx series injectors. When performing larger injections, the Aspirate and Dispense speeds for the 2700 system must be slowed down to ensure that there is no cavitation causing air gaps to be injected into the system. This is especially important when using sealed vials and solvents such as DMSO. Hardware Setup 206

207 Note: Pre-slit vials work better than sealed tops. Note: Use slower aspirate and dispense speeds for injections greater than 500 µl. The following examples from the Inlet Editor are typical for large-volume injections. Injection Configuration Tab A Figure A-17 Inlet Editor, Injection Configuration Tab The configuration above allows the following: A 2-mL injection volume A suitable air gap between the solvent and sample (50 µl in this example) No over-sampling Note: Ensure that the transfer tubing between the probe and the syringe is capable of holding the injection volumes required in the system. Hardware Setup 207

208 Dilutor Configuration Tab A Figure A-18 Inlet Editor, Dilutor Configuration Tab Loop Volume for Injector Systems Analytical systems running with small (e.g., 10 µl) loops will typically over-sample and therefore overfill the loop when performing full loop injections. This ensures that the loop is cleared of any previous sample carryover. For full loop injections, MassLynx will over-sample by twice the volume. For example, in preparative systems the injector can be fitted with a 2-mL loop and a 5-mL syringe. If the user had 2 ml of sample and wanted to inject the full 2 ml, this would result in the system trying to draw 4 ml of sample giving rise to the potential to inject 2 ml of air into the system. If MassLynx sees an injection volume equivalent to the loop volume, then it assumes full loop injection and automatically performs the over-sampling. To avoid this, select Partial Loop and enter a slightly larger loop volume than actually exists in the system. Hardware Setup 208

209 Wash Parameters Tab A Figure A-19 Inlet Editor, Wash Parameters Tab When the needle rinse volume is <800 µl, the wash cycle uses the syringe and shallow wash station. If the needle rinse wash volume is >800 µl, the mini-wash pump and deep wash station are used. The rinse volume can be set as high as 30 ml. The volume depends on the injection volume, but 5000 or 6000 is a good number to start with. * Mini-wash prime: wash time is the duration of the mini-wash pump priming. Hardware Setup 209

210 Sampler Configuration Tab A Figure A-20 Inlet Editor, Sampler Configuration Tab A.4 Connecting the Waters Fraction Collector II or lll Cable Connections The WFC II or III requires a 9-pin female to 9-pin female null modem cable to connect it to the PC. Cables and adapters are supplied with the Equinox card when installing multiple units. The pin connections for this null modem cable are as follows: 9-Pin Female 9-Pin Female Hardware Setup 210

211 A.5 Connecting the Gilson 215 Collector Cable Connections The Gilson 215 Collector is connected via Com2 on the MassLynx PC. Use the standard RS-232 cable, supplied with the Gilson 215 unit (9-pin female to 25-pin female) to connect the units. A Hardware Configuration The rear panel of the Gilson 215 unit has two rotary dials. SW1 SW2 Provides the instrument ID. Add 20 to the number indicated on the dial and use this in the FractionLynx Editor when configuring the software. Determines the baud rate and clock synchronization of the 215 unit. Select option and GSIOCMaster/Break Active. A.6 Connecting the Gilson 204 Fraction Collector Cable Connections In order for MassLynx to control the Gilson 204 Fraction Collector, it must be connected via the 506C interface unit. PC to 506C unit 506C to 204 collector RS-232, 9-pin female to 25-pin male GSIOC communications Both the RS-232 and GSIOC cables are provided with the 506C interface unit. Additional cables are available through Gilson or their local suppliers. Other important notes for configuration are as follows: A UniPoint Key is not needed. In systems with Gilson Inlets and Gilson Fraction Collectors, separate 506C interface units are required. This is because the Inlet Kernel and Fraction Kernel require separate COM ports on the PC. Hardware Configuration Each fraction collector in the system must have a unique ID address. This can be set/determined using the keypad on the front of the fraction collector. Hardware Setup 211

212 To determine the unit ID: 1. Select Edit > 2 on the front panel of the 204 Fraction Collector. 2. Press until the Device ID is displayed. If this is not a unique ID, then enter a new/unique ID number for the unit. A This is the ID number that MassLynx requires when the fraction collector is configured into the system. This section presents procedures to prepare a UV- or MS-directed system for operation. Ensure that each module that makes up the UV- or MS-directed system is integrated according to the previous chapters, in addition to the installation and operator s guides provided with each instrument. A.7 Configuring System Modules to MassLynx The Inlet Editor window links the system modules to MassLynx and enables them for use. To configure the Waters 2525 BGM, C/FO, and 2767 Sample Manager, set the MassLynx software to recognize the modules connected to the system. In addition to setting the communications, you must also set each module s parameters. Click (Inlet Editor). The Inlet Editor Status window appears (Figure A-21). The Inlet Editor has many toolbar buttons that contain the controls for the system components. Toolbar Figure A-21 Inlet Editor Window, Status Tab Hardware Setup 212

213 A.8 Inlet Configuration Dialog Box The Inlet Configuration dialog box (Figure A-22) contains three categories of instruments for configuration. Check boxes are provided next to each instrument that allow the MassLynx application to link and communicate with a selected instrument. Inactive check boxes are grayed out. A The Inlet Configuration dialog box also contains several tabbed pages that allow you to set up properties for optional system modules, should they require relay contact closures, event triggers, or GPIB (IEEE-488) or HP communications to become enabled. The tabs are labeled as follows: Contact Closures Triggers GPIB communications HPIB (Hewlett-Packard Interface Board) communications To access the Inlet Configuration dialog box (Figure A-22), select Tools > Instrument Configuration from the Inlet Editor. Figure A-22 Inlet Configuration Dialog Box Hardware Setup 213

214 Contact Closure Tab The Contact Closure tab (Figure A-23) contains switches that become active when a module requiring an Event Start input and output is connected to the system. The properties in this tab are linked to properties in the Triggers tab. See the MassLynx User Guide for additional details. A Event must be active for MS-directed systems Figure A-23 Contact Closure Tab Triggers Tab When non-waters detectors or pumps are integrated into a system configuration, the module may require a means to trigger an event. The Triggers tab (Figure A-24) contains switches selections to select a trigger type. Trigger options are either a software-generated trigger or a hardware trigger (generated by relay contact closures). Contact closure triggers are related to the assignment of the switches selected from the Contact Closure tab. Note: When using Waters detectors and pumps, select Trigger by Software as your default selection. Hardware Setup 214

215 A GPIB Communication Tab Figure A-24 Triggers Tab The GPIB Communication tab (Figure A-25) contains switches used to configure modules integrated to the system through the general purpose interface board (GPIB). The GPIB lists IEEE-488 compatible devices, such as the 996 PDA Detector. Drop-down lists allow you to select the address assignment of the connected module(s). The I/O Timeout field provides the reply time in seconds. Note: The recommended I/O Timeout is 10 seconds. Hardware Setup 215

216 A Address Assignment Drop-Down Lists Recommended Setting of 10 Seconds Drop-Down List HPIB Communication Tab Figure A-25 GPIB Communication Tab The HPIB (Hewlett-Packard Interface Board) Communication tab is a MassLynx interface for other applications and is not used for Waters AutoPurification (Inject to Collect) system configurations. A.9 Scan Command The Scan command in MassLynx polls and scans each module connected to the system through Ethernet or IEEE-488 (GPIB) and sets up a communication link between a connected device and the MassLynx application. The Scan command does not set up the communication link to modules connected to the system through RS-232 serial ports. The Scan command also retrieves embedded serial number information from linked Waters modules. Note: Before a scan can be performed, each module must have its address assigned to establish a communication link. Hardware Setup 216

217 To scan the connected components: 1. Click (Inlet Editor). 2. Select View > Waters2525 Pump (Figure A-26) to open the Modify Instrument Method dialog box (Figure A-27). A Instrument List Figure A-26 Inlet Editor, View Menu 3. Click (Configure Autopurification Modules). The Configure Autopurification Modules drop-down list appears (Figure A-27). Configure Autopurification Modules Button Figure A-27 Modify Instrument Method Dialog Box Hardware Setup 217

218 4. Select Configure Autopurification Modules to open the Autopurification System Configuration dialog box (Figure A-28). A Figure A-28 Autopurification System Configuration Dialog Box 5. Click Scan to retrieve serial number information for all the instruments that are connected to the PC. The Waters Instruments dialog box appears (Figure A-29). Note: The Waters 2767 Sample Manager will not be displayed on the dialog box because it is connected to the system through an RS-232 serial port. Figure A-29 Waters Instruments Dialog Box Hardware Setup 218