user’s guide - waters corporation

FractionLynx SoftwareUser’s Guide

34 Maple StreetMilford, MA 01757

71500054302, Revision A

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.

© 2003 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.

Table of Contents

Preface ....................................................................................... 18

Chapter 1 Introduction ...................................................................................... 23

1.1 UV-Directed System Test ...................................................... 24

1.1.1 Instrument Setup ....................................................... 24

1.1.2 Required Materials .................................................... 24

1.2 Setting Up the Chromatography Test .................................... 26

1.3 MS-Based Chromatography Test Procedure......................... 31

1.4 Accessing Reports Using the FractionLynx Browser ............ 38

1.4.1 Creating Report Files................................................. 38

1.4.2 Accessing the FractionLynx Browser ......................... 38

1.4.3 Setting Report Scheme Parameters .......................... 39

Chapter 2 Installing FractionLynx Software as an Option ................................. 42

2.1 Installing FractionLynx Software ........................................... 42

Chapter 3 Using the FractionLynx Editor .......................................................... 48

3.1 Accessing the FractionLynx Editor ........................................ 48

3.2 Enabling Fraction Collection.................................................. 50

3.3 Resetting Communications ................................................... 50

3.4 Exiting the FractionLynx Editor.............................................. 51

Table of Contents 3

3.5 Selecting Fraction Collection Parameters ............................. 51

3.5.1 Fraction File Editor..................................................... 51

3.5.2 General Page............................................................. 52

3.5.3 Timing Page............................................................... 54

3.5.4 Volume Page.............................................................. 55

3.5.5 Positive and Negative Pages ..................................... 57

3.5.6 Analog Page .............................................................. 59

3.5.7 PDA Page .................................................................. 59

3.5.8 UV Page..................................................................... 60

3.5.9 Timed Events Page.................................................... 61

3.5.10 Mixed (Boolean) Triggers Editor............................... 62

3.6 Fraction Collection Setup ...................................................... 80

3.6.1 Instrument Setup ....................................................... 80

3.6.2 Collector Configuration .............................................. 81

3.6.3 Gilson Comms Dialog Box......................................... 85

3.6.4 Gilson Positions ......................................................... 85

3.6.5 Bed Layout................................................................. 86

3.6.6 Plate Generator ......................................................... 88

3.6.7 Manual Control .......................................................... 92

3.7 Resetting Beds...................................................................... 94

Chapter 4 Collection Modes ............................................................................. 96

4.1 Definition of Terms ................................................................ 96

4.2 Overview of Each Mode........................................................ 96

Table of Contents 4

4.3 Fraction Collector Tube Numbering Schemes....................... 97

4.3.1 Single Plate Per Fraction Collector ............................ 97

4.3.2 Multiple Plates Per Fraction Collector ........................ 98

4.3.3 Multiple Fraction Collectors...................................... 100

4.4 Method of Operation for Each Mode ................................... 101

4.4.1 Sequential Collection ............................................... 102

4.4.2 Enable User Starts .................................................. 105

4.5 One for One Collection........................................................ 105

4.5.1 One for One Collection Overrides the FractionLynx Parameters......................................... 106

4.5.2 Sample Plate Size Versus Fraction Collector Rack Size ................................................................ 106

4.5.3 Sample Plate Numbering Versus Fraction Collector Rack Numbering....................................... 108

4.5.4 Logging Multiple Batches......................................... 110

4.5.5 Sample Running Order ............................................ 111

4.6 Multiple Fractions Per Tube ................................................. 112

4.7 Reserved Tubes .................................................................. 114

4.7.1 OpenAccess Implementation................................... 114

Chapter 5 Running FractionLynx Samples ..................................................... 117

5.1 Setting Up Projects ............................................................. 117

5.1.1 Root Directory.......................................................... 117

5.1.2 Acqudb Subdirectory ............................................... 117

5.1.3 Data Subdirectory.................................................... 118

5.1.4 Sampledb Subdirectory ........................................... 118

Table of Contents 5

5.2 Sample List ......................................................................... 118

5.3 Starting a Run from the Sample List ................................... 123

5.4 Displaying Fractions in MassLynx Chromatograms ............ 125

5.5 Real-Time Display of Fractions in Chromatogram .............. 127

Chapter 6 FractionLynx Browser .................................................................... 128

6.1 Introduction ......................................................................... 128

6.2 FractionLynx Browser.......................................................... 129

6.2.1 FractionLynx Browser Toolbar.................................. 130

6.3 Browser Files ...................................................................... 131

6.4 View Options ....................................................................... 133

6.4.1 Spectrum Page ....................................................... 133

6.4.2 Chromatogram Page................................................ 135

6.4.3 Default Plate Page ................................................... 136

6.4.4 Colors Page ............................................................. 138

6.4.5 Copy Control Page................................................... 140

6.4.6 Column Pages ......................................................... 141

6.4.7 Other Display Options.............................................. 144

6.5 Other Menu Options............................................................ 145

6.5.1 File Menu ................................................................. 145

6.5.2 View Menu ............................................................... 147

6.5.3 Window Menu .......................................................... 148

6.6 Sample Plate Pane ............................................................. 149

6.7 Fraction Plate Pane............................................................. 150

6.8 Sample Description Pane.................................................... 151

Table of Contents 6

6.9 Fraction Collection Results Pane ........................................ 151

6.9.1 Fraction Trigger and Fraction Tube Export Options .................................................................... 153

6.10 Results Table Pane ........................................................... 154

6.11 Spectrum Pane ................................................................. 156

6.12 Fraction Tube Spectra Pane .............................................. 157

6.13 Chromatogram Pane......................................................... 158

6.13.1 Chromatogram Fraction Display ............................ 158

6.14 Elemental Composition / Library Search Pane ................. 160

6.15 Report Schemes ............................................................... 160

6.15.1 Report Scheme Settings........................................ 160

6.15.2 Print Control Page ................................................. 161

6.16 Report Column Selection Pages....................................... 164

6.16.1 Sample Summary Page......................................... 165

6.16.2 MS Peaks Results Summary Page........................ 166

6.16.3 Report Header Page.............................................. 166

6.16.4 Sample Header Page............................................. 166

6.16.5 Chromatogram Report Page.................................. 166

6.16.6 Fraction Tube Summary Page................................ 166

6.16.7 Fraction Trigger Summary Page ............................ 167

6.16.8 Decimal Places Page............................................. 167

6.16.9 Producing Tab-Delimited Files ............................... 168

Chapter 7 FractionLynx with OpenLynx ......................................................... 171

7.1 Setting Up the Walk-up Page .............................................. 171

Table of Contents 7

Chapter 8 Troubleshooting FractionLynx ........................................................ 176

8.1 Uncollected Samples .......................................................... 176

8.2 Minimum Intensity Threshold (MIT) .................................... 179

8.3 Raw Data Files.................................................................... 181

8.4 Plumbing Diagrams............................................................. 184

8.5 UV-Directed System Test .................................................... 187

8.5.1 Instrument Setup ..................................................... 187

8.5.2 Delay Timing Dye Test ............................................. 187

8.6 MS-Directed System Test.................................................... 189

8.6.1 Measuring Flow to the Mass Spectrometer ............. 191

8.6.2 MS-Directed System Dye Test ................................. 191

8.7 Troubleshooting Tips ........................................................... 193

8.8 OpenLynx Troubleshooting.................................................. 194

Appendix A Hardware Setup.............................................................................. 197

A.1 Equinox Setup for Windows XP Platform Systems ........... 197

A.2 Equinox Setup for Windows NT Platform Systems ........... 199

A.2.1 Setting Up the Equinox Card .................................. 199

A.2.2 Equinox Driver......................................................... 199

A.3 Post-Installation System Performance Enhancements ..... 206

A.4 Connecting the Waters Fraction Collector II or lll .............. 210

A.5 Connecting the Gilson 215 Collector ................................. 211

A.6 Connecting the Gilson 204 Fraction Collector ................... 211

A.7 Configuring System Modules to MassLynx ....................... 212

Table of Contents 8

A.8 Inlet Configuration Dialog Box ........................................... 213

A.9 Scan Command ................................................................. 216

A.10 BGM and C/FO Configuration ......................................... 219

A.11 2767 Sample Manager Configuration .............................. 219

Appendix B Peak Detection ............................................................................... 220

B.1 Analytical ........................................................................... 220

B.1.1 Mass Detection ....................................................... 220

B.1.2 Analog Detection ..................................................... 221

B.1.3 PDA Detection......................................................... 221

B.2 Preparative ........................................................................ 222

B.2.1 Mass Detection ....................................................... 222

B.2.2 Analog Detection ..................................................... 223

B.2.3 PDA Detection......................................................... 223

Index ..................................................................................... 225

Table of Contents 9

List of Figures

1-1 Load Sample List Format Dialog Box ............................................ 251-2 Customize Field Display Dialog Box .............................................. 261-3 Starting Data Acquisition in MassLynx .......................................... 281-4 Enable Fraction Collection Dialog Boxes ....................................... 281-5 General and Timing Pages ............................................................ 291-6 Volume and Analog Pages............................................................. 291-7 PDA and UV Pages ....................................................................... 301-8 UV-Directed Dye Test Chromatograms.......................................... 301-9 Starting Data Acquisition in MassLynx .......................................... 341-10 Enable Fraction Collection Dialog Boxes ....................................... 351-11 General Page................................................................................. 351-12 Timing and Volume Pages ............................................................. 361-13 Positive and Negative Pages ......................................................... 361-14 MS-Directed Dye Test Chromatograms ......................................... 371-15 FractionLynx Browser Window....................................................... 381-16 Setting Print Controls in the Edit Report Scheme Settings

Dialog Box ..................................................................................... 40

2-1 MassLynx V4.0 Installation Wizard................................................ 432-2 Modifying MassLynx 4.0 Program.................................................. 432-3 Accepting the License Agreement ................................................. 442-4 Selecting the FractionLynx Software Option .................................. 442-5 Installing OpenLynx Options.......................................................... 452-6 Starting the Program Installation ................................................... 452-7 Selecting the FractionLynx Key Disk Path...................................... 462-8 Selecting the OpenLynx Key Disk Path ......................................... 462-9 Program Installation Complete ...................................................... 47

List of Figures 10

3-1 FractionLynx Editor ........................................................................ 483-2 Enable Fraction Collection Dialog Box........................................... 503-3 Reset Communications Command ................................................ 503-4 Collection Modes Dialog Box......................................................... 513-5 Fraction File Editor, General Page ................................................. 523-6 Fraction File Editor, Timing Page................................................... 543-7 Fraction File Editor, Volume Page.................................................. 553-8 Fraction File Editor, Positive and Negative Adducts Pages............ 573-9 Fraction File Editor, Analog Page................................................... 593-10 Fraction File Editor, PDA Page....................................................... 593-11 Fraction File Editor, UV Page......................................................... 603-12 Fraction File Editor, Timed Events Page........................................ 613-13 Illustrated Example of Mass A AND WaveLength B ...................... 623-14 Illustrated Example of Mass A OR Mass B.................................... 633-15 Illustrated Example of Mass A NOT Mass B.................................. 633-16 Illustrated Example of Mass A XOR (Exclusive OR) Mass B......... 643-17 MassLynx Boolean Trigger, Boolean Column Dialog Box.............. 643-18 FractionLynx Mixed Triggers Editor Dialog Box.............................. 653-19 FractionLynx Mixed Triggers Editor Dialog Box, Expanded ........... 653-20 Selecting from the Trigger Source List........................................... 673-21 Adding an Expression.................................................................... 673-22 Selecting an Operator.................................................................... 683-23 Selecting Wavelength A................................................................. 683-24 Adding the Wavelength to the Expression ..................................... 693-25 Saving and Closing the FractionLynx Mixed Triggers

Dialog Box ..................................................................................... 693-26 Sample List Page, Browse............................................................. 703-27 Selecting and Saving a Mixed Trigger............................................ 703-28 Mixed Trigger Entered in the Boolean Logic Column..................... 713-29 Entering Components into the Boolean Expression ...................... 723-30 Validation Error Message............................................................... 733-31 Mix Displayed on the Fraction Chromatogram............................... 74

List of Figures 11

3-32 Mix Displayed in the Fract.txt Screen............................................. 743-33 Selecting a Trigger from the Sample List ....................................... 753-34 Delete Mixed Trigger Warning Box ................................................ 753-35 Expression Deleted from the Mixed Trigger Editor......................... 763-36 Viewing Trigger in the Mixed Trigger Editor and Mixed

Triggers Dialog Box........................................................................ 763-37 Boolean Logic INI File.................................................................... 773-38 Deleting a Trigger from the Boolean Logic Section and

Boolean Logic File Section ............................................................ 783-39 Saving Changes ............................................................................ 793-40 Display of INI with Trigger Removed.............................................. 793-41 Triggers Removed from Mixed Triggers Editor and Mixed

Triggers Dialog Boxes .................................................................... 803-42 Collector Configuration Page (Waters Fraction Collector III) ......... 803-43 Selector Valve Page....................................................................... 823-44 Collector Configuration (Waters 2757)........................................... 823-45 Typical Gilson Fraction Collector Setups ....................................... 843-46 Gilson Comms Dialog Box............................................................. 853-47 Positions Dialog Box ...................................................................... 853-48 Bed Layout Dialog Box, Select/Create Bed Layout Page .............. 863-49 Bed Layout Dialog Box, Modify Bed Layout Tab ............................ 873-50 Plate Description Dialog Box ......................................................... 883-51 Rack Generator Window................................................................ 893-52 Example of Rotated Rack .............................................................. 913-53 Scale Rack Dialog Box .................................................................. 923-54 Manual Control Dialog Box ............................................................ 92

4-1 Plate Generator Types ................................................................... 984-2 30-Tube Plate Layout ..................................................................... 994-3 60-Tube Plate Layout ..................................................................... 994-4 96-Tube Plate Layout ................................................................... 1004-5 Plate Layouts for Multiple Fraction Collectors .............................. 100

List of Figures 12

4-6 Plate Layout for Four Fraction Collectors..................................... 1014-7 Collection Modes Dialog Box....................................................... 1024-8 Example of How Samples and Batches Collect........................... 1034-9 Unable to Start Queue Warning Message Box............................ 1044-10 Sample Plate Size Equals Collector Rack Size ........................... 1064-11 Unequal Sample and Collection Racks ....................................... 1074-12 Sample Plates Larger than Collection Plates .............................. 1074-13 Mapped Fraction Collector Positions for Each Well Location....... 1094-14 Four Injection Plates Running One to One .................................. 1104-15 MassLynx Not Enough Vessels Error Message Box.................... 1104-16 Mapping for Single Shot Samples ............................................... 1114-17 Plate Defined with Horizontal First Priority .................................. 1124-18 Sequential, One to One, and MultiFraction Mapping ................... 1134-19 Mini-Racks ................................................................................... 1154-20 Modified Bed Layout for Mini-Racks or Fraction Blocks ............... 1154-21 GeneVac Rack............................................................................. 116

5-1 Sample List .................................................................................. 1195-2 Sample List Example: Fewer Mass Triggers than Masses .......... 1225-3 Start Sample List Run Dialog Box ............................................... 1245-4 Fraction Display Parameters Dialog Box...................................... 126

6-1 FractionLynx Browser .................................................................. 1296-2 Print Control Dialog Box ............................................................. 1316-3 Set Margins Dialog Box ............................................................... 1326-4 View Options Dialog Box, Spectrum Page................................... 1336-5 View Options Dialog Box, Chromatogram Page .......................... 1356-6 View Options Dialog Box, Default Plate Page.............................. 1376-7 View Options Dialog Box, Colors Page........................................ 1396-8 View Options Dialog Box, Copy Control Page ............................. 1406-9 View Options Dialog Box, List Columns Page ............................. 1426-10 View Options Dialog Box, Fraction Display Page......................... 143

List of Figures 13

6-11 Program Not Found Dialog Box ................................................... 1466-12 Failed Samples Options Dialog Box ............................................ 1466-13 View Menu ................................................................................... 1476-14 Window Menu .............................................................................. 1486-15 Sample Plate Pane ...................................................................... 1496-16 Fraction Plate Pane...................................................................... 1506-17 Sample Description Pane ............................................................ 1516-18 Fraction Collection Results Pane................................................. 1516-19 View by Trigger: Selecting NO For Export Trigger ....................... 1536-20 View by Tubes: All Fraction Tubes Set to NO for Trigger.............. 1536-21 View by Tubes: Changing One Tube to YES................................ 1546-22 View by Trigger: Corresponding Fraction Reverting to YES ........ 1546-23 Results Table Pane ...................................................................... 1546-24 Spectrum Pane ............................................................................ 1566-25 Fraction Tube Spectra Pane......................................................... 1576-26 Chromatogram Pane with Fractions Displayed on all

Chromatograms ........................................................................... 1596-27 Chromatogram Pane with Fractions Not Displayed on all

Chromatograms ........................................................................... 1596-28 Chromatogram Pane with View Fractions by Tube ...................... 1596-29 Edit Report Scheme Settings Dialog Box, Print Control Page..... 1616-30 Edit Report Scheme Settings Dialog Box, Sample Summary

Page ............................................................................................ 1656-31 Edit Report Scheme Settings Dialog Box, Decimal Places

Page ............................................................................................ 167

7-1 Setting Up the Walk-up Page....................................................... 1727-2 Adding and Deleting Input Fields................................................. 1747-3 OpenLynx Login Window............................................................. 175

8-1 Sample Chromatogram Using Dye 01 ......................................... 1778-2 Display View ................................................................................ 1788-3 Normalized Data Set to Lowest Point .......................................... 178

List of Figures 14

8-4 Fraction File Editor....................................................................... 1798-5 MIT Example Set to 3 Million ....................................................... 1798-6 Example of MIT Set to 30 Million ................................................. 1808-7 Absorbance Value Data Points .................................................... 1808-8 Displayed List of Data Points ....................................................... 1818-9 Raw Data File Accessed via Explorer.......................................... 1828-10 Typical Inject-to-Collect, Mass-Directed AutoPurification

System......................................................................................... 1858-11 Typical Inject-Collect Mass-Directed AutoPurification System..... 1868-12 Fraction File Editor, General Tab.................................................. 194

A-1 Found New Hardware Wizard ..................................................... 197A-2 System Setting Changes Dialog Box .......................................... 198A-3 Installation Completed Page ....................................................... 198A-4 Start Up Screen .......................................................................... 199A-5 Selecting the Driver Option ......................................................... 200A-6 Selecting the Appropriate Driver ................................................. 200A-7 Install It Now Screen ................................................................... 201A-8 Help Screen ................................................................................ 201A-9 Network Dialog Box, Adapters Tab ............................................. 202A-10 Select Network Adapter Dialog Box ............................................ 203A-11 Insert Disk Dialog Box ................................................................ 203A-12 Select OEM Option Dialog Box ................................................... 204A-13 Setup Message Dialog Box ........................................................ 204A-14 Equinox SST Configuration Dialog Box ...................................... 204A-15 EquiView Plus Datascope Path Dialog Box ................................ 205A-16 Network Adapters Tab Showing the Equinox Board Installed .... 206A-17 Inlet Editor, Injection Configuration Tab ...................................... 207A-18 Inlet Editor, Dilutor Configuration Tab ......................................... 208A-19 Inlet Editor, Wash Parameters Tab ............................................. 209A-20 Inlet Editor, Sampler Configuration Tab ...................................... 210A-21 Inlet Editor Window, Status Tab ................................................. 212

List of Figures 15

A-22 Inlet Configuration Dialog Box .................................................... 213A-23 Contact Closure Tab ................................................................... 214A-24 Triggers Tab ............................................................................... 215A-25 GPIB Communication Tab .......................................................... 216A-26 Inlet Editor, View Menu ............................................................... 217A-27 Modify Instrument Method Dialog Box ........................................ 217A-28 Autopurification System Configuration Dialog Box ..................... 218A-29 Waters Instruments Dialog Box .................................................. 218

B-1 Analytical Peak Detection ........................................................... 221B-2 Analytical Peak Termination ....................................................... 222B-3 Preparative Peak Detection ........................................................ 223B-4 Preparative Peak Termination .................................................... 224

List of Figures 16

List of Tables 17

1-1 UV Test Sample Dye Compounds ................................................ 241-2 MS Test Sample Dye Compounds ........................................... 311-3 FractionLynx Browser Panes ................................................... 391-4 Print Reports........................................................................... 411-5 Report Formats ....................................................................... 41

3-1 FractionLynx Mixed Triggers Editor Properties ............................. 66

4-1 Bed Scheme Defined as Two Columns of Five Rows ................ 116

5-1 Acqu Database Control Files ...................................................... 1185-2 Sample List Properties .......................................................... 1205-3 Sample List with Fewer Mass Triggers than Masses .............. 122

6-1 Sample Summary ....................................................................... 1686-2 MS Peak Results and Fraction Tube Summary ...................... 169

8-1 System Pressure Readings ........................................................ 190

A-1 Equinox Ports ............................................................................. 205

List of Tables

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.

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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.

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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 http://www.waters.com.

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.

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http://www.waters.com

Documentation Conventions

The following conventions can be used in this guide:

Notes

Notes call out information that is helpful to the operator. For example:

Note: Record your result before you proceed to the next step.

Convention Usage

Purple Purple text indicates user action such as keys to press, menu selec-tions, and commands. For example, “Click Next to go to the next page.”

Italic 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 Courier indicates examples of source code and system output. For example, “The SVRMGR> prompt appears.”

Courier Bold 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.”

Underlined Blue 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 origi-nating 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.

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Attentions

Attentions provide information about preventing damage to the system or equipment. For example:

Cautions

Cautions provide information essential to the safety of the operator. For example:

STOPAttention: To avoid damaging the detector flow cell, do not touch the flow cell window.

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.

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1
Chapter 1Introduction
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 716000765, 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

1

• 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 8.

1.1 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.

1.1.1 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.

1.1.2 Required Materials

The Sample Dye Kit (part number 716000765) 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

UVmax 590 nm 418 nm 590 nm

Formula C12H9N3S.C2H4O2 C17H19ClN2S C25H30ClN3

Sigma part number

T3387 T3516 C3886

Introduction 24

1

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.

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

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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® 19 × 50.

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

Introduction 26

1

• Start Conditions: 95:5 A:B

• Run Time: 10 minutes

2. 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).

Time A% B% Curve Number

T = 0 minutes 95 5 6

T = 1 minute 95 5 6

T = 7 minutes 57 43 6

T = 7.5 minutes 5 95 6

T = 8.5 minutes 5 95 6


T = 10 minutes 95 5 6

Setting Up the Chromatography Test 27

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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

Start Icon

Introduction 28

1

Click Collection Parameters to display the Fraction File Editor.

Figure 1-5 General and Timing Pages

Figure 1-6 Volume and Analog Pages

Setting Up the Chromatography Test 29

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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

1

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.

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 716000765) 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.

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 (C2H4O2) to give this [M+H] ion:

Thionin ([M+H] = 228.1), Lambda Max. = 590 nm

Table 1-2 MS Test Sample Dye Compounds

Item Thionin Acetate Thioflavin T Crystal Violet

m/z 228.1 283.1 372.2

UVmax 590 nm 418 nm 590 nm

Formula C12H9N3S.C2H4O2 C17H19ClN2S C25H30ClN3

Sigma part number

T3387 T3516 C3886

MS-Based Chromatography Test Procedure 31

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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

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

2. 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.

Time A% B% Curve Number


T = 1 minute 95 5 6

T = 7 minutes 57 43 6

T = 7.5 minutes 5 95 6

T = 8.5 minutes 5 95 6


T = 10 minutes 95 5 6

Introduction 32

1

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 1-9.

6. 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 1-9.


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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.

Start Icon

Introduction 34

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Figure 1-10 Enable Fraction Collection Dialog Boxes

Figure 1-11 General Page


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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

1

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.

Figure 1-14 MS-Directed Dye Test Chromatograms


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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.

1.4.1 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.

1.4.2 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

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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.

1.4.3 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.

Table 1-3 FractionLynx Browser Panes

Pane Description

Plate Displays the plate layout and compounds found.

Sample Description Displays sample information and indicates if a search desig-nated the compound as found, found tentative, or not found.

Fraction Collection Results List

Displays fraction collection information. This pane appears only if you collected fractions.

Results Table Lists detected compounds.

Spectrum Displays the spectrum for the highlighted sample well and the selected retention time.

Elemental Composition Results List and Library Search Results List

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.

Chromatogram Displays the integrated chromatogram for the highlighted sample well.

Accessing Reports Using the FractionLynx Browser 39

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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

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Table 1-4 Print Reports

Plate summary Produces a plate report. This is a picture of the plate showing:1 for found compounds? for found tentative compounds0 for compounds not found+ if no search was performed on the compound– for unused wells

Sample summary Produces a summary report for a sample. Define the number of samples printed in the report on the Print Control dialog box.

Sample Report Produces a more detailed sample report.

Sample on a New Page

Starts each sample in the sample report on a new page.

Table 1-5 Report Formats

Spectra height (mm) Prints the spectra associated with the sample. Select the check box, then enter the required height.

Chromatogram height (mm)

Prints the chromatogram associated with the sample. Select the check box, then enter the required height

All Chromatograms On One Axis

Uses one axis for all chromatograms displayed.

Mass Chromatogram/Axis

Select, then enter the number of chromatograms to display on each axis. Use this option to make overlaid mass chromato-grams easier to view. The default of 1 displays each chromatogram on a different axis.

Side by Side Chromatograms

Prints two chromatograms per line.

Note: The Peak Information table is not displayed if you select this option.

Side by Side Spectra 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

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Chapter 2Installing 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.

2.1 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

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The Welcome to the Wizard for MassLynx v4.0 page appears (Figure 2-1).

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

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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.

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


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9. Select OpenLynx Global Server if this option is to be installed. If not, leave the check box empty and click Next.

Figure 2-5 Installing OpenLynx Options

10. Click Install to start the program installation (Figure 2-6).

Figure 2-6 Starting the Program Installation


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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

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).


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13. Click Finish to close the wizard.

Figure 2-9 Program Installation Complete


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Chapter 3Using 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).

Figure 3-1 FractionLynx Editor

Fraction Kernel State Area

Communication 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.

Collection Enabled

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 frac-tion collection.

Accessing the FractionLynx Editor 48

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Fraction Collection Area

Peak Detection Area

Collection Mode

The currently selected collection mode appears, which can be Sequential, One to One, or Reserve tubes Per Injection. See Chapter 4 for further details.

State Current state of the fraction collector.

Location Location of the vessel currently used for collection in the format Fraction Collector Number:Vessel Number.

Next Collection Site

Location of the vessel used for collecting the next fraction using the format Fraction Collector Number:Vessel Number.

State 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.

Peak Start Time Start time of the peak being collected. This is the time since the injection, i.e., real retention time. All times referenced in the Frac-tionLynx system relate to the time that the peak was detected in the mass spectrometer, UV detector, or analog device.

Trigger Mass or wavelength of the fraction trigger, TIC, or analog.

Additional Information

Ionization mode.

Using the FractionLynx Editor 49

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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).

Figure 3-3 Reset Communications Command

Note: Communications cannot be reset when the instrument is acquiring data.

Enabling Fraction Collection 50

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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.

Figure 3-4 Collection Modes Dialog Box

For further details about this dialog box, see Section 4.4.1 on page 102.

3.5.1 Fraction File Editor1. 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.


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2. Define the parameters as needed (see Section 3.5.2 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.

3.5.2 General Page

Use the General page to enable the general parameters of Fraction Detection, Multiple Fractions, Rinse Between Fractions, and Span (Figure 3-5).

Figure 3-5 Fraction File Editor, General Page

The following topics describe the General page parameters.

Selecting Fraction Collection Parameters 52

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Fraction Detection Area

Multiple Fractions Area

Rinse Between Fractions Area

Fraction Collection

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.

Peak Type 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.

Max. Fractions per Injection

Enter the maximum number of fractions to be collected for a single sample in the Sample List. Range: 1 to 1000. (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).

Max. Tubes per Injection

Enter the maximum number of tubes to be collected for a single sample in the Sample List. Range 1 to 1000.

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.


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Span Area

3.5.3 Timing Page

Use the Timing page to set the parameters for Trace Monitoring and Peak Timing (Figure 3-6).

Figure 3-6 Fraction File Editor, Timing Page

Trace Monitoring 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.

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.


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Peak Timing Area

3.5.4 Volume Page

Use the Volume page to define fraction width and collection capacity (Figure 3-7).

Figure 3-7 Fraction File Editor, Volume Page

Split/Collector Delay (secs)

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.

MS/Aux. Delay (secs)

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 spectrom-eter 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 spec-trometer 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.


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Fraction Sizes Area

Collection Capacity Area

MinimumFraction 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.)

MaximumFraction Width

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.)

MaximumTube 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.


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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).

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 = −1This 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 nega-tive ionization scan. Use M + 23 for sodium adducts produced in + ion mode.


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Min. Intensity Threshold (MIT)

Enter the value above which peak intensities must be before they will be considered for peak detection. It is used to avoid false trig-gers 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., 250000 or 2.5e5. Range: 0 to 134217728 or 1.34e8.

Peak Start 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 1000.

0% 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.

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.


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3.5.6 Analog Page

Figure 3-9 Fraction File Editor, Analog Page

3.5.7 PDA Page

Figure 3-10 Fraction File Editor, PDA Page


Set the value according to the parameters in Section 3.5.5.

Peak Start Set the value according to the parameters in Section 3.5.5.

Terminate Peak Set the value according to the parameters in Section 3.5.5.


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3.5.8 UV Page

Figure 3-11 Fraction File Editor, UV Page

Span (+ / – nm) 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 3.5.5.

Peak Start Set the value according to the parameters in Section 3.5.5.

Terminate Peak Set the value according to the parameters in Section 3.5.5.


Set the value according to the parameters listed in Section 3.5.5.

Peak Start Set the value according to the parameters listed in Section 3.5.5.

Terminate Peak Set the value according to the parameters listed in Section 3.5.5.


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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 999.0 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).

Figure 3-12 Fraction File Editor, Timed Events Page


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3.5.10 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.

AND Operator Example

Figure 3-13 Illustrated Example of Mass A AND WaveLength B

Enable/Disable Collect

Turns on and off the Fraction Collection functionality of the soft-ware 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.

Start/Stop Collect 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.

MIT Settings 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.

Mass A

Wavelength B

Mass A AND Wavelength BCollects


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OR Operator Example

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

Figure 3-15 Illustrated Example of Mass A NOT Mass B

Collects A Collects BCollectsA and B

Mass A NOT Mass B

Collects AOnly

Mass A Mass B


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XOR Operator Example

Figure 3-16 Illustrated Example of Mass A XOR (Exclusive OR) Mass B

3.5.10.1 Using Boolean Triggers1. 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).

Figure 3-17 MassLynx Boolean Trigger, Boolean Column Dialog Box

Mass A XOR Mass B

Collects A Collects B

Mass A Mass B


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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.

Figure 3-19 FractionLynx Mixed Triggers Editor Dialog Box, Expanded


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c. Enter a trigger description in the Description field (Figure 3-19).

d. Select Mass A from the Trigger Source list (Figure 3-20).

Table 3-1 FractionLynx Mixed Triggers Editor Properties

Property Description

Description Lists Boolean trigger expression descriptions. It is this description that is entered into the Fraction Boolean Logic column in the MassLynx Sample List.

OK Exits the dialog box and saves any changes to the Boolean expressions.

Delete Removes a description from the list.

Cancel Exits the dialog box without saving any changes to the Boolean expression.

More Switches to the expanded view.

Less Switches to the reduced view.

Trigger Source 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.

Trigger Expression

Displays the Boolean trigger expression.

Add Adds the Trigger Source to the Trigger Expression field.

AND Adds the Boolean AND operator to the Trigger Expression field.

OR Adds the Boolean OR operator to the Trigger Expression field.

NOT Adds the Boolean NOT operator to the Trigger Expression field.

XOR Adds the Boolean XOR operator to the Trigger Expression field.

Open Brace Adds an open brace to the Trigger Expression field.

Close Brace Adds a close brace to the Trigger Expression field.

Backspace Removes the last Trigger Source or operand from the Trigger Expression field.

Clear Clears the Trigger Expression field.


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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).

Figure 3-21 Adding an Expression

f. Select the operator (Figure 3-22).

Syntax Warning


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Figure 3-22 Selecting an Operator

g. Select WaveLength A (Figure 3-23).

Figure 3-23 Selecting Wavelength A

h. Click Add to include the chosen wavelength to the editor file (Figure 3-24).


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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.

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).


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Figure 3-26 Sample List Page, Browse
4. Select the mixed trigger and click OK (Figure 3-27).

Figure 3-27 Selecting and Saving a Mixed Trigger


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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.


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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).


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Figure 3-30 Validation Error Message
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).


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


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3.5.10.2 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).

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).


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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).

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.


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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).


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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).


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Figure 3-39 Saving Changes

The trigger is now removed from the .ini file (Figure 3-40).

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).


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Figure 3-41 Triggers Removed from Mixed Triggers Editor and Mixed Triggers Dialog Boxes

3.6 Fraction Collection 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).

Figure 3-42 Collector Configuration Page (Waters Fraction Collector III)

Fraction Collection Setup 80

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3.6.2 Collector Configuration

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.

Number Of Collectors

Enter the number of fraction collectors that are being used. Range 1 to 9.

Collector Model Select the fraction collector model that is being used.If you select WFC II/III, the configuration is as shown in Figure 3-42.Variations for the Waters 2757 and the Gilson 215 and Gilson 204 are shown in Figure 3-44.

Collection Type 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 frac-tion 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.

Comm Port Enter the name of the comm port that the fraction collector is connected to (COM1, COM2, etc.).

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.


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Figure 3-43 Selector Valve Page

Communications (Waters 2757)

Figure 3-44 Collector Configuration (Waters 2757)


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Gilson Setup

Gilson 215

Gilson 204


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Figure 3-45 Typical Gilson Fraction Collector Setups

ID Enter the ID number displayed at the back of the instrument. For more information, see Section A.5 on page 211.

Gilson Comms Click this button to open the Gilson Comms dialog box (Figure 3-46).

Positions Click this button to open the Positions dialog box (Figure 3-47).

Gilson 215Inject/Collect


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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.

3.6.4 Gilson Positions

Use this dialog box to define the integer position (Figure 3-47).

Figure 3-47 Positions Dialog Box


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3.6.5 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.

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 2757 13x100 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.

Rinse Station for Collector 1

Specifies the coordinates of the rinse station from the home posi-tion of the needle (i.e., the position of the needle when not in use).

Travel Height Defines the travel height above the plate that the rack will sit on.

Dispensing Height

Defines the dispense height above the plate that the rack will sit on.


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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).

Figure 3-49 Bed Layout Dialog Box, Modify Bed Layout Tab


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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 1/10th 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.

3.6.6 Plate Generator

Select Collector Setup > Plate Generator from the FractionLynx Editor to open the custom Rack Generator window (Figure 3-51).


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Figure 3-51 Rack Generator Window

Rack Name Enter the name of the plate to edit.

Rows 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.

Columns 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.

Vial (These parameters do not affect fraction collection.)

Offsets Specify alternate vial rows or columns to be offset.

Origin Specify the corner of the rack from which the vial grid referencing starts.

Grid Reference Indicate how to reference the vial rows and columns, e.g. whether the rows are alphabetical or numerical.


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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.

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 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.

Plate Size Define the size of the plate to its outside edges.

Top Left Vial Offset

Define the measurement to the center of the first vial from the top-left corner of the plate.


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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).

Figure 3-52 Example of Rotated Rack

Scaling Plates

1. Select View > Scale Rack to open the Scale Rack dialog box (Figure 3-53).


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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 3-51.

3.6.7 Manual Control

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


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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


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).

Moving to an XY Coordinate


2. Enter values in the X and Y fields and click Move XY. The needle moves to the required position.

STOPAttention: Do not move in the XY direction before changing Z. This will damage the needle.


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Moving to a Z Coordinate

Note: This is a Gilson 215 only command.


2. Enter a value in the Z field and click Move Z. The needle moves to the required position.

Moving to the Home Position


2. Click Move Home. The needle moves to the Home position.

Moving to the Rinse Station Position


2. Click Rinse Station. The needle moves to the Rinse Station position.

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.

STOPAttention: Do not move in the XY direction before changing Z. This will damage the needle.

Resetting Beds 94

3

• 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.


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Chapter 4Collection 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

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.

Vessel / Well / Tube / Location Collection tube or bottle.

Rack / Plate Physical holder or tray for fraction vessels.

Bed / Bed Layout Configuration of a fraction collector in terms of the number and types of racks present.

Definition of Terms 96

4

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

4.3.1 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 4-1.

Collection Modes 97

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Figure 4-1 Plate Generator Types

4.3.2 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).

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

1

20

21

40

41

60

61

80

81

100

101

120

1 16 31 46 1 17 33 49 65 81

1

13

26

37

49

61

73

85

1 9 17 25 33 41 49 57 65 73 81 89

Fraction Collector Tube Numbering Schemes 98

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Figure 4-2 30-Tube Plate Layout


Each plate holds 30 locations/tubes.In FractionLynx, the tubes are reported as:Actual plate/Reported as1. 1…302. 31…603. 61…90

Each plate holds 60 locations/tubes.In FractionLynx, the tubes are reported as:Actual plate/Reported as1. 1…602. 61…120

1 2 3

1 2

Collection Modes 99

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4.3.3 Multiple Fraction Collectors

Where multiple fraction collectors are used in a single system, the location of each vessel is indicated by FractionCollector:VesselPositionOnCollector.

Figure 4-5 Plate Layouts for Multiple Fraction Collectors

Each plate holds 96 locations/tubes.In FractionLynx, the tubes are reported as:Actual plate/Reported as1. 1…962. 97…1923. 193…2884. 289…3845. 385…480

1 2 3 4 5

1 2 a b c

Fraction Collector Tube Numbering Schemes 100

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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…1:120

2. 2:1…2:120

3. 3:1…3:120

4. 4:1…4:120

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.

TP01606A TP01606A

TP01606A TP01606A

1 2

3 4

Collection Modes 101

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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

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

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Figure 4-8 Example of How Samples and Batches Collect

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).

Batch Sample Num Fractions

1 123

531

2 12

20

3 1 1

Samples continue to be run and the fraction collector bed will fill until it becomes full…

n 1 3


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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 Ctrl+A

Reset Collector 1 Ctrl+1

Reset Collector N Ctrl+N (where N is the number of collectors in the system)

Reset All Beds 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.

Reset Collector N 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.

Method of Operation for Each Mode 104

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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.

User start locations could be used to force specified samples to start collecting on a new plate.

4.5 One for One Collection

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.

Injection Fraction StartNumber of Fractions

Tubes Filled

1 10 4 10 – 13

2 12 14 – 25a

a. If a Fraction Start is not specified, collection continues into the next sequential tube.

3 30 6 30 – 35

4 2 36 – 37a

5 4 38 – 41a

6 50 4 50 –53

STOPAttention: 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).


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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.

4.5.2 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.

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.

Inject

Collect

One for One Collection 106

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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.

Figure 4-12 Sample Plates Larger than Collection Plates

Inject Collect

Wasted

#1

#2

#1 #2

#1

#2


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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.


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


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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.)

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).

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.

#1 #2 #3 #4

Collector PlatesInjector Plates

#1 #2

#3 #4


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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.




Figure 4-16 Mapping for Single Shot Samples

The fraction collector is now full despite only six actual samples being run.

4.5.5 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

Inject

Collect

#1 #2 #3 #4


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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.

Multiple Fractions Per Tube 112

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Consider a sample where the target mass elutes more than once from the column (Figure 4-18).

Figure 4-18 Sequential, One to One, and MultiFraction Mapping

Where the targetequals

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).


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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.

4.7.1 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).

Reserved Tubes 114

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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.

Figure 4-20 Modified Bed Layout for Mini-Racks or Fraction Blocks


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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

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 http://www.genevac.com/brochure/genevacaccessories.pdf.

Figure 4-21 GeneVac Rack

Table 4-1 Bed Scheme Defined as Two Columns of Five Rows

Row Column 1 Column 2

1 #1: 1−6 #6: 31−36

2 #2: 7−12 #7: 37−42

3 #3: 13−18 #8: 43−48

4 #4: 19−24 #9: 49−54

5 #5: 25−30 #10: 55−60

Reserved Tubes 116

http://www.genevac.com/brochure/genevacaccessories.pdf

5

Chapter 5Running 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.

5.1.1 Root Directory

The project root directory (<PROJECT_NAME>.PRO) contains seven subdirectories of which Acqudb, Data, and Sampledb are the most important.

5.1.2 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.

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5.1.3 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.

5.1.4 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).

Table 5-1 Acqu Database Control Files

Mass spectrometer tune ZMD*.dbf, ZQ*.ipr

Mass spectrometer acquisition *.mdb

Fraction collection parameters *.frc

HPLC pump control:Waters 515Waters 600Waters 1525Waters 2525Waters 2690/5Waters 2700Waters CapLCWaters 2790/5HP1100GilsonShimadzuJasco 900Jasco 1500Ultimate

*.w51*.w60*.w25*.bgm*.wat*.w27*.clc*.w29*.h11*.gil*.szu*.jas*.j15*.ult

Running FractionLynx Samples 118

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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

Sample List 119

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Table 5-2 Sample List Properties


File Name Raw data file name under which the data will be stored.

MS File Method containing the parameters that will control the mass spec-trometer during data acquisition.

Inlet File Inlet method file that will control the HPLC gradient and injection parameters.

Bottle Location of the sample in the autosampler tray.

Inject Volume Volume of sample to be injected.

Process 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).

Parameter File Contains the name of the OpenLynx parameter file (*.olp), which defines the OpenLynx processing to be performed.

Fraction File 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.

Mass A to Mass T 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.

Wavelength A to Wavelength J

(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.


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• UV1 to UV4

• 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.

Fraction Trigger 1 to Fraction Trigger 10

Specify which trigger type (up to 10) to use as the trace to deter-mine 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 A to Mass T 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.

Table 5-2 Sample List Properties (Continued)


Sample List 121

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Figure 5-2 Sample List Example: Fewer Mass Triggers than Masses

Table 5-3 Sample List with Fewer Mass Triggers than Masses


Wavelength A to Wavelength J

Wavelength-derived fraction collection will use the relevant wave-lengths 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).

Analog Takes the Analog Detection parameters from the Fraction param-eter file together with the relevant analog input to determine when fractions should be collected.

Mass TIC 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.

PDA TAC Enter PDA TAC in the Fraction Trigger field. PDA TAC collection uses the PDA detection parameters.


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5.3 Starting a Run from the Sample List

Select Start from the Run menu or click . The Start Sample List Run dialog box opens (Figure 5-3).

Fraction Start 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.

Fraction Boolean Logic

Browse for a named Boolean expression by right-clicking in this column and selecting Browse. For details, see Section 3.5.10 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).

Project 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).

Acquire Sample Data

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.

Auto Process Samples

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 Param-eters fields (.olp) in the Sample List.

Auto Quantify Samples

Automatically quantifies the results and is not applicable to the FractionLynx process.

Run From Sample n To Sample m

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.

Table 5-3 Sample List with Fewer Mass Triggers than Masses (Continued)


Starting a Run from the Sample List 123

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Figure 5-3 Start Sample List Run Dialog Box

Priority Flags the Sample List as a priority process and put it above all nonpriority lists in the queue.

Night Time Process

Runs the Sample List at night.

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

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

Displays the Lines and Fill Colors on the chromatogram.

Fill Fractions Displays the region of the chromatogram from which the fraction was collected, using the fill colors described below.

Show On All Chromatograms

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.

Lines 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 fractionFor 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.

Fill Fraction Display

Displays the region of the chromatogram where the fraction was collected, using the fill colors described below.

Displaying Fractions in MassLynx Chromatograms 125

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Figure 5-4 Fraction Display Parameters Dialog Box

First Colour Select the required colour from the list. The area of the chromato-gram where the first fraction was collected will be displayed in this colour. If there are more than two fractions collected from a chro-matogram, this colour will also be used to show the 1st, 3rd, 5th, 7th, etc. fractions.

Second Colour Select the required colour from the list. The area of the chromato-gram 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 2nd, 4th, 6th, 8th, etc. fractions.

Start Vessel Displays the location of the vessel where the collection of the frac-tion 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:


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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.

Last vessel on first plate 1:96

First vessel on second plate 1:97

First vessel on third plate 1:193

Number Of Vessels

Displays the number of vessels used to collect the fraction.

Fraction Trigger Displays the mass that triggered the fraction.

Chromatogram Type Displayed

TIC TIC

Diode Array wavelength nm

Mass m/z = mass

Real-Time Display of Fractions in Chromatogram 127

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Chapter 6FractionLynx Browser
6.1 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

6

6.2 FractionLynx Browser

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

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6.2.1 FractionLynx Browser Toolbar

Button Menu Equivalent Purpose

File > Open Open an existing FractionLynx Browser report.

Edit > Copy Copy the selection and put it on the Clipboard.

File > Print 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 between Fractions by Tube and Fractions by Trigger.

Swap Plate View Swap between normal plate view and multiple injection plate view.

View > Swap List View

Swap between the Elemental Composition and the Library Search Results views.

Help > About FractionLynx Results Browser

Display program information, version number, and copyright.


6

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.

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 6.4.5 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).


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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

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6.4 View Options

A number of options are available to change the appearance of the Browser screen. To access them, select View > Options.

6.4.1 Spectrum Page

Figure 6-4 View Options Dialog Box, Spectrum Page


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Peak Annotation Area

Horizontal Axis Area

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.

Decimal Places 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.

Accurate Mass Error Reporting

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.

Decimal Places Changes the number of decimal places displayed on the hori-zontal axis. Click the arrows to change this value. Range: 0 to 4 decimal places.

WaveLength Label Enter the text to appear as the label on the horizontal axis for diode array data.

Daltons Label Enter the text to appear as the label on the horizontal axis for mass spectral data.

Peak/Tube Number 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.

Retention Time Displays the Retention Time in the Spectrum header.

Process Description Displays the Process Description in the Spectrum Header.

Set DAD Axis Label Displays a label on the DAD Axis. Enter the label to display in the adjacent text box.

Select all Peak Spectra

Displays all spectra for the selected well.

Default range to data If selected, displays the default mass display range of a spec-trum (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.

View Options 134

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6.4.2 Chromatogram Page

Figure 6-5 View Options Dialog Box, Chromatogram Page

Peak Integration Displays the integrated baselines on the chromatogram trace.

Chromatogram Purity

Displays the chromatogram peak purity on the chromatogram peak tops.

Peak Numbers Displays the peak number above a peak on the chromato-gram peak tops.

Found Mass 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.


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6.4.3 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).

Spectrum Purity Displays the spectrum purity on the chromatogram peak top of a found peak.

Base Peak Mass 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.

Retention Time Displays the retention time values on the chromatogram peak tops. If selected, the Decimal Places option becomes avail-able. Click (arrows) or enter a value for the number of decimal places to display.

Threshold (%) Value that peaks must be above in order to be annotated on the chromatogram.

Horizontal Axis Decimal Places

Change the number of decimal places displayed on the hori-zontal axis. Click the arrows to change this value.

Label Enter the text to appear as the label on the horizontal axis.

Fill Peaks 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.

Fill Background Select this check box to fill the area under integrated peaks in the Unused color, specified on the Colors page.

Process Description Displays the process description on the chromatogram.

Replace DAD 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.

Set DAD Axis Label Select this check box to display a label on the DAD Axis and enter the label to display in the adjacent text box

View Options 136

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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 Enter the number of rows on the plate to be used.

Columns Enter the number of columns on the plate to be used.

Origin 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.


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6.4.4 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.

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.

STOPAttention: 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 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.

Vertical 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.

Horizontal priority

Select this check box if samples are to be acquired horizontally across the plate.If Referencing = X, Y, Horizontal = Letter, Vertical = Number and Hori-zontal 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.

View Options 138

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Figure 6-7 View Options Dialog Box, Colors Page

Sample Plate Area

Found The intensity of the peaks is above all threshold values.

Found (Tentative) 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.

Not Found There were no peaks for the mass entered.

Not Searched No mass was entered.

Unused There was no sample defined for this position on the plate.

Multiple Injection Multiple injections were made from the same well or vial.


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Fraction Plate Area

6.4.5 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

Current Sample Select the color to be displayed for fractions belonging to the currently selected sample.

Fraction Collected Select the color to be displayed for the vials where fractions were collected.

No Fraction Select the color to be displayed if no fraction was collected.

View Options 140

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6.4.6 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.

Sample Plate 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.

Fraction Plate

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.

Sample Summary

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.

Spectrum List

Select this check box to copy details of each peak for each sample.


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Figure 6-9 View Options Dialog Box, List Columns Page

Fraction Display 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

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Figure 6-10 View Options Dialog Box, Fraction Display Page

Show Fractions Select this check box to display the Lines and Fill Colours on the Chromatogram pane.

Show on all Chromatograms 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.


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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.

6.4.7 Other Display Options

Displaying Two Documents Simultaneously

1. Select File > Open. This displays a new window over the top of the previous one.

Start Line Shown at the start of each fraction.

End Line Shown at the end of each fraction.

Current A set of lines displayed at each end of the fraction selected in the Frac-tion Results List pane.

Fill Fraction Display Select this check box to display the region of the chromatogram that the fraction was collected from, using the fill colours described below.

First Colour 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 chro-matogram, this colour will also be used to show the 1st, 3rd, 5th, 7th, etc. fractions.

Second Colour 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 2nd, 4th, 6th, 8th, etc. fractions.

Current Colour 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.

View Options 144

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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.

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

6.5.1 File Menu

Load on startup

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.

Send To If this option is selected, an e-mail message is created with the currently selected report attached to it. Enter the address to send it to and click Send.

STOPAttention: To view the entire report the recipient of the e-mail 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 e-mail 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):


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Other Menu Options 146

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 E-Mailing of Report File

Select this check box to e-mail the *.rpt file (formed when the *.olb file is processed) to the recipient specified in the E-Mail Address field. The *.olb file is processed when the user submits it to the MassLynx queue.

Create File In Enter the location to which you want the failed samples *.olb file to be saved.

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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.

6.5.2 View Menu

Figure 6-13 View Menu

Refresh 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.

Swap List View 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.

Fractions by Tube Select this option to view fractions by Tube. Unselected fractions will be viewed by Trigger.

Multiple Injec-tions Per Vial

When this option is selected the user can swap between normal plate view and multiple injection plate view.


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6.5.3 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

Toolbar 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.

Status Bar 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.

New Window 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

Cascade Select this option to arrange document windows so that the title bar of each window is visible

Tile 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.

Arrange Icons

Select this option to arrange all iconized windows into rows.

Split Selects the splitter bar on the selected pane.

Other Menu Options 148

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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.

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.


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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.

6.7 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 6.4.4 on page 138).

Figure 6-16 Fraction Plate Pane

Fraction Plate Pane 150

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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.

Figure 6-17 Sample Description Pane

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

Submitter The username entered on the first page of the OpenLynx Login program.

Sample The file name of the sample, as entered in the Login dialog box or Sample List.

Vial The plate number followed by the well column and row references.

ID The sample ID as entered in the Login dialog box or Sample List.

Description The file description, as entered in the Login dialog box or Sample List.


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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:

View by Tube

If you do not select View > Fractions by Tube, the following columns are displayed within the Fraction Collection Results pane:

Fraction Trigger 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 corre-sponding peak on the Chromatography pane.

Original Target The mass minus any adducts.

Ion Mode The ion mode of the collected fraction.

Start Time The retention time at the beginning of the fraction collection.

End Time The retention time at the end of the fraction collection.

Collection Site The vial numbers of the fraction collected.

No. Of Tubes The number of tubes the fraction collection required.

Export Fraction 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.

Pool Tubes 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.

Tube Trigger 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.

Original Target The mass minus any adducts.

Fraction Collection Results Pane 152

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6.9.1 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).

Ion Mode The ion mode of the collected fraction.

Start Time The retention time at the beginning of the tube collection.

End Time The retention time at the end of the tube collection.

Collection Site The vial number of the tube collected.

Export Tube 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).


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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 6.16.7), then only fraction triggers with YES in their export field will be included in the exported file. The same is true for fraction tubes.

6.10 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

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Peak Number 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.

Compound If an entered mass is detected in a particular Peak Number then this column will display 'Found'.

Mass 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.

Function This column states the MS function and ionization technique in which the chromatographic peak was detected.

Time The retention time of the spectrum in decimal minutes.

Trace Description of the chromatogram used to locate this spectrum. This may be blank if a specific retention time was used to locate spectrum. The informa-tion listed here can be, among others, TIC, DAD, or a mass chromatogram.

% 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 The height of a particular peak.

RT Index The retention index of a particular peak.

RT Log P The measure of the hydrophobicity of a particular peak.

Concentration 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.

Amount 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.

State 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.


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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.

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.

6.11 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

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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.

6.12 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).


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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.

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.

6.13 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.

6.13.1 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

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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.


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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.

6.15 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.

6.15.1 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

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6.15.2 Print Control Page

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


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Print Reports Area

Peak Information Tables Area

To display peak information tables below a chromatogram, select the check box relevant to the type of information required.

Sample Plate summary

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.

Fraction Plate summary

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.

Sample summary 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.

Delimit Sections 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 6.16.9, Producing Tab-Delimited Files).

New Line Per Peak

Select this check box to start each peak on a new line.

New Line Per Trigger

Select this check box to start each trigger on a new line.

New Line Per Tube

Select this check box to start each tube on a new line.

Sample Report Select this check box to produce a more detailed report for a sample.

Sample on New Page

Select this check box to start each sample of the Sample Report on a new page.

Report Schemes 162

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Format Sample Report Area

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.

Spectra height (mm)

Select this check box and enter the required height, to print the spectra associated with a sample.

Chromatogram height (mm)

Select this check box and enter the required height, to print the chromatograms associated with a sample.

All Chromato-grams On One Axis

Select this check box to use one axis for all chromatograms.

Mass Chromato-grams/Axis

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.

Maximum Spectra Displayed

To limit the number of spectra printed on a page, select this check box and specify the maximum number of spectra to print.

Side by Side Chromatograms

Select this check box to print two chromatograms per line.

Note: The Peak information table is not displayed if this option is chosen.

Side by Side Spectra

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.

Page Orientation Click Portrait or Landscape to print the report with the required page orientation.


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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.

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

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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.

Figure 6-30 Edit Report Scheme Settings Dialog Box, Sample Summary Page

6.16.1 Sample Summary Page

Select Include Sample Summary Fields in Summary Text File to select or clear all Summary fields at once.


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6.16.2 MS Peaks Results Summary Page1. 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.

6.16.3 Report Header Page1. 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.

6.16.4 Sample Header Page1. 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.

6.16.5 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.

6.16.6 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).


6

6.16.7 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).

6.16.8 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


6

6.16.9 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.

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 n]


…….. ……..

SampleSummary[sample n] MSPeaksSummary[sample n, peak 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.

FRACTION TRIGGER RESULTS

SampleSummary[sample 1] FractionTriggerSummary[sample 1, trigger 1]


……. ……..

SampleSummary[sample 1] FractionTriggerSummary[sample 1, trigger n]


…….. ……..

SampleSummary[sample n] FractionTriggerSummary[sample n, trigger n]

FRACTIONTUBERESULTS

SampleSummary[sample 1] FractionTubeSummary[sample 1, tube 1]


……. ……..

SampleSummary[sample 1] FractionTubeSummary[sample 1, tube n]


…….. ……..

SampleSummary[sample n] FractionTubeSummary[sample n, tube n]

Table 6-2 MS Peak Results and Fraction Tube Summary

PEAKSUMMARYRESULTS



……. ……..

SampleSummary[sample 1] MSPeaksSummary[sample 1, peak n]


…….. ……..

SampleSummary[sample n] MSPeaksSummary[sample n, peak n]

Table 6-1 Sample Summary (Continued)

6
FRACTIONTUBERESULTS


……. ……..

SampleSummary[sample 1] FractionTubeSummary[sample 1, tube n]


…….. ……..

SampleSummary[sample n] FractionTubeSummary[sample n, tube n]

Table 6-2 MS Peak Results and Fraction Tube Summary (Continued)


7

Chapter 7FractionLynx 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.

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

7

Figure 7-1 Setting Up the Walk-up Page

Parameter Description

Description Displays a description of the method.

Input Fields Lists the fields that are displayed on the Login PC for user input. See “Adding or Deleting Input Fields” on page 173 for informa-tion on adding or deleting input fields.

Maximum Samples Enter the maximum number of samples to be processed in one OpenLynx login session.

Priority Process 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.

Night Time Process Defines the job submitted from the OpenLynx Login program as a night-time process.

FractionLynx with OpenLynx 172

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).

Note: All samples processed using this method become night- time processes. For more information, see the MassLynx User Guide.

HPLC File Allows you to define an LC method when logging in a sample.

Time of Analysis (mins)

Enter the time, in minutes, to analyze one sample.

Create Failed Samples Files

Saves failed sample data as an *.olb file.

MS Tune Select the tune parameters file from the list.

MS Method Select the scanning parameters file from the list.

Inlet Method Select the LC parameters file from the list. File extensions vary depending on the type of inlet you selected.

Injection Volume (µL) Enter the volume of sample to be injected.

Pre-Run Method Select the pre-run parameters file from the list to condition the column. File extensions vary depending on the type of inlet you selected.

Post-Run Method 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.

Switch Mode 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.

Fraction Method Select the fraction collection parameters file from the list.

Loop Mode Performs a loop injection, which you should only use for samples requiring no chromatography.

Loop Time (mins) Enter the retention time at which to acquire spectral data during processing.

Parameter Description


7
Figure 7-2 Adding and Deleting Input Fields
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.

Input Fields Description

Available Fields Lists available fields.

Order of Fields to Use Lists the fields displayed on the Login PC for users.

Append Adds a highlighted field to the end of the list.

Insert Adds a field immediately before that highlighted in the Order of Fields to Use list.

Remove Deletes a highlighted field.

Clear Deletes all fields.

Field Alias Enters a description of the field.

FractionLynx with OpenLynx 174

7

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.

Figure 7-3 OpenLynx Login Window


8

Chapter 8Troubleshooting 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.

8.1 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

8
Figure 8-1 Sample Chromatogram Using Dye 01
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

8
Figure 8-2 Display View
Under Normalize Data To, click Lowest Point.

Figure 8-3 Normalized Data Set to Lowest Point

Uncollected Samples 178

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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.

Figure 8-5 MIT Example Set to 3 Million


8

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.

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

8

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

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).


8

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:

Collected Fractions

Number 2

Trigger1 228.80

OrgTrigger1 227.80

IonMode1 +ve ion

StartTime1 2.97

EndTime1 3.35

CollectionSite1 1:5 to 1:6

NumOfTubes 2

Trigger2 372.90

OrgTrigger2 371.90

IonMode2 +ve ion

StartTime2 5.97

EndTime2 6.56

CollectionSite2 1:7 to 1:8

NumOfTubes 2

Raw Data Files 182

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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

CollectedFractions

CollectedTubes

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

DetectorMethod Intern

Analog Triggers Not Used

trigger1 trigger2 trigger3 trigger4

227.8 371.9 0.0 0.0

;PosAdd1 PosAdd2 PosAdd3 PosAdd4 PosAdd5

1.0 23.0 0.0 0.0 0.0

NegAdd1 NegAdd2 NegAdd3 NegAdd4 NegAdd5

–1.0 0.0 0.0 0.0 0.0

Trigger OrgTarget IonMode StartTime EndTimeCollection

SiteNumTubes

228.8 227.8 +ve ion 2.975 3.346 1:5 to 1:6 2

372.9 372.9 +ve ion 5.970 6.561 1:7 to 1:8 2

FractionNumber

TubeNumber

CollectorNumber

Well StartTime StopTime Plate

1 1 1 1, 1:1 2.985 3.305


8

• RinseTime = 0.0

• MassSpecToFracCollectorDelay = 15.00

• AuxToMSDelay = -4.00

• SolventFrontDelay = 0.00

• MinFractionValue = 3.00

• MaxFractionValue = 120.00 (If the sample is greater than 120 seconds wide, the collector would stop collecting.)

• MinFractionType = Time

• MaxFractionType = Time

• MaxTubeFill = 85.00

• 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 = 2500000.00 (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 = 80.00

• PDASpan = 3.00

• PDAThreshold = 1000.00 (If the threshold was set too high, then the wavelength would not be identified by the computer.)

8.4 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

8
Figure 8-10 Typical Inject-to-Collect, Mass-Directed AutoPurification System
GradientPump

Autosampler

Column19 x 50 mm

MakeupPump

1000:1Splitter

Splitter

ZQ MS

UV-VisPurifiedFraction

WasteCollectionExcess

Waste


8

Figure 8-11 Typical Inject-Collect Mass-Directed AutoPurification System

T118

T104

2487

T117

T103

T116

T106

T108

7

9

5

31

T113

T111

P-2

T112

T107T114P-1

2767

LFTINJ

RGTINJ

T101

2

6

1

54

3T105

515

T115

Fra

ctio

n F

low

Spl

itter

T120

WASTE

WASTE

ZQ

T121T122

Notes:1. Rheodyne pin plugs (289000677) are required and supplied

in the UV-directed Startup Kit (200000144).

2. Replace T105, T106, T107, T108, and T111 with “HF” equivalentsfor 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.

5. T117 is needed for 2996 PDA, 2487 Inlet connects directly

6. T120 connects to the outlet port of 2996 PDA, or to the outlettube 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.

into the detector flow splitter.

Port closest to the adjustment dial connects to the ZQ Detector.

4

56

78

3

Plumbing Diagrams 186

8

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.


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.

8.5.2 Delay Timing 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 1-1). 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 716000765 (in System Startup Kit)

• Stopwatch

• Mobile phases:

– A – Water with 0.1% V:V TFA

– B – Acetonitrile with 0.1% V:V TFA


8

UV-Directed System Test 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 1-5.

3. 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 = 30 − 15 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.

8

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 8-1.

2. 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 0.010 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

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.

Table 8-1 System Pressure Readings

System Pressure Readings

Prep side before disconnection: __________psi

Delta: __________psi

Prep side after disconnection: __________psi

Delta ofPrep and

Make-up Sides:__________psi

Make-up flow side before disconnection: __________psi

Delta: __________psi

Make-up flow side after disconnection: __________psi

MS-Directed System Test 190

8

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.

8.6.2 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 716000765 (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


8

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 = 30 − 15 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.

MS-Directed System Test 192

8

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

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.

OpenLynx Troubleshooting 194

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.

Error Message Field


8

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.

Note: For more information on OpenLynx troubleshooting, see the OpenLynx Software User’s Guide.

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Appendix AHardware Setup
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® 2000- 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

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Figure A-2 System Setting Changes Dialog Box

Figure A-3 Installation Completed Page

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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.

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).

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Figure A-5 Selecting the Driver Option

3. Select SST Driver (Figure A-6).

Figure A-6 Selecting the Appropriate Driver

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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.

Figure A-7 Install It Now Screen

Figure A-8 Help Screen

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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.

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.

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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

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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

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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).

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.

Table A-1 Equinox Ports

Labeled Port Number

Software (MassLynx) Port for Configuration

1 3

2 4

3 5

4 6

5 7

6 8

7 9

8 10

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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.

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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

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.

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Dilutor Configuration Tab

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.

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Wash Parameters Tab

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.

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Sampler Configuration Tab

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

2 3

3 2

4 6

5 5

6 4

7 8

8 7

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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.

Hardware Configuration

The rear panel of the Gilson 215 unit has two rotary dials.

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.

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.

SW1 Provides the instrument ID. Add 20 to the number indicated on the dial and use this in the FractionLynx Editor when configuring the software.

SW2 Determines the baud rate and clock synchronization of the 215 unit. Select option 6 – 19200 and GSIOCMaster/Break Active.

PC to 506C unit RS-232, 9-pin female to 25-pin male

506C to 204 collector GSIOC communications

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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.

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.

Figure A-21 Inlet Editor Window, Status Tab

Toolbar

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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.

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

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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.

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.

Event must be activefor MS-directed systems

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Figure A-24 Triggers Tab

GPIB Communication 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.

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Figure A-25 GPIB Communication Tab

HPIB 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.

AddressAssignmentDrop-Down

Lists

Recommended Setting of

10 SecondsDrop-Down

List

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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).

Figure A-26 Inlet Editor, View Menu

3. Click (Configure Autopurification Modules). The Configure Autopurification Modules drop-down list appears (Figure A-27).

Figure A-27 Modify Instrument Method Dialog Box

Instrument List

ConfigureAutopurification

ModulesButton

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4. Select Configure Autopurification Modules to open the Autopurification System Configuration dialog box (Figure A-28).

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

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Note: Do not click Close until the system finishes retrieving embedded information, such as serial numbers and addresses.

When the system finishes retrieving embedded information, the Autopurification System Configuration dialog box reappears (Figure A-28).

6. Click OK to save the scanned information.

A.10 BGM and C/FO Configuration

See the Waters 2525 Binary Gradient Module Installation and Maintenance Guide and the Waters Column/Fluidics Organizer Installation and Maintenance Guide for initial setup requirements.

A.11 2767 Sample Manager Configuration

See the Waters 2767 Sample Manager, Injector and Collector Installation and Maintenance Guide for comprehensive instructions on how to configure the 2767 Sample Manager to the AutoPurification system.

Hardware Setup 219

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Appendix BPeak Detection

This appendix describes how a peak is detected using the analytical and preparative peak types.

B.1 Analytical

B.1.1 Mass Detection

Positive ion acquisition with:

Target Mass = 69.0

Additional peak detection parameters include:

Span = 0.5

Adducts = 1 and 18

In this example, three independent Mass Traces will be monitored for eluting peaks. These will be:

This results in the following windows being set up, based on the Span parameter:

As data is acquired, any points that fall within a window will be summed. At the end of the scan, an average intensity for each window will be produced, for example:

69.0 70.0 87.0

(Target) (Target + Adduct 1) (Target + Adduct 18)

68.5 to 69.5 69.5 to 70.5 86.5 to 87.5

(69.0 +/– 0.5) (70.0 +/– 0.5) (87.0 +/– 0.5)

WindowNumSampsIn

Intensitymass

mass∑

5.69

5.68

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Each of these averaged intensities then gets put into a 3-point sliding window (Figure B-1 and Figure B-2). Again, there is a separate 3-point window for each active trace. The purpose of this window is to build up a profile of the trace. The three points represent the summed intensities for the last three scans. As a new scan is performed, the oldest point (A) in the window will be discarded and the new trace value tagged onto the end in the newest position (C).

B.1.2 Analog Detection

Analog detection uses the same idea of the 3-point sliding window and keeps separate traces for each analog input that is being acquired.

B.1.3 PDA Detection

PDA detection uses the same idea of the 3-point sliding window and keeps separate traces for each input wavelength that is being acquired. Adducts do not apply to wavelengths.

Figure B-1 Analytical Peak Detection

A B C

MIT

A peak has started once the two most recent samples, B and C, are abovethe minimum intensity threshold (MIT) AND the leading edge of the peak has a suitable gradient. This is identified in one of two ways 1. C > aA 2. (B > aA) AND (aB < C) The 2nd part avoids single scan ‘spikes’

aB = B * Leading Edge Gradient % aA = A + ( A * Leading Edge Gradient %)

Peak Detection

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Figure B-2 Analytical Peak Termination

B.2 Preparative

B.2.1 Mass Detection

When the user is trying to recover as much sample as possible, it is not uncommon for the column to be overloaded. So long as the components can be separated, the peak shape is often sacrificed.

These (semi-) preparative peaks frequently have a more gradual leading edge that consists of smaller gradient increases.

In these applications, a wider window is required to determine when the peak is detected. The selection of a preparative peak shape uses a 5-point sliding window (Figure B-3 and Figure B-4). Slightly different detection algorithms are used to determine the gradient across the window (see below).

The rules used to define traces and window values in the analytical system are still used here, i.e., separate mass traces for each defined mass.

A B C

MI

A peak has terminated when one of the following conditions is met C < MIT aA > B

aA = A * Trailing Edge Gradient %

Peak Termination

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B.2.2 Analog Detection

Again the rules applied to analytical peaks are used here, but the sliding window is 5 points wide.

B.2.3 PDA Detection

PDA detection uses the same idea of the 5-point sliding window and keeps separate traces for each input wavelength that is being acquired. Adducts do not apply to wavelengths.

Figure B-3 Preparative Peak Detection

A C EB D

A peak has started once the three most recent samples (C, D and E) are above the minimum intensity threshold (MIT) AND the leading edge of the peak has a suitable gradient. This is identified as

X > aA

X = (C + D + E) / 3 aA = A + ( A * Leading Edge Gradient %)

Peak Detection

MIT

Peak Detection 223

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Figure B-4 Preparative Peak Termination

A peak has terminated when one of the following conditions is met 1. E < MIT 2. aA > X

X = (C + D + E) / 3 aA = A * Trailing Edge Gradient %

Peak Termination

A C EB D

MIT

Peak Detection 224

I NDEX

Index

Symbols*.olb file 173*.ors file 39*.rpt file 38

AAcqudb directory 117Adding

events 61fields 164

BBed layout

creating 86deleting 87

Bed Layout dialog box 86Beds, resetting 94Boolean logic 123Boolean triggers 64Browser

menu options 145report file creation 38View Options dialog box 133

Browser filecopying 131opening 131printing 131

Browser window 129

CChromatogram page 135Chromatogram pane 158

Fraction Display 158Chromatogram Report page 164, 166

Chromatogramslines displayed 144print order 163

Clearing Event table 61Collection Modes dialog box 51, 102Collector Setup page 80Colors page 138Column pages 141Communications, resetting 50Configuring system modules 212Connecting

Gilson 204 Fraction Collector 211Gilson 215 Collector 211Waters Fraction Collector II or lll 210

Conventions, documentation 21Copy Control page 140Copying Browser file 131Creating

bed layout 86list of failed samples 147plates 90

DData directory 118Decimal Places page 164, 167Defining

HPLC file 173Walk-up page parameters 171

Deletingbed layout 87plates 90timed events 61

DialogsCollection Modes 51, 102Failed Samples Options 146Gilson Comms 85Gilson Positions 85Manual Control 92Modify Bed Layout 87

Index 225

INDEX

I

Not Enough Vessels 110Plate Description 88Print Control 131Program Not Found 146Scale Rack 92Set Margins 132View Options 133

DirectoriesAcqudb 117Data 118Root 117Sampledb 118

Display options 144Displaying lines 144Diverting sample 94Documentation

conventions 21related 18

EEdit Report Scheme Settings dialog box

Chromatogram Report page 166Decimal Places page 167MS Peaks Results page 166Print Control page 161Report Header page 166Sample Header page 166Sample Summary page 165

Elemental Columns page 141Elemental Report page 164Enabling fraction collection 50Equinox setup

for Windows NT systems 199for Windows XP systems 197

Exiting FractionLynx Editor 51

FFailed Samples Options dialog box 146Fields

alias 164, 174available 174editing 174

order of 174width 165

Files*.olb 173*.ors 39

Format Sample Report 163Fraction Boolean Logic 123Fraction Collection Results pane 151Fraction collection, enabling 50Fraction Collector

Multiple Plates 98Single Plate 97

Fraction Columns page 141Fraction Display page 142Fraction File Editor 51

General page 52PDA page 59Timed Events page 61Timing page 54Volume page 55

Fraction mode 173Fraction Plate pane 150Fraction Results Summary page 164Fraction Tube Spectra pane 157FractionLynx Editor

exiting 51opening 48

GGeneral page, Fractionn File Editor 52Gilson 204 Fraction Collector, connecting

211Gilson 215 Collector, connecting 211Gilson Comms dialog box 85Gilson positions 85Gilson setups 84, 85

HHardware, setting up 197Home position, moving to 94HPLC file, defining 173

Index 226

INDEX

IInjection volume 173Inlet method, selecting 173Input fields

editing 173listing 172

Instrument setup 80

LLibrary Columns page 141Lines on chromatograms 144List Columns page 141Load on Startup option 145Logging multiple batches 110Loop

mode 173time 173volume 208

MManual Control dialog box 92Manual Control option 92MassLynx software

2525 BGM configuration 2192767 Sample Manager configuration

219C/FO configuration 219configuring system modules 212Inlet Configuration dialog box 213Inlet Editor 212scan command 216setting up event start properties 214setting up event trigger properties 214setting up GBIP properties 215

Maximum samples 172Menu options 145

I

MenusView 147Window 148

Modesloop 173switch 173

Modify Bed Layout 87Moving

to Home position 94to Rinse Station position 94to vessel 93to XY coordinate 93to Z coordinate 94

MS method, selecting 173MS Peaks Results Summary Page 164MS Peaks Results Summary page 166MS tune file, selecting 173Multiple fraction collectors 100Multiple Fractions Per Tube 112Multiple Fractions Per Tube mode 96Multiple Plates Per Fraction Collector 98

NNight-time process 172Numbering Scheme

Fraction Collector 108Sample Plate 108

Numbering schemes 97

OOLB file 173One for One Collection mode 96OpenAccess Mode 174Opening

Browser file 131FractionLynx Editor 48report schemes 160

OpenLynx Browseraccessing 38report file creation 38

Index 227

INDEX

Optionsdisplay 144Manual Control 92

Order of fields to use 174ORS files 39

PPages

Column 141Decimal Places 167Print Control 161

PanesChromatogram 158Fraction Collection Results pane 151Fraction Plate 150Fraction Tube Spectra 157Results Table 154Sample Description 151Sample Plate 149Spectrum 156

PDA page 59Peak detection 220

analytical 220preparative 222

Peak information tables 162Plate Description dialog box 88Plate Generator dialog box 88Plate size versus rack size 106Plates

creating 90rotating 91scaling 91

Positions dialog box 85Post-installation system performance

enhancements 206Post-run method 173Pre-run method 173Print Control dialog box 131Print Control page 161Print order

chromatograms 163spectrum 163

I

Printing Browser file 131Priority process 172Projects 117

RRack size versus plate size 106Related documentation 18Removing fields 164Report Header page 164, 166Report schemes 160

opening 160saving 160settings 160

Reserved Tubes mode 96Resetting

beds 94communications 50

Results Table pane 154Root directory 117Rotating plates 91RPT file 38

SSample Description pane 151Sample Header page 164, 166Sample Plate pane 149Sample running order 111Sample Summary page 164Sample Summary Report page 165Sample, diverting 94Sampledb directory 118Samples, creating list of failed 147Saving report schemes 160Scale Rack dialog box 92Scaling plates 91Selecting

fraction collection parameters 51MS method 173MS tune file 173

Index 228

INDEX

Sequential collection 102Sequential Collection mode 96Set Margins dialog box 132Single plate per fraction collector 97Software, setting up 197Spectrum page 133Spectrum pane 156Spectrum print order 163Spectrum Report page 164Spectrum Search Report page 164Splitter/Probe Delay experiment 220Startup Kit, Sample Dye Kit 191Swap List View option 147Switch mode 173System-level tests

MS-directed chromatography test procedure 31, 193

required supplies for MS-directed system test 191

required supplies for UV-directed system test 187

UV-directed system test 24, 187

TTime of analysis 173Timed events

adding 61deleting 61

Timed Events page 61Timing page 54Triggers, Boolean 64

VVessel, moving to 93View by Trigger

Fraction Collection Results pane 152Fraction Tube Spectra pane 158

View by TubeFraction Collection Results pane 152Fraction Tube Spectra pane 158

I

View menu 147View Options dialog box 133

Colors page 138Column pages 141Copy Control page 140Fraction Display page 142Spectrum page 133

Volume page 55

WWalk-up page, defining parameters 171Waters Fraction Collector II or III, connecting

210Window menu 148

Browser 148

XXY coordinate, moving to 93

ZZ coordinate, moving to 94

Index 229