prep 15 sfc system guide - waters corporation · 2014. 10. 14. · considerations specific to the...

Prep 15 SFC SystemSystem Guide

715004396 / Revision A

Copyright © Waters Corporation 2014All rights reserved

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General InformationCopyright notice

© 2014 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA AND IN IRELAND. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER. 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, its use. For the most recent revision of this document, consult the Waters Web site (waters.com).

TrademarksWaters and Viridis are registered trademarks of Waters Corporation. Prep 15 SFC, MassLynx and THE SCIENCE OF WHAT’S POSSIBLE are registered trademarks.Microsoft, Internet Explorer, Windows, and Windows 7 are registered trade-marks of Microsoft Corporation in the United States and/or other countries.Other registered trademarks or trademarks are the sole property of their owners.

Customer commentsWaters’ Technical Communications organization invites you to report any errors that you encounter in this document or to suggest ideas for other-wise improving it. Help us better understand what you expect from our documentation so that we can continuously improve its accuracy and usab-ility.

March 14, 2014, 715004396 Rev. A iii

We seriously consider every customer comment we receive. You can reach us at [email protected].

Contacting WatersContact Waters® with enhancement requests or technical questions regard-ing the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail.

Waters contact information:

Internet The Waters Web site includes contact information for Waters locations worldwide. Visit www.waters.com

Telephone and fax

From the USA or Canada, phone 800 252-HPLC, or fax 508 872 1990.For other locations worldwide, phone and fax numbers appear in the Waters Web site.

Conventional mail

Waters Corporation34 Maple StreetMilford, MA 01757USA

Safety considerationsSome reagents and samples used with Waters instruments and devices can pose chemical, biological, or radiological hazards (or any combination thereof). You must know the potentially hazardous effects of all substances you work with. Always follow Good Laboratory Practice, and consult your organization’s safety representative for guidance.

Considerations specific to the Prep 15 SFC SystemAlways keep in mind these safety practices:

l Familiarize yourself with proper handling, storage, and disposal of all chemicals used with your SFC system. Refer to the Material Safety

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mailto:[email protected]://www.waters.com/

Data Sheet (MSDS) for each solvent you use, and know its chemical properties.

l Wear the appropriate personal protective equipment when working with any type of hazardous chemical.

l Wear eye protection while near the instrument to protect eyes from possible failure of column seals or fittings.

l Ensure that the system is depressurized before loosening any fittings in the CO2 flow path.

l Use care when removing and opening the column. Loosening an in-line column's fitting with liquid CO2 in the system can cause a high-pressure release of sample and solvent or damage the column.

Warning: To avoid burn injuries, use care when handling the column or other components heated to high temperatures. Wait until the hot components have sufficiently cooled before you handle them. Warning: The Prep 15 SFC system poses a potential rapid decom-pression hazard. Exposure to rapidly expanding fluids can cause injuries, including frostbite. To avoid this hazard, take these precau-tions: l Always depressurize the system before changing or working on

the bulk CO2 supply. l Use only bulk CO2 supply lines constructed of appropriate mater-

ials. l To avoid a high-pressure release of sample and solvent or damage

to the column, do not attempt to loosen a column's fitting when liquid CO2 is in the system. You must first completely depres-surize the system using the ABPR needle control setting, and then slowly loosen the column outlet fitting to allow the column to depressurize to ambient conditions. To do this without drain-ing the dampening vessel of CO2, before you depressurize the system, close the MV-2 valve.

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Warning: To avoid causing a hazardous condition in which a collec-tion bottle can explode should the bottle become pressurized, avoid these conditions: l Use of alternately-sized collection bottles without Waters

approval l Use of bottles that are not safety coated l For the exhaust tubing, any size reductions, sharp bends, kinks

or other conditions that can restrict flow of the CO2 exhaust l Modifications to the collection bottle inlet l Opening of collection bottles while the system is running

FCC radiation emissions noticeChanges or modifications not expressly approved by the party responsible for compliance, could void the users authority to operate the equipment. This device complies with Part 15 of the FCC Rules. Operation is subject to the following two conditions: (1) this device may not cause harmful inter-ference, and (2) this device must accept any interference received, including interference that may cause undesired operation.

Canada spectrum management emissions noticeThis class A digital product apparatus complies with Canadian ICES-003.Cet appareil numérique de la classe A est conforme à la norme NMB-003.

Electrical power safety noticeDo not position the instrument so that it is difficult to operate the discon-necting device.

Safety hazard symbol notice

Documentation needs to be consulted in all cases where the symbol is used to find out the nature of the potential hazard and any actions which have to be taken.

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Equipment misuse noticeIf the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired.

Safety advisoriesConsult Appendix A for a comprehensive list of warning and caution advisories.

Operating the Prep 15 SFC SystemWhen operating the Prep 15 SFC system, follow standard quality-control (QC) procedures and the guidelines presented in this section.

Applicable symbols

Symbol Definition

Manufacturer

Authorized representative of the European Community

Confirms that a manufactured product complies with all applicable European Community directivesAustralia C-Tick EMC compliant

Confirms that a manufactured product complies with all applicable United States and Canadian safety require-mentsConsult instructions for use

Contact Waters Corporation for the correct disposal and recycling instructions

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Audience and purposeThis system guide is for personnel who install, operate, and maintain the Prep 15 SFC System. It provides most of the information required to config-ure, operate, and maintain the system's hardware and software components. The only information it does not provide is that explaining the operation of the Thermo Scientific Accel 500 Laboratory Chiller unit, the mass flow meter (which is set up by a Waters Service Representative) and additional operation and maintenance details for the Waters 2998 Photodiode Array (PDA) detector and 2545 Quaternary Gradient Module (QGM). For details about the chiller unit, refer to the Thermo Scientific Accel 250/500 Labor-atory Chillers Installation and Operation Guide. For details about the 2998 PDA detector and 2545 QGM, refer to the operator's guide for each instru-ment.

Intended use of the Prep 15 SFC SystemWaters designed the Prep 15 SFC System to serve as a high-pressure puri-fication system for quantitative recovery of purified products such as enan-tiomers, complex synthetic chemicals, and natural products, or for research. It is not intended for use in diagnostic or biologically hazardous applic-ations. Use the system as a preparative-scale, high-pressure purification system, or as an investigative tool, to perform supercritical fluid chro-matography of compounds in these environments:

l Pharmaceutical development l Quality assurance and quality control l Chemical materials l Environmental sciences l Food safety environments

The Prep 15 SFC is for research use only and is not intended for use in diagnostic applications.

CalibratingTo calibrate SFC systems, follow acceptable calibration methods using at least five standards to generate a standard curve. The concentration range for standards should include the entire range of QC samples, typical speci-mens, and atypical specimens.

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In cases where an overview and maintenance guide, not operator’s guide, accompanies the instrument, consult the instrument’s online Help system for calibration instructions.

Quality-controlRoutinely run three QC samples that represent subnormal, normal, and above-normal levels of a compound. Ensure that QC sample results fall within an acceptable range, and evaluate precision from day to day and run to run. Data collected when QC samples are out of range might not be valid. Do not report these data until you are certain that the instrument performs satisfactorily.

ISM classification

ISM classification: ISM Group 1 Class BThis classification has been assigned in accordance with IEC CISPR 11 Industrial Scientific and Medical (ISM) instruments requirements. Group 1 products apply to intentionally generated and/or used conductively coupled radio-frequency energy that is necessary for the internal functioning of the equipment. Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage, power-supply network.

EC authorized representative

Waters Wilmslow Stamford AvenueAltrincham RoadWilmslow SK9 4AX United Kingdom

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Telephone:Fax:Contact

+44-161-946-2400+44-161-946-2480Quality manager

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Table of Contents

General Information iii

Copyright notice iii

Trademarks iii

Customer comments iii

Contacting Waters iv

Safety considerations ivConsiderations specific to the Prep 15 SFC System ivFCC radiation emissions notice viCanada spectrum management emissions notice viElectrical power safety notice viSafety hazard symbol notice viEquipment misuse notice viiSafety advisories vii

Operating the Prep 15 SFC System viiApplicable symbols viiAudience and purpose viiiIntended use of the Prep 15 SFC System viiiCalibrating viiiQuality-control ix

ISM classification ixISM classification: ISM Group 1 Class B ix

EC authorized representative ix

1 Getting Help 19

Prep 15 SFC system 19

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Purpose 19Audience 19

Observe Good Laboratory Practice 20

2 Reviewing System Components 21

Reviewing Prep 15 SFC capabilities 21

View components: UV-directed system 22

View components: mass-directed system 23

Review component descriptions 24

View flow diagram: UV-directed system 29

View flow diagram: mass-directed system 30

Gas-liquid separator 31Description of operation 32Specifying the GLS MBPR pressure 34Troubleshooting 35

3 Getting Started with System Setup 39

Configure the inlet 39

Specify column names and solvent names 40

Configure system components 41

Software requirements 42

4 Getting Started with MassLynx Software 43

Review MassLynx software workflow 43

Review online Help for control software 46

Start and exit MassLynx software 46

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Tour the MassLynx software interface 47

Work with MassLynx software projects and sample lists 48Project files 49Sample lists 49

Activate FractionLynx software 51

5 Managing Inlet Methods 53

Create, edit, and load an inlet method 53

Specify settings for the fluid delivery module 56Split ratio 57Isocratic runs 57Solvent selection 58Makeup flow rate 58Run gradient 58Run time 59

Configure the photodiode array detector 59

Specify pressure settings and column oven settings 60

Specify settings for the sample manager 62

Set FractionLynx method timing parameters 63Suggested timing parameters 65Detector configurations and delay parameters 66Determine the offset between two detectors 67

Review terms and settings for purification runs 69

6 Preparing the System for Use 71

Prepare the system: checks and procedures 71

Supply CO2 and nitrogen 81Carbon dioxide 82Nitrogen 82

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Prepare solvent vessels for use 82

Fill solvent vessels 84

Determine fluid delivery module status 85

Prime the FDM 88

Load samples 88

Prepare the fraction collection bed 90

Activate the Collection Control application in FractionLynx soft-ware 90

Specify GLS vent-outlet pressure 91

7 Executing Purification Runs 95

Review purification procedures 95

Conduct a typical purification run 97

Specify injection vial in a sample list 99

Select or modify the bed layout 101

Monitor system status in the Inlet Method editor 102Status tab 102Thar Inlet Additional Status tab 104

Determine status of the PDA detector 106

Verify system status during purification runs 107System equilibrating 108System ready 108System running 109System idle 110

Monitor CO2 pressure and temperature 110

View the results of a purification run 111

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8 Maintaining the System 113

Create a cleaning method 113

Shut down the system 113

Clean flow lines 114

Replace a flow line filter 115

Maintain the FDM 116Reviewing FDM operation 117Maintaining the CO2 pump 119Maintaining the co-solvent pump 129

Replace CO2 cylinder 137External CO2 supply connections 137Connecting the CO2 supply cylinder to the CO2 inlet fitting 138

Clean fraction collection tubes 139

Replace component fuses 140

Maintain the ABPR and heat exchanger module 141

9 Troubleshooting the System 143

Correct CO2 flow problems 143

Resolve system problems 145

Troubleshoot fluid delivery module 146

Pressure test the system 148

Troubleshoot the column oven 149

Review audit log 151

10 Reviewing Column Oven Procedures 153

Review the operation of the column oven 153

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Review safety information for the column oven 154General safety 154Electrical safety 155Chemical safety 155Integrated safety features 156

Control the column oven from the front panel 157

Replace a column in the column oven 158

Clean oven surfaces 160

11 Ensuring a Proper Installation and Operating Envir-onment 161

Review requirements for CO2 and co-solvents 161

Review environmental requirements 162

Review power requirements 163

Review system and component dimensions and weights 164

Confirm the integrity of tubing and connections 166Tubing 166Fittings 167

A Safety Advisories 169

Warning symbols 169Specific warnings 170

Caution advisory 173

Warnings that apply to all Waters instruments and devices 173

Warnings that address the replacing of fuses 180

Electrical and handling symbols 180Electrical symbols 180

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Handling symbols 181

B Specifications 183

Review system specifications 183

Review equipment control settings 187

Consumables and spare parts 189

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1 Getting HelpContents:

Prep 15 SFC system 19Observe Good Laboratory Practice 20

Prep 15 SFC systemYou can use the Prep 15 SFC system as a chromatographic system, or as a small-scale preparative purification system, to isolate compounds for research and analysis. The system uses supercritical CO2 as the primary solvent for the mobile phase. The core system relies on an ultraviolet detector. Adding a mass spectrometer or an ELS detector expands the system's capabilities.

PurposeThis guide explains how to operate and maintain the Prep 15 SFC system. Specifically, it addresses these topics:

l Reviewing system capabilities l Preparing for purification runs l Starting and operating components l Initiating, monitoring, and completing purification runs l Maintaining the system for reliable performance and long life l Diagnosing and correcting errors or other problems

AudienceThis guide provides information for chemists, technicians, application engin-eers, and other individuals who operate or maintain the Prep 15 SFC system.

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See also:

l Review component descriptions

Observe Good Laboratory PracticeObserve Good Laboratory Practice (GLP) when you operate the Prep 15 SFC:

l Familiarize yourself with proper handling, storage, and disposal of all chemicals used with your SFC system. Refer to the Material Safety Data Sheet for each solvent you use, and know its chemical prop-erties.

l Wear the appropriate personal protective equipment when working with any type of hazardous chemical.

l Wear eye protection while near the instrument to protect eyes from possible failure of column seals or fittings.

l Ensure that the system is depressurized before loosening any fittings in the CO2 flow path.

l Exercise caution when accessing internal oven components. Surfaces of the components can become hot during normal operation, and direct contact with them could cause injury. The internal oven compartment can attain temperatures high enough to cause burn injuries, and the internal surfaces do not spontaneously cool when you open the door. Internal surfaces such as extraction vessels, valves, tubing, and fit-tings can be hot.

Warning: To avoid burn injuries, use care when handling columns or other components heated to high temperatures in the column oven. Wait until the oven and its components have sufficiently cooled before you open the oven door and handle internal compon-ents.

See also:

l Warning symbols

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1 Getting Help

2 Reviewing System ComponentsContents:

Reviewing Prep 15 SFC capabilities 21View components: UV-directed system 22View components: mass-directed system 23Review component descriptions 24View flow diagram: UV-directed system 29View flow diagram: mass-directed system 30Gas-liquid separator 31

Reviewing Prep 15 SFC capabilitiesPurification laboratories use the Prep 15 SFC to benefit from the advant-ages of supercritical fluid chromatography (SFC). The Prep 15 SFC uses supercritical CO2 as its main solvent, which enables you to conduct prepar-ative-scale purification at a low cost of operation.Specifically, the Prep 15 SFC uses high-pressure CO2 as the primary mobile phase for preparative-scale purifications. This technology enables rapid equi-libration and faster run times than conventional preparative chro-matographic LC techniques. SFC is versatile in its application to a large variety of compounds. You can configure the system for both mass-directed and UV-directed purification.

Prep 15 SFC capabilities:

Description Capability

Maximum flow rate 15 mL/min Suggested working flow range 5 to 15 mL/min total flow for puri-

ficationsCo-solvent gradient range 5% to 55%

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Description Capability

Optimal column size 4.6-mm ID analytical columns for method development; 10-mm ID preparative columns for purification

Suggested maximum crude mixture concentration

2 mg/injection

Suggested working crude mixture concentration

0.01 to 1.00 mg/injection

Additional capabilities:

l Triggering fraction collection from a Waters 2998 photodiode array, and from a Waters mass spectrometer or ELS detector

l Automating sample injection l Collecting fractions in an open bed

See also:

l Review component descriptions l View components: UV-directed system l View flow diagram: UV-directed system l View components: mass-directed system l View flow diagram: mass-directed system

View components: UV-directed systemThe table below lists components in the UV-directed version of the Prep 15 SFC.

UV-directed system components:

Abbreviation Component

FDM Fluid delivery moduleHE1 Post-ABPR heat exchangerGLS Gas-liquid separator

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2 Reviewing System Components


ABPR Automatic back-pressure regulatorSM 2767 sample managerHPLC 515 Collection makeup pumpPDA 2998 photodiode array detectorN/A Chiller unitN/A Column ovenN/A Stacked injection module (optional)N/A Solvent reservoirs

See also:

l Review Prep 15 SFC capabilities l Review component descriptions l View flow diagram: UV-directed system l View flow diagram: mass-directed system

View components: mass-directed systemThe table below lists components in the mass-directed version of the Prep 15 SFC.

Mass-directed system components:


MS Mass spectrometerSplitter SFC flow splitterPDA 2998 photodiode array detectorABPR Automatic back-pressure regulatorHE1 Post-ABPR heat exchangerSM 2767 sample managerHPLC 515 Collection makeup pump

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View components: mass-directed system


HPLC 515 MS makeup pumpGLS Gas-liquid separatorFDM Fluid delivery moduleN/A Chiller unitN/A Column ovenN/A Stacked injection module (optional)N/A Solvent reservoirs

See also:

l Review Prep 15 SFC capabilities l Review component descriptions l View flow diagram: UV-directed system l View flow diagram: mass-directed system

Review component descriptionsThe following two tables describe Prep 15 SFC components:

l Components included in both UV-directed and mass-directed systems l Components included in mass-directed systems only

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Components included in both UV-directed and mass-directed systems:

Component Description

Fluid delivery module The fluid delivery module contains two pumps. One delivers high-pressure, super-critical CO2 to the Prep 15 SFC system. The second pumps co-solvent to the 2767 sample manager. l Maintains steady flow for liquid CO2,

and for co-solvents. l Total flow = 15 mL/min (CO2 and co-

solvent). l Co-solvent flow rate is 5% to 55% of

total flow. l Pump head is cooled to maintain liquid

CO2. CO2 at the FDM is liquid, and not yet supercritical.

l Solvent selector for up to six co-solvents.

Chiller unit The chiller unit chills a water and ethyl-ene glycol mixture (anti-freeze) in the circulating bath. The circulating bath cools and liquefies the CO2 before it enters the fluid delivery module's CO2 pump. Circulating coolant also removes heat of compression from CO2 pump heads.

Post-ABPR heat exchanger Post-ABPR heat exchanger reduces the effects of CO2 expansion, such as freezing, dry ice formation, and sample precip-itation. To accomplish that, it maintains correct temperature for the mobile phase as it flows to the gas-liquid separator.The column oven's External Zone 2 temper-ature controller regulates the temperature set point for the heat exchanger.

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Review component descriptions


2767 sample manager Manages sample injection and fraction collection. The sample manager has separ-ate sampling and fraction dispensing probes. The dual-probe robotic arm has access to all positions on the worktable. l Under control of MassLynx and Frac-

tionLynx software. l Self-venting probe that performs accur-

ate sample injections from tightly covered containers.

l High velocity wash pump with a flow rate greater than 30 mL/min, to flush the probe and tubing, and maintain high sample throughput.

l Fume hood, for ventilation of haz-ardous vapors.

l (Optional) Stacked injection module manages stacked injections for increased throughput. It supplants the injection mechanism in the 2767 sample manager.

Column oven Column oven's primary purpose is to main-tain column temperature for separation.Note: The critical temperature for CO2 is 30.98 °C. Typical set points are between 35 °C and 50 °C. The column oven's full temperature range is 5 °C above ambient temperature to 90 °C ± 0.5 °C. User sets temperature set points. The column oven holds multiple columns of different sizes, depending on the config-uration of the racks inside the oven.

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2998 photodiode array detector Integrated software and optics deliver chro-matographic and spectral sensitivity: l Maximum noise less than 10 µAU. l Quantifies high-level and low-level

components within a single chro-matographic separation.

l Flexible sampling rates for both nor-mal and fast SFC separation.

l Definitive compound identification and co-elution detection.

l Optimized signal strength across the full range of wavelengths.

Automatic back-pressure regu-lator

Electronically controlled needle valve in the ABPR maintains system pressure.Note: The critical pressure for CO2 is 73.77 bar (1070 psi). System pressure is typically set between 100 bar and 200 bar (1450 psi and 2900 psi).

Collection makeup pump Supplies additional flow to the solvent stream to maintain the set flow rate required for the GLS, to provide optimal fraction collection, and to maintain sample solubility.

Gas-liquid separator Separates CO2 gas from the liquid portion of the mobile phase near the end of the purification process.

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Review component descriptions

Components included in mass-directed systems only:


Mass spectrometer Mass spectrometer detects ions, allowing the system's control system to determine whether or not to collect a particular compound. Since mass spectrometry is a destructive technique, the SFC flow split-ter is an essential component in its imple-mentation.

Mass spectrometer (MS) makeup pump

515 HPLC pump adds conditioning solvent to the mobile phase as it flows through SFC flow splitter to the mass spec-trometer. The conditioning solvent helps to maintain peak shape, promotes solu-bility, and aids ionization. You can add some ionization agents such as formic acid, water, or both to the makeup solvent, to enhance ionization, if necessary.

SFC flow splitter Splits a small portion of the mobile phase from the main preparative flow, and redir-ects it to the mass spectrometer. After the split, the MS makeup pump adds condi-tioning solvent to the redirected flow as it moves toward the mass spectrometer for analysis. Most of the flow from the splitter travels through the PDA to the gas-liquid separator, and then to the fraction collector.

See also:

l Review Prep 15 SFC capabilities l View components: UV-directed system l View flow diagram: UV-directed system l View components: mass-directed system l View flow diagram: mass-directed system

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View flow diagram: UV-directed systemThe diagram below illustrates components and direction of flow for the UV-directed version of the Prep 15 SFC.

Prep 15 SFC UV-directed system:

See also:

l Review Prep 15 SFC capabilities l View components: UV-directed system

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View flow diagram: UV-directed system

l View components: mass-directed system l Review component descriptions

View flow diagram: mass-directed systemThe diagram below illustrates components and direction of flow for the mass-directed version of the Prep 15 SFC.

Prep 15 SFC mass-directed system:

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See also:

l Review Prep 15 SFC capabilities l View components: UV-directed system l View components: mass-directed system l Review component descriptions

Gas-liquid separatorThe GLS is an integral part of the fraction collection section of the Prep 15 SFC system. It enables you to collect fractions into open-bed collection vessels at atmospheric pressure.The fraction collection section of the system comprises a set of components in which the pressurized supercritical fluid mobile phase is separated into its depressurized gas and liquid constituents. After the mobile phase flow passes through the ABPR, the liquid portion of the mobile phase, along with the fractions to be collected, is directed via the GLS to the collection tubes, while the CO2 gas is safely vented. In the post-ABPR section of the collection path, a phase change occurs as the liquid CO2 depressurizes, resulting in a non-linear flow rate that is orders of magnitude faster than the chromatographic flow rate. Additionally, when the system is operating in gradient mode, a variable mix of solvent and CO2 gas flows through the collection path. As the compressed CO2 expands into the gaseous state, it possesses high kinetic energy that, if not controlled, could lead to undesired aerosolization of the co-solvent during collection of fractions of the eluting mixed gas and liquid solvent stream. The fraction collection components of the Prep 15 SFC are designed to provide the fine control needed to collect the fractions into open-bed tubes without aerosol formation, and to effectively maintain optimal collection performance while safely venting the CO2 portion of the mobile phase. Frac-tion collection delay times are predictable and reproducible across an SFC gradient, ensuring a high level of fraction purity and recovery performance, across the entire operating range of the instrument.The following sections further describe the GLS:

l Description of operation l Specifying the GLS MBPR pressure

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Gas-liquid separator

l Troubleshooting

Description of operationThe system's collection section includes all component parts and tubing, from the ABPR, through the collector, to the waste vessel. These are the major components, in the order of the flow path:

l ABPR l Input tee for the GLS makeup solvent flow (with associated GLS

makeup pump) l Post-ABPR heater l GLS with manual back-pressure regulator (MBPR) l Collection valve l Gas and liquid waste lines

The dimensions of these components in the collection flow path and their connecting tubing are fixed by design. Collection delays, from the UV detector to the fraction collection valve, are determined by these dimensions within the context of the system’s operating flow rate range. Therefore, to maintain the pre-determined, calibrated, collection delay values, you must not alter any of the components in the collection flow path. Furthermore, components or tubing that you replace must match the original parts.

GLS module

The GLS consists of these parts: l Initiator (or dripper) tube l Glass, V-bottomed cyclone housing l Exit port l MBPR

At the center of the module, the initiator, or dripper, tube transfers the expanding mobile phase flow into the glass, V-bottomed cyclone housing. That housing is contained within a glass, pressure container, itself contained within a stainless-steel safety housing. The exit port is at the bottom and center of the glass V tube. Liquid flows to the exit port and then downward to the fraction collector.

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Important: The position of the initiator tube is set and tested at the factory for optimal performance. The initiator tube is then held in position by a clamp. Do not loosen the clamp, for doing so will change the factory setting. Likewise, when attaching incoming tubing to the GLS, take care to avoid changing the position of the initiator tube.The MBPR is a pressure regulator and gauge placed in the CO2 vent line. It sets and maintains the pressure in the GLS for driving the liquid flow to the fraction collector. A pressure relief valve, set at 100 psi (6.9 bar), is also fitted to the GLS, to prevent overpressuring in the event of a blockage.Between the V-bottomed tube and the pressure container is a void space. It is within the GLS’s pressurized area but not in the system’s flow path. A drain tube with a stopcock, affixed to this void area between the glass tubes, drains from the pressure container any liquid from condensation that accumulates. During system operation, this stopcock must remain closed, to maintain proper pressure in the GLS. If liquid accumulates in the void area, you can slowly open the stopcock to drain the liquid. Some accu-mulated liquid in this area will not affect system performance, and is gener-ally of no concern unless it accumulates significantly, at which point it can be drained to waste.Requirement: Open the stopcock slowly. Otherwise, the pressurized liquid accumulated in the void area can drain quickly and possibly spray.

Makeup flow

The Make-Up Flow setting of the MassLynx Inlet Method determines the minimum total liquid solvent flow through the GLS. The makeup pump does not flow at this setting; rather, the software determines, in real time, the flow rate required for the makeup pump to maintain the minimum liquid flow rate in the GLS. A makeup flow is necessary to maintain optimal collection performance at any point in an SFC gradient. For UV-directed systems, the GLS makeup flow rate equals the GLS Make-Up setting minus the co-solvent flow rate.As the sum of liquid flows, from the co-solvent pump and potentially other pumps, achieves the GLS Make-up Flow setting, the GLS makeup pump shuts down. During gradient operation, the GLS makeup pump runs a reverse gradient.

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Typically, at the end of an SFC gradient, after all analytes are eluted and collected, the system quickly resets to starting conditions and a low co-solvent percentage. During this gradient reset, you can sometimes observe an accumulation of liquid in the GLS that remains until the increased CO2 flow eventually pushes it out. Because all fraction collections for the run are completed before gradient reset, this liquid is of no concern. It is, in fact, useful, for it rinses the GLS in preparation for the next gradient run. The accumulated liquid drains from the GLS before the start of the next run. If the liquid does not completely clear before the next injection, verify the MBPR pressure setting, and inspect the collection flow path to ensure it is not obstructed.Note: Makeup flow does not start until the ABPR reaches its pressure set point. The delay reduces liquid buildup in the GLS during system startup.

Specifying the GLS MBPR pressureThe MBPR maintains the appropriate pressure balance within the GLS, for optimal collection performance. Having been set, this pressure rarely, if ever, requires adjusting. Yet it is good practice to verify the GLS pressure, as reported by the pressure gauge, when the system achieves equilibrium after each start.

To specify MBPR pressure:

1. Open or create a method, and adopt these nominal system parameters: l 15 mL/min total flow l 5% co-solvent l GLS makeup flow of 4 mL/min l ABPR setting of 120 bar

2. Start the system, and allow it to equilibrate. 3. Verify that the ABPR is holding consistent pressure at the set point. 4. Set the MBPR to 60 psi, if methanol is the co-solvent, or to 80 psi for

the more viscous isopropanol.Note: For co-solvents of intermediate viscosity, specify the isopropanol setting.

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After setting the MBPR to its nominal pressure, revise the inlet method for high co-solvent flow, and download it to the system. Usually, you would specify a 50% co-solvent method. Nevertheless, if you expect to collect frac-tions that contain as much as 55% co-solvent, then specify a 55% method.Download the high co-solvent percent method. While the system equi-librates, view inside the GLS through the sight glasses. When the ABPR settles to its set point at these conditions, no liquid should accumulate at the bottom of the GLS. If liquid is accumulating, increase the MBPR, in increments of 2 psi, until the liquid stops accumulating. If previously accu-mulated liquid is present, allow two minutes for it to clear under the higher MBPR setting, before increasing the MBPR by another two psi.When you determine the lowest MBPR setting at which liquid does not accu-mulate, download again the 5% co-solvent method. Allow the system to re-equilibrate, and then record the MBPR’s pressure. This setting is the start-up setting for the MBPR. Thus for each subsequent system start-up, start the system first at a 5% method. Then, when it equilibrates, view the MBPR pressure gauge to verify the pressure setting. This setting remains stable without further adjustment for day-to-day operations. For subsequent system start-up and stand-by methods, which are not to be used for collec-tion runs, you can reduce the GLS Make-up setting to zero to save solvent. Doing so does not affect the MBPR reading that you must verify under star-tup conditions.As the co-solvent percentage increases, and therefore CO2 flow rate decreases, the reading on the GLS’s MBPR decreases consistent with the decreased CO2 flow rate. This expected decrease is part of the GLS design. The MBPR setting is calibrated only at 5% co-solvent. You do not need to recalibrate the MBPR when you use other co-solvent percentages.

TroubleshootingThe MBPR setting and GLS flow characteristics give rise to certain troubleshooting considerations.

MBPR setting

If, when setting the MBPR, you note that a pressure greater than approx-imately 20 psi above the nominal pressure is needed to prevent accu-mulation of liquid in the GLS, the collection path between the GLS and liquid waste container may be blocked.

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To identify and remove a blockage:

1. Beginning with the fitting at the bottom of the GLS, verify that all fittings are seated and that the gasket at the exit port at the bottom of the GLS is centered properly in the port.

Caution: To avoid breaking the fraction collection tubing, handle it with extreme care.

2. Inspect the fraction collection tubing for kinks or restrictions. 3. If kinks or restrictions are present, replace the tubing with one of

equivalent length.Requirement: Ensure the replacement tube is exactly as long and of the same diameter as the original one.

4. If the tubing is not kinked or restricted, continue inspecting along the collection flow path for blockages.

Increased back-pressure along the waste line can also cause a system's oper-ator to increase the MBPR setting, to maintain proper flow. Thus, when configuring the waste flow path, avoid creating kinks and restrictions in the tubing, for they can disrupt the GLS’s pressure balance. If you cannot identify the source of the elevated back-pressure, contact Waters' Technical Service department.

GLS flow characteristics

The mixed gas and liquid phases enter the GLS via the initiator tube at a high velocity. You can observe the flow through the site glasses of the GLS.Having specified the MBPR pressure, with the system equilibrated at 15 mL/min, 5% co-solvent, and a 4 mL/min GLS makeup setting, observe the liquid flow inside the GLS. Expect to see a slow stream and drops, with little or no splashing of solvent. Some drops or a stream fall directly down-ward from the initiator, rather than in an initial vortex around the wall. Such behavior is acceptable because the solvent flow is exiting the GLS quickly. Occasionally, small droplets can bounce about in the vortex. Never-theless, enough drops to cause an accumulation of solvent above the initi-ator outlet is not acceptable. Similarly, sheeting of solvent on the walls of the GLS at a height above the initiator outlet is not acceptable. All liquid flow must channel downward, either as drops, stream, or a vortex, to the GLS outlet.

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If the flow does not appear satisfactory, contact Waters' Technical Service department.If the GLS performs as expected at 5% co-solvent (a maximum CO2 flow rate of 95%), it will perform properly as co-solvent flow is increased and CO2 decreased, so no further evaluation beyond 5% co-solvent is necessary.

See also:

l Specify GLS vent-outlet pressure

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3 Getting Started with System Setup

Contents:

Configure the inlet 39Specify column names and solvent names 40Configure system components 41Software requirements 42

Configure the inlet n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

Before you configure the inlet, install Waters Instrument Control Software (ICS). To do so, request assistance from your Waters field service engineer.

To configure the inlet:

1. In the Inlet Method editor, from the top menu, select Tools > Instru-ment Configuration.

2. In the Inlet Configuration pane, click Configure. 3. In the Inlet Configuration Wizard, click Next. 4. Select Thar Pump, for the pumping device, and then click Next. 5. Select Waters 2767, for the autosampler, and then click Next. 6. Select your detector or detectors. 7. Click the arrow to add detectors to the Configured Detectors list on

the right-hand side, and click Next. 8. On the Configure Communications page, click Scan For Instruments,

and when the list of instruments appears, click OK. 9. On the Configure Communications page, from the drop-down list next

to the detector, select the detector's ID, and then click Next.

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10. On the Finished Configuring page, click Finish, to exit the Inlet Con-figuration Wizard.

11. In the Inlet Configuration pane, click Events & Triggering. 12. In the Events & Triggering Wizard, click Next. 13. On the Choose Events page, for a system that includes a mass spec-

trometer, under Event In, select 1, and then click Next. 14. On the Choose Triggering Method page, select Trigger by Software for

the pump, Trigger by Contact Closure for the detector, and click Next.

15. On the Finished Configuring page, click Finish. 16. In the Inlet Configuration pane, click Finish.

See also:

l Configure major components l Configure SFC components

Specify column names and solvent names n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

Specify column names and solvent names in the Columns & Solvents tab of the TharSFC Component Configuration pane.

To specify column and solvent names:

1. In the Inlet Method editor, select Thar Pump from the top menu.

2. In the Columns & Solvents tab of the TharSFC Component Configuration pane, enter a descriptive name for each column in use in the column oven.

3. Enter a descriptive name for each co-solvent available on the inlet side of the fluid delivery module.Note: The fluid delivery module can pump up to six co-solvents, one at a time.

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4. After you enter all column names and co-solvent names for your current purification runs, click OK.

See also:

l Configure the inlet l Configure SFC components l Specify settings for the FDM l Specify pressure settings and column oven settings

Configure system components n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

Your Waters service representative configures system components during installation and testing of the Prep 15 SFC system. Open the TharSFC Component Configuration pane to view these settings.

To view system and component settings:

1. In the Inlet Method editor, select Thar Pump from the top menu. 2. In the TharSFC Component Configuration pane, select one of five

tabs: l Columns & Solvents l General l Defaults l ABPR l Thar Oven

3. Click OK to close the TharSFC Component Configuration pane.

See also:

l Configure the inlet l Configure major components l Review equipment control settings

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Configure system components

Software requirements n MassLynx main page

The standard software configuration for the Prep 15 SFC system is MassLynx 4.1 running on Windows 7. Your Waters field service engineer installs the current MassLynx Software Change Note (SCN) for your system.

See also:

l Tour MassLynx software interface l Start and exit MassLynx software

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4 Getting Started with MassLynx Software

Contents:

Review MassLynx software workflow 43Review online Help for control software 46Start and exit MassLynx software 46Tour the MassLynx software interface 47Work with MassLynx software projects and sample lists 48Activate FractionLynx software 51

Review MassLynx software workflowMassLynx software controls system instruments, devices, and their auto-mation. It manages and reports data, and provides workflows for developing methods and purifying samples. Via its FractionLynx Application Manager, MassLynx software controls fraction collection.

Use MassLynx software to accomplish these tasks:

l Selecting a column in the column oven. l Specifying co-solvent gradients. l Creating, editing, and storing inlet methods and sample lists. l Configuring and controlling system components. l Executing and monitoring purification runs. l Tracking fractions and maintaining fraction data. l Managing and analyzing separation data. l Setting chromatogram offsets. l Setting delay for the FractionLynx software method.

For general information about MassLynx software, from the application's top menu, select Help.

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The diagram below illustrates a typical MassLynx software workflow.

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MassLynx software flow diagram:

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Review MassLynx software workflow

See also:

l Start and exit MassLynx software l View the results of a purification run

Review online Help for control softwareOnline Help for MassLynx software and FractionLynx software contain detailed instructions about routine operations:

l MassLynx software online Help explains how to set up your mass spec-trometer, and how to use MassLynx control software.

l FractionLynx software online Help explains how to set up sample lists and how to create fraction files to control fraction collection. Refer particularly to these sections in FractionLynx software online Help:

l Getting Started with FractionLynx software l Configuring FractionLynx software l Creating Methods l Running Samples l Viewing Results

See also:

l Review MassLynx software workflow l Work with MassLynx software projects and sample lists

Start and exit MassLynx softwareIf the workstation requires you to log in to MassLynx software, follow regis-tration prompts to establish your user name and password. If your install-ation does not require login credentials, launch the software without a user name or password.Requirement: Always wait at least 30 seconds after you power on the last system component before you start MassLynx software, to ensure system components communicate properly with the software interface.

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To start the application and log in to MassLynx software:

1. On the Windows desktop, double-click the shortcut icon for MassLynx software.Alternative: Select All Programs from the Windows start menu, and then click MassLynx.

2. If necessary, enter your user name and password, and then press Enter.

To exit MassLynx software:

On the MassLynx main page, from the top menu, select File > Exit.

See also:

l Review MassLynx software workflow

Tour the MassLynx software interface n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

After you launch MassLynx software, you configure instruments and oper-ate the system via the controls represented in the top menu bar, the toolbar below it, and the lozenges in the left-hand pane. The first step of many component-related tasks is to select Inlet Method in the left-hand pane of the MassLynx main page. When you select Inlet Method, the Inlet Method editor appears.

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Tour theMassLynx software interface

Inlet Method editor:

Use the tools available in this editor to set up, execute, and monitor puri-fication runs. For example, after you specify settings for an inlet method, click Load Method (the right-most button in the toolbar) to set the compon-ents to the values specified in the method.See also: MassLynx Getting Started Guide

See also:

l Review MassLynx software workflow l Work with MassLynx software projects and sample lists

Work with MassLynx software projects and sample lists n MassLynx main page

Before using MassLynx software to control the Prep 15 SFC, you should become familiar with project files and sample lists.

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Project filesMassLynx software stores sample lists, inlet methods, instrument config-uration settings, and run data in project files.

To create a new project:

1. On the MassLynx main page, from the top menu, select File > Project Wizard.

2. Follow the prompts in the new project wizard.Tip: Each project contains numerous files. To ensure that your MassLynx software data is well organized, give your projects and inlet files descriptive names.

To open an existing project:

1. On the MassLynx main page, from the top menu, select File > Open Project.

2. Select the appropriate MassLynx software project, and click OK.Note: The specific procedure used to open a project file depends on the file type. Identify the file type by its three-letter suffix. A sample list, for instance, ends with .SPL.

Sample listsControl your purification runs from the appropriate sample list.

To open a sample list:

1. In the MassLynx main page, from the top menu, select File > Open. 2. Browse to the MassLynx software project that contains the appro-

priate sample list, and open the project. 3. From the list of saved sample lists, double-click the appropriate

sample list.Tip: Each list has the extension .SPL.

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Work with MassLynx software projects and sample lists

To open an inlet file from the sample list:

1. In the Inlet File column, select the inlet file you want to edit. 2. Right-click the highlighted file, and then, from the context menu,

select Edit. 3. In the Inlet Method editor, select these options:

l Inlet, to configure the fluid delivery module, the ABPR, or the column oven.

l Autosampler, to configure the 2767 sample manager. l Waters 2998, to configure the PDA detector.

To conduct a run from the sample list:

1. Select the lines in the sample list you intend to run. 2. Click the Run arrow in the toolbar above the sample list. 3. Use the status message above the sample list to monitor progress of

the run. 4. Click the Pause button in the toolbar to pause the run, if necessary. 5. Click the Run button in the toolbar to restart a paused run. 6. Click Chromatogram above the sample list, then Display > Real-Time

Update in the top menu, to watch separation data arrive from the detector in real time.

Default sample list format

The left-most column in the sample list contains the line number. Scroll to the right to see all the columns in the list. The first five columns in the default sample list contain the following information.

Sample list initial columns:

Sample list column

Description

File Name Name of the data file associated with the run.File Text Brief description of the run.

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Sample list column

Description

Tray:Vial Tray and vial the sample manager injector will use for the sample injection. The vial address contains three numbers: l The first number designates the rack. l The second number designates the tray or plate

within the rack. l The third number designates the vial in the tray.

Inject Volume Indicates the volume to be injected from the sample vial, in microliters.

Inlet File File name of the inlet method used for the run.

Recommendations: l Monitor progress of a run at the sample list, as you check other sys-

tem status indicators available in MassLynx software. l Return to the sample list to queue more runs. Sample list lines

already in the queue run automatically. l If necessary, modify run parameters in the inlet file from the sample

list.See also: MassLynx Getting Started Guide and topics under Running Samples in FractionLynx online Help

See also:

l Review MassLynx software workflow l Tour the MassLynx software interface l Specify injection vial in a sample list

Activate FractionLynx software n MassLynx main page

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Activate FractionLynx software

FractionLynx software runs as an application manager under MassLynx software. If you plan to collect fractions, you must open the Collection Control pane and activate FractionLynx before you start a purification run.Requirement: Do not activate FractionLynx software until the inlet has completed initializing. To verify that initialization is complete, note the system status. To do so, in the sample list's toolbar, click Status. All indic-ator lights must be green.

To activate FractionLynx software:

1. On the MassLynx main page, click the FractionLynx tab. 2. In the left-hand pane, click Collection Control. 3. In the Collection Control pane, in the toolbar, click Activate.

Results: l Activating FractionLynx software loads the collection bed layout and

sets up communication between MassLynx software and the fraction collector in the sample manager.

l When FractionLynx software is activated, a blue, double-arrow icon appears in the system tray. When FractionLynx software is not activ-ated, the same icon appears with color faded.

See also:

l Activate the Collection Control application in FractionLynx

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5 Managing Inlet MethodsContents:

Create, edit, and load an inlet method 53Specify settings for the fluid delivery module 56Configure the photodiode array detector 59Specify pressure settings and column oven settings 60Specify settings for the sample manager 62Set FractionLynx method timing parameters 63Review terms and settings for purification runs 69

Create, edit, and load an inlet method n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

Inlet methods control the purification runs you perform on the Prep 15 SFC. You can create and save as many inlet methods as necessary to control settings for a run. Manage your inlet methods from MassLynx soft-ware:

l Create a new inlet method l Edit an existing inlet method l Load an inlet method

When you change instrument settings in MassLynx software, save your changes. The new settings take effect at the beginning of the next run. If you enter new settings but do not save the modified inlet method, MassLynx software uses saved settings from the method specified in the sample list, not the settings currently displayed in the method editor.The following procedures apply to general work flows. For specific inform-ation, refer to topics that explain settings for the fluid delivery module, the PDA detector, and the sample manager.

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To create a new inlet method:

1. In the Inlet Method editor, select File > New.Tip: Often, only a few settings change from method to method. To save time, select File > Open, to open an existing method. Then select File > Save As, to save the method under a new name. Specify settings required for the new method. Select File > Save, to save the new settings.

2. Select Inlet, to edit pump flows, oven settings, and pressure settings. 3. Select Autosampler, to enter settings for the sample manager.

Requirement: Identify which loop you intend to use for sample injec-tion. For the Prep 15 SFC system, select the left sample loop and injec-tion valve.

4. Select 2998 Detector, to specify settings for the PDA detector. 5. Select File > Save or File > Save As, to save the method.

To complete the inlet method, specify the total run time in three locations: l Modify Instrument Method for CO2 and co-solvent pumps l Modify Instrument Method for PDA detector l Mass spectrometer Experiment Setup pane

For gradient runs, sum the run time for each gradient to calculate the total run time. Specify the duration of each gradient in the Modify Instrument Method dialog box for the CO2 pump.Important: Specify all mobile phase parameters for isocratic or gradient runs in the Modify Thar Mobile Phase Method dialog box.Tips:

l A typical setting for the ABPR is 120 bar. l You can control the temperature of the mobile phase at key points in

the flow. The column oven temperature is typically to 40 °C. The post-ABPR temperature is typically to 25 °C.

To edit an existing inlet method:

1. In the Inlet Method editor, select File > Open. 2. Browse to the appropriate inlet method, and click Open.

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5 Managing Inlet Methods

Alternative: In the sample list, find the inlet file that you want to modify. Right-click the file name, and then, from the menu that appears, select Edit.

3. Specify the required settings in the appropriate component panes. 4. From the top menu, select File > Save, to save your changes.

MassLynx software does not update the inlet method until you save the new settings. To ensure your changes take effect, download the modified method between runs. You cannot change the method of a run already in progress. You can, however, save a modified method while a sample list is running. The run in the sample list adopts the updated method. A typical change of inlet conditions is an increase or decrease in co-solvent concentration. To change isocratic or gradient parameters for co-solvent concentration, open the Modify Instrument Method dialog box for the CO2 pump.By means of test runs, determine the best settings and gradients for co-solvent concentration and the corresponding CO2 flow, system temperature and pressure, co-solvent selection, sample composition, run time, type of column, etc.

To load an inlet method:

1. On the MassLynx main page, select File > Open. 2. Browse to the appropriate method, and click Open. 3. Click the Load Method icon.

Alternative: In the Inlet Method editor, from the top menu, select LC > Load Method.

See also:

l Specify settings for the FDM l Specify ABPR and column oven settings l Configure the photodiode array detector l Specify settings for the sample manager l Review terms and settings for purification runs l Create a cleaning method

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Create, edit, and load an inlet method

Specify settings for the fluid delivery module n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

You must specify mobile phase parameters for the pumping system.Note: The fluid delivery module (FDM) pumps one co-solvent at a time. You can, however, plumb six different co-solvents to the FDM, and select one of six co-solvents for each purification run.Specify settings for these mobile phase parameters in the Modify Thar Mobile Phase Method dialog box:

l Split ratio l Isocratic parameters for isocratic runs l Co-solvent l Gradient parameters for gradient runs l Run time

To open the Modify Thar Mobile Phase Method dialog box, in the Inlet Method editor, click Inlet. The Modify Thar Mobile Phase Method dialog box opens to the Pumping System tab.

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Pumping System tab of the Modify Thar Mobile Phase Method dialog box:

Split ratioSelect the flow rate for the MS makeup pump in the Split Ratio drop-down list. The MS makeup pump adds conditioning solvent to the portion of the mobile phase that flows from the SFC flow splitter to the mass spec-trometer.

Isocratic runs

To set up an isocratic run:

1. Select Run Isocratic. 2. Enter the total flow in milliliters per minute, and the co-solvent per-

centage for the run.

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Specify settings for the fluid delivery module

Solvent selectionSelect the co-solvent you intend to use from the Solvent Selection drop-down list. The list shows the co-solvent names you entered in the TharSFC Component Configuration panel.

Makeup flow rateThe control system automatically ensures a minimum flow rate through the gas-liquid separator. The minimum flow rate equals the Make-up Flow Rate setting, nominally 4 mL/min.

Run gradientIf you plan a non-isocratic run, on the Pumping Systems tab, select Run Gradient. Typically, you determine gradient percentages and flow rates during the method development process. Enter these settings in the Gradi-ent Parameters table.

To set up the Gradient Parameters table:

l Click Add Gradient, to add a new row to the table. l Click Edit Gradient, to modify an existing row. l Click Remove Gradient, to delete a row from the table.

The figures in the Start Perc and End Perc columns of the Gradient Para-meters table refer to the proportion of co-solvent in the total flow. Subtract these figures from 100 to determine the proportion of CO2 at the start and end of each gradient.When you set up the Gradient Parameters table, ensure all gradient changes are continuous, with no jumps between rows. That is, the end percent in one row of the gradient table must equal the start percent in the next row.The same rule of continuity, from one gradient to the next, holds for the total flow rates under Start Flow and End Flow. The flow rates in the Gradi-ent Parameters table are in milliliters per minute.

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Run timeSpecify the total run time in the Run Time field. The total run time in the dialog box above is 5.0 minutes.Run Time fields do not populate automatically. When you specify the run time in the Modify Thar Mobile Phase Method dialog box for the fluid deliv-ery module, navigate to the appropriate panel to specify the same run time for these two instruments:

l PDA detector l Mass spectrometer

See also:

l Monitor system status l Prime the FDM

Configure the photodiode array detector n MassLynx main page > Instrument tab > Waters 2998 > General tab

The photodiode array (PDA) detector uses variable wavelength light and a photodiode array to detect separated compounds.

To configure the PDA detector:

1. In the General tab of Modify 2998 PDA Detector, in the Run Time field, specify the time required for your run.

2. Select Lamp On. 3. Select Enable 3D Data, if appropriate. 4. Specify settings consistent with your inlet method on the lower half of

the General tab, and click OK, to save your settings. 5. Specify settings, as necessary, in the tabs 2D Channels, Analog Out,

and Events. 6. Modify your settings if a new inlet method requires modifying.

See also: 2998 Photodiode Array Detector Operator's Guide, Revision C.

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Configure the photodiode array detector

See also:

l Configure SFC components l Review equipment control settings

Specify pressure settings and column oven settings n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

You must specify pressure settings and column oven settings. Specify these settings in the Modify Thar Mobile Phase Method dialog box, on the Addi-tional Options tab.

To open the Modify Thar Mobile Phase Method dialog box, Additional Options tab:

1. In the Inlet Method editor, select Inlet. 2. In the Modify Thar Mobile Phase Method dialog box, click the Addi-

tional Options tab.

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Additional Options tab of the Modify Thar Mobile Phase Method dialog box:

Specify pressure settings on the Additional Options tab:

Settings Description

System back-pres-sure, in bar

Specify the appropriate back-pressure setting, typic-ally 120 bar.

System back-pressure alarm, in bar

Specify the pressure that triggers the system's back-pressure alarm.

External heater temperature, in °C

Not applicable to the Prep 15 SFC system.

External heater temperature alarm, in °C

Not applicable to the Prep 15 SFC system.

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Specify pressure settings and column oven settings

Specify column oven settings on the Additional Options tab:

Setting Description

Column oven temper-ature, in °C

Specify the appropriate column oven temperature setting, typically 40 °C.

Post-BPR temper-ature, in °C

Specify the appropriate post-ABPR temperature setting, typically 25 °C.

Column From the drop-down list, select the column in the column oven to use for current purification runs. The list shows the column names you entered in the TharSFC Component Configuration panel.

See also:

l Review the operation of the column oven l Maintain ABPR and post-ABPR heat exchanger

Specify settings for the sample manager n MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor

You must specify settings for the sample manager in the Autosampler section of the Inlet Method editor.

To specify settings for the sample manager:

1. In the Inlet Method editor, select Autosampler. 2. On the Injection tab, specify the appropriate settings.

Example: Typical settings specify that the injection pump use the left-hand injection valve and sample loop, with a 1000.0 μL capacity, and to center the sample in the loop.

Caution: For mass-directed systems, you must choose the left-hand injection valve, or the system will over pressure.

3. Specify settings, as necessary, for injection type, syringe speeds, and air gaps.

4. Verify that settings in all Autosampler tabs are correct: Injection, Wash, Auxiliary, Fraction Mixing, and Stacked Injections.

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See also: Waters 2767 Sample Manager, Injector, and Collector Installation and Maintenance Guide, Revision B, and Using the SFC Stacked Injection Module.

See also:

l Load samples l Create, edit, and load an inlet method

Set FractionLynx method timing parameters n MassLynx main page > Chromatogram window

n MassLynx main page > FractionLynx tab > FractionLynx Method editor

When you collect fractions, you must set the time delay between peak detec-tions and fraction collections in the FractionLynx Method editor. The timing values you enter depend on the detector or detectors in your system, and on the flow rate for a particular run.

Terms related to setting FractionLynx method timing parameters:

Term Definition

PDA Photodiode array detectorDAD Diode array detector (Alternate name for

photodiode array detector)ELS detector Evaporative light scattering detectorMS Mass spectrometerEIC Extracted ion chromatogramSplit/Collector Delay The time between peak detection and frac-

tion collectionMS/Analog Delay Time offset between the mass spectrometer

and the PDA’s 2D channels, or between the ELS detector and the PDA's 2D channels, if the channels are in use

MS/DAD Delay Time offset between the mass spectrometer and the PDA’s 3D channels

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Set FractionLynx method timing parameters

In the FractionLynx Method editor, click the Timing tab. Three parameters appear in the Peak Timing section:

l Split/Collector Delay (secs) l MS/Analog Delay (secs) l MS/DAD Delay (secs)

The figures below show the FractionLynx Method editor Timing tab for two detector configurations: PDA with a mass detector, and PDA with an ELS detector.

Peak Timing parameters for PDA with a mass detector at 15 mL/min:

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Peak Timing parameters for PDA with an ELS detector at 15 mL/min:

The Split/Collector Delay is the primary delay value. Specify MS/Analog Delay and MS/DAD Delay parameters to apply time offsets between detect-ors, for those systems that integrate multiple detectors. Do not specify these two parameters for systems that use only a PDA detector.

Suggested timing parametersThe table below lists delay reference values and peak timing parameters for various detector configurations and flow rates.Notes:

l The suggested collection delay timing values are appropriate for most applications, but they can vary with different splitter configurations.

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Depending on the detectors incorporated in a given system, the split-ter can affect collection delays. For some atypical applications, addi-tional offset determinations or delay testing may be necessary.

l All system variants are evaluated at 5, 10, and 15 mL/min for the optimum timing value across a 5% to 50% gradient.

FractionLynx method delay reference values and peak timing parameters:

Detector con-figurations and flow rates

PDA-to-col-lector delay ref-erence value

Split/Collector Delay

MS/Analog Delay

MS/DAD Delay

PDA only15 mL/min 9 sec 9 sec 0 sec 0 sec10 mL/min 10 sec 10 sec 0 sec 0 sec5 mL/min 11 sec 11 sec 0 sec 0 secPDA with mass detector15 mL/min 9 sec 9 sec 0 sec 0 sec10 mL/min 10 sec 10 sec 0 sec 0 sec5 mL/min 15 sec 15 sec 0 sec 0 secPDA with ELS detector15 mL/min 9 sec 10 sec -1 sec N/A10 mL/min 10 sec 11 sec -1 sec N/A5 mL/min 16 sec 17 sec -1 sec N/A

Detector configurations and delay parametersTo understand the role of the three Peak Timing parameters, start with the simplest configuration: a system that has only a PDA detector. Volume of the flow path from the PDA to the collection valve is fixed in this config-uration, so the collection delay varies only with the flow rate. Refer to the Split/Collector Delay column in the table above. For a system that contains 1 PDA, the collection delay increases 1 second when the flow rate decreases 5 mL/min. For UV-directed systems, set the MS/Analog Delay and MS/DAD Delay parameters to 0.

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For systems that incorporate a mass spectrometer in addition to a PDA, specify the offset between traces from the two detectors in the MS/DAD field of the Peak Timing section. The MS/DAD Delay represents the time interval that elapses between the peak's arrival at the PDA, and the peak's arrival at the mass spectrometer. The offset is due to the addition of a split leg to the mass spectrometer in the flow path, via the SFC flow splitter.The MS/DAD Delay can vary from suggested values because of variations in the MS makeup pump flow rate. At higher flow rates, the inclusion of a split leg for a second detector does not substantially affect the PDA-to-collector delay time. At low flow rates, however, the split leg for a second detector potentially increases the main flow path's delay time.For systems with an ELS detector, specify the time offset between the PDA and the ELS detector in the MS/Analog Delay field of the Peak Timing section.

Determine the offset between two detectorsYou can determine the offset between two detectors in the MassLynx soft-ware Chromatogram window. The MS EIC and PDA traces of the target peak are overlaid in the Chromatogram window. Determine the time offset between the peak start, or peak rise, for each of the two detectors, and then enter the difference as decimal minutes in the Align Chromatogram Time dialog box. Note that the alignment value applies only to the PDA trace; the MS Traces Align value must remain 0. Tip: To open the Align Chromatogram Time dialog box, select Display > Range > Align in the Chromatogram window. Recommendation: Perform the alignment such that the start of the MS EIC peak aligns with or slightly precedes the PDA’s corresponding peak trace for the target compound, so that the MS EIC trace encompasses the PDA peak.You can determine offsets most accurately by using peaks that are on-scale, and that elute under conditions similar to the target compound. Test injec-tions yield the best data for determining these offsets. High-intensity, off-scale peaks can appear wider than on-scale peaks, whereas low-intensity peaks can appear narrower, because the bottom portion of the peak is not visible above baseline noise. Both types of peak distortions can skew the apparent time offset in the Chromatogram window.

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Requirement: Once you determine the MS/DAD offset via overlaid chro-matograms, enter the value, in seconds, in the FractionLynx Method editor's Timing tab. You must maintain the positive or negative sign of the determined offset.

To calculate the Split/Collector Delay parameter:

Split/Collector Delay, in seconds = (PDA-to-collector delay reference value) minus (the determined detector offset, in seconds)Tip: When determined values fall between integers, choose the value that results in the lower collection delay, to ensure that the front of a target peak is collected. As the determined offset value increases, the overall collec-tion delay decreases.The determined detector offset equals:

l The MS/DAD Delay for systems with a PDA and a mass spectrometer. l The MS/Analog Delay for systems with a PDA and an ELS detector.

For both the MS/DAD Delay and the MS/Analog Delay, maintain the posit-ive or negative sign of offsets, as determined from overlaid chromatogram traces.Requirement: Specify all collection delay values in the Timing tab, Peak Timing section of the FractionLynx Method editor.For the MS/DAD Delay and the MS/Analog Delay offset parameters:

l The MS/DAD Delay indicates the time offset between the mass spec-trometer and the PDA’s 3D channels.

l The MS/Analog Delay indicates the time offset between the mass spec-trometer and the PDA’s 2D channels, or between the ELS detector and the PDA's 2D channels, if the channels are in use.

Therefore, when a system includes a PDA and a mass spectrometer, the MS/DAD Delay and MS/Analog Delays are equal, because both apply to the PDA detector.When a system contains an ELS detector, the MS/Analog channel indicates the offset between the PDA and the ELS detector. In this setup, use a 3D channel of the PDA for peak timing and triggering.

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See also:

l Create, edit, and load an inlet method

Review terms and settings for purification runsThe following table lists default settings for the Prep 15 SFC system.

Default settings:

Setting Value

Total flow 15 mL/minMakeup flow setting 4 mL/min (minimum liquid flow through the gas-

liquid separator)Back pressure 120 bar (1740 psi)Oven temperature 40 °CPost-ABPR temper-ature

25 °C

The following table lists key terms that you may encounter as you prepare for a run.

Terms and definitions:

Term Definition

Sample injection vial Vial that contains the sample to be injected at the beginning of the run.

Fraction collection tube

Test tube designated to collect the fraction, if any, that results from the run.

Co-solvent Solvent mixed with CO2 before mobile phase enters the column. Enter co-solvent names in the TharSFC Component Configuration pane.

Co-solvent flow or percent

Co-solvent flow rate, in mL/min, or as a percent of total flow.

CO2 flow or percent CO2 flow rate in mL/min, or as a percent of total flow.

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Review terms and settings for purification runs

Term Definition

Oven temperature The temperature set point for the column oven, in °C.Range: Ambient to 90 °C. The "Off" setting affords no temperature control.

System pressure System pressure, specified at the automatic back-pressure regulator (ABPR).

Makeup flows Flow rates, in mL/min, for the system's makeup pumps: l Collection makeup pump: UV and mass-directed

systems l MS makeup pump: mass-directed systems only

See also:

l Review MassLynx software workflow l Create, edit, and load an inlet method l Specify settings for the FDM

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6 Preparing the System for UseContents:

Prepare the system: checks and procedures 71Supply CO2 and nitrogen 81Prepare solvent vessels for use 82Fill solvent vessels 84Determine fluid delivery module status 85Prime the FDM 88Load samples 88Prepare the fraction collection bed 90Activate the Collection Control application in FractionLynx software 90Specify GLS vent-outlet pressure 91

Prepare the system: checks and proceduresAfter all electrical, communication, and plumbing connections are in place, you can operate the system. Refer to the following procedures when conduct-ing purification runs using the Prep 15 SFC system.To prepare the system for operation, you must perform these preliminary tasks:

l Power-on the CO2 chiller unit. Open the valve to the CO2 supply. After one half hour, confirm the temperature of the coolant delivered from the chiller to the fluid delivery module reflects the set temperature of 4 °C, and the CO2 inlet pressure is 55 to 76 bar (798 to 1100 psi).

l For each system component that requires electrical power, move the power switch to the On position.

l Power-on the computer workstation. Launch MassLynx software. MassLynx software opens to the last project and sample list in use.

l Inspect solvent vessels and supply tubes, to ensure they are ready for use.

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l Prime the co-solvent pump in the fluid delivery module. Prime the col-lection makeup pump, if necessary.

l Open and inspect the column oven, to ensure that columns are con-nected properly.

l Inspect the sample injection vials and fraction collection tubes in the sample manager. Ensure the bed layout in the sample manager matches the bed layout specified in MassLynx software and Frac-tionLynx software.

The preparation procedures in the following table assume a cold start. Observe them when starting a system that has been shut down, or restor-ing one after a loss of electrical power.

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6 Preparing the System for Use

Preparation procedures:

Preparation Procedure

Power on system components (except mass spectrometer).

Power-on all system components moving the On/Off switches located on their front or rear panels.Tips: l When powering on the ABPR,

make sure the red light on the back panel is blinking before you start the MassLynx software, to ensure correct communication with the software.

l Make sure the status indicators on the column oven, the fluid delivery module, and other components do not show an error.

l The gas-liquid separator and the manual back-pressure regulator do not require power.

Prepare the MassLynx software. 1. Turn on the system workstation. 2. Launch MassLynx software.

Result: The system's control inter-face opens.

3. Find the appropriate MassLynx project.

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Prepare the system: checks and procedures


Prepare the mass spectrometer. 1. From the MS Tune panel in MassLynx software, start the API gas.

2. Put the mass spectrometer in Oper-ate mode.

Note: If the instrument is configured and tuned properly, it remains ready for use from session to session.If it is not configured and tuned prop-erly, you must perform those tasks. When doing so, refer to the operator's guide for the mass spectrometer in your system.

Confirm that the correct column is installed.

1. Open the oven and ascertain that the column type is correct for your run.

2. Inspect all column connections, ensuring that they are secure.

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Put the Waters 515 pumps in remote mode.When you power the Waters 515 pumps off and power them on again, they are in local mode. For normal operation, you must put these two pumps in remote mode (system-controlled mode).

To put the pumps in remote mode: 1. On the pump's front panel, press

the Menu button. 2. Press arrow keys, to open the

Mode menu. 3. Press the Edit button, to change

pump mode. 4. Press the arrow keys, to select

Rem mode. 5. Press the Edit button, to exit the

Mode menu. 6. Press the Run/Stop button to

engage remote mode.Tip: The mode indicator in the display's upper-right alternates between Rem and Run.

See also: 515 HPLC Pump Operator's Guide

Determine solvent levels.

You must confirm all the containers contain sufficient solvent before you start a run. Refill vessels as required. If a vessel empties during system oper-ation, pause the run immediately, and refill the vessel.

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Start the CO2 flow. 1. Ensure that the CO2 supply is suf-ficient for the run.

2. Open the tank or bulkhead valve, allowing CO2 to flow to the FDM's CO2 pump inlet.

3. After the pump is running, when the CO2 in the system has reached its normal operating pressure, inspect fittings and valves for leaks, and repair as necessary.

Tip: A typical setting for the ABPR is 120 bar (1740 psi). The FDM's CO2 pump pressure,which depends on flow rate, co-solvent, and column pressure, is properly higher than the ABPR setting.

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Prime the FDM. 1. Open the front of the FDM. 2. Open the black priming valve by

turning it counter-clockwise. 3. Insert a syringe in the black prim-

ing valve. 4. Pull the syringe plunger, until the

syringe stops pulling air. 5. Remove the syringe from the black

priming valve. 6. Close the black priming valve by

turning it clockwise. 7. Close the front of the FDM.Recommendation: Prime the FDM if the system is idle for more than three hours.Note: Do not open the prime valve with a syringe inserted. Residual pres-sure is sometimes present after the system sits idle.

Prime the makeup pumps. In addition to the FDM, the Prep 15 SFC contains two other pumps: l Conditioning makeup pump for the

mass spectrometer l Collection makeup pump for the

gas-liquid separatorBoth pumps are Waters 515 HPLC pumps. Prime and purge these pumps of unwanted gas when the system has been idle for an extended period.

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Load the inlet method for your run.

1. Select the line you intend to run in the sample list.

2. Verify that the inlet file in the selected line of the sample list is the one that you want to use for your run.Recommendation: Verify gradi-ents and other key parameters for your run. To do so, refer to the settings for the FDM's CO2 pump in the Inlet Method editor's Modify Thar Mobile Phase Method dialog box.

3. In the MassLynx main page, select Inlet Method.

4. In the Inlet Method editor, click the Load Method icon.Result: The system loads the inlet file open in the method editor. The method settings take effect for the next run.Recommendation: If the inlet method specified in the sample list differs from the inlet method open in the method editor, the sample list method overrides the inlet method settings. Verify the inlet file specified in the sample list before you initiate a run.

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Prime and wash the sample manager.When the inlet is ready and the system is at pressure, prepare the sample manager.

Caution: To avoid broken collection tubes, unsuccessful purification runs, and damage to the sample manager's components, prepare the sample manager carefully.

1. From the MassLynx main page, select Inlet Method in the left-hand pane.

2. From the Inlet Method editor, from th

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