Prep 15 SFC System System Guide / Revision A Copyright Waters Corporation 2014 All rights reserved

Transcription

1 Prep 15 SFC System System Guide / Revision A Copyright Waters Corporation 2014 All rights reserved

2 ii March 14, 2014, Rev. A

3 General Information Copyright 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). Trademarks Waters 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 trademarks of Microsoft Corporation in the United States and/or other countries. Other registered trademarks or trademarks are the sole property of their owners. Customer comments Waters Technical Communications organization invites you to report any errors that you encounter in this document or to suggest ideas for otherwise improving it. Help us better understand what you expect from our documentation so that we can continuously improve its accuracy and usability. March 14, 2014, Rev. A iii

4 We seriously consider every customer comment we receive. You can reach us at Contacting Waters Contact Waters with enhancement requests or technical questions regarding 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 Telephone and fax Conventional mail The Waters Web site includes contact information for Waters locations worldwide. Visit From the USA or Canada, phone HPLC, or fax For other locations worldwide, phone and fax numbers appear in the Waters Web site. Waters Corporation 34 Maple Street Milford, MA USA Safety considerations Some 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 System Always keep in mind these safety practices: Familiarize yourself with proper handling, storage, and disposal of all chemicals used with your SFC system. Refer to the Material Safety iv March 14, 2014, Rev. A

5 Data Sheet (MSDS) for each solvent you use, and know its chemical properties. Wear the appropriate personal protective equipment when working with any type of hazardous chemical. Wear eye protection while near the instrument to protect eyes from possible failure of column seals or fittings. Ensure that the system is depressurized before loosening any fittings in the CO 2 flow path. Use care when removing and opening the column. Loosening an inline column's fitting with liquid CO 2 in the system can cause a highpressure 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 decompression hazard. Exposure to rapidly expanding fluids can cause injuries, including frostbite. To avoid this hazard, take these precautions: Always depressurize the system before changing or working on the bulk CO 2 supply. Use only bulk CO 2 supply lines constructed of appropriate materials. 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 CO 2 is in the system. You must first completely depressurize 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 draining the dampening vessel of CO 2, before you depressurize the system, close the MV-2 valve. March 14, 2014, Rev. A v

6 Warning: To avoid causing a hazardous condition in which a collection bottle can explode should the bottle become pressurized, avoid these conditions: Use of alternately-sized collection bottles without Waters approval Use of bottles that are not safety coated For the exhaust tubing, any size reductions, sharp bends, kinks or other conditions that can restrict flow of the CO 2 exhaust Modifications to the collection bottle inlet Opening of collection bottles while the system is running FCC radiation emissions notice Changes 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 interference, and (2) this device must accept any interference received, including interference that may cause undesired operation. Canada spectrum management emissions notice This 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 notice Do not position the instrument so that it is difficult to operate the disconnecting 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. vi March 14, 2014, Rev. A

7 Equipment misuse notice If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Safety advisories Consult Appendix A for a comprehensive list of warning and caution advisories. Operating the Prep 15 SFC System When 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 directives Australia C-Tick EMC compliant Confirms that a manufactured product complies with all applicable United States and Canadian safety requirements Consult instructions for use Contact Waters Corporation for the correct disposal and recycling instructions March 14, 2014, Rev. A vii

8 Audience and purpose This system guide is for personnel who install, operate, and maintain the Prep 15 SFC System. It provides most of the information required to configure, 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 Laboratory Chillers Installation and Operation Guide. For details about the 2998 PDA detector and 2545 QGM, refer to the operator's guide for each instrument. Intended use of the Prep 15 SFC System Waters designed the Prep 15 SFC System to serve as a high-pressure purification system for quantitative recovery of purified products such as enantiomers, complex synthetic chemicals, and natural products, or for research. It is not intended for use in diagnostic or biologically hazardous applications. Use the system as a preparative-scale, high-pressure purification system, or as an investigative tool, to perform supercritical fluid chromatography of compounds in these environments: Pharmaceutical development Quality assurance and quality control Chemical materials Environmental sciences Food safety environments The Prep 15 SFC is for research use only and is not intended for use in diagnostic applications. Calibrating To 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 specimens, and atypical specimens. viii March 14, 2014, Rev. A

9 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-control Routinely 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 B This 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, powersupply network. EC authorized representative Waters Wilmslow Stamford Avenue Altrincham Road Wilmslow SK9 4AX United Kingdom March 14, 2014, Rev. A ix

10 Telephone: Fax: Contact Quality manager x March 14, 2014, Rev. A

11 Table of Contents General Information Copyright notice Trademarks Customer comments Contacting Waters Safety considerations Considerations specific to the Prep 15 SFC System FCC radiation emissions notice Canada spectrum management emissions notice Electrical power safety notice Safety hazard symbol notice Equipment misuse notice Safety advisories Operating the Prep 15 SFC System Applicable symbols Audience and purpose Intended use of the Prep 15 SFC System Calibrating Quality-control ISM classification ISM classification: ISM Group 1 Class B EC authorized representative iii iii iii iii iv iv iv vi vi vi vi vii vii vii vii viii viii viii ix ix ix ix 1 Getting Help 19 Prep 15 SFC system 19 March 14, 2014, Rev. A xi

12 Purpose 19 Audience 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 31 Description of operation 32 Specifying the GLS MBPR pressure 34 Troubleshooting 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 xii March 14, 2014, Rev. A

13 Tour the MassLynx software interface 47 Work with MassLynx software projects and sample lists 48 Project files 49 Sample 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 56 Split ratio 57 Isocratic runs 57 Solvent selection 58 Makeup flow rate 58 Run gradient 58 Run 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 63 Suggested timing parameters 65 Detector configurations and delay parameters 66 Determine 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 81 Carbon dioxide 82 Nitrogen 82 March 14, 2014, Rev. A xiii

14 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 software 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 102 Status tab 102 Thar Inlet Additional Status tab 104 Determine status of the PDA detector 106 Verify system status during purification runs 107 System equilibrating 108 System ready 108 System running 109 System idle 110 Monitor CO2 pressure and temperature 110 View the results of a purification run 111 xiv March 14, 2014, Rev. A

15 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 116 Reviewing FDM operation 117 Maintaining the CO2 pump 119 Maintaining the co-solvent pump 129 Replace CO2 cylinder 137 External CO2 supply connections 137 Connecting 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 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 Reviewing Column Oven Procedures 153 Review the operation of the column oven 153 March 14, 2014, Rev. A xv

16 Review safety information for the column oven 154 General safety 154 Electrical safety 155 Chemical safety 155 Integrated safety features 156 Control the column oven from the front panel 157 Replace a column in the column oven 158 Clean oven surfaces Ensuring a Proper Installation and Operating Environment 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 166 Tubing 166 Fittings 167 A Safety Advisories 169 Warning symbols 169 Specific 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 180 Electrical symbols 180 xvi March 14, 2014, Rev. A

17 Handling symbols 181 B Specifications 183 Review system specifications 183 Review equipment control settings 187 Consumables and spare parts 189 March 14, 2014, Rev. A xvii

18 xviii March 14, 2014, Rev. A

19 1 Getting Help Contents: Prep 15 SFC system 19 Observe Good Laboratory Practice 20 Prep 15 SFC system Purpose You 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 CO 2 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. This guide explains how to operate and maintain the Prep 15 SFC system. Specifically, it addresses these topics: Reviewing system capabilities Preparing for purification runs Starting and operating components Initiating, monitoring, and completing purification runs Maintaining the system for reliable performance and long life Diagnosing and correcting errors or other problems Audience This guide provides information for chemists, technicians, application engineers, and other individuals who operate or maintain the Prep 15 SFC system. March 14, 2014, Rev. A 19

20 1 Getting Help See also: Review component descriptions Observe Good Laboratory Practice Observe Good Laboratory Practice (GLP) when you operate the Prep 15 SFC: 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 properties. Wear the appropriate personal protective equipment when working with any type of hazardous chemical. Wear eye protection while near the instrument to protect eyes from possible failure of column seals or fittings. Ensure that the system is depressurized before loosening any fittings in the CO 2 flow path. 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 fittings can be hot. See also: 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 components. Warning symbols 20 March 14, 2014, Rev. A

21 2 Reviewing System Components Contents: 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 31 Reviewing Prep 15 SFC capabilities Purification laboratories use the Prep 15 SFC to benefit from the advantages of supercritical fluid chromatography (SFC). The Prep 15 SFC uses supercritical CO 2 as its main solvent, which enables you to conduct preparative-scale purification at a low cost of operation. Specifically, the Prep 15 SFC uses high-pressure CO 2 as the primary mobile phase for preparative-scale purifications. This technology enables rapid equilibration and faster run times than conventional preparative chromatographic 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 Suggested working flow range 15 ml/min Co-solvent gradient range 5% to 55% 5 to 15 ml/min total flow for purifications March 14, 2014, Rev. A 21

22 2 Reviewing System Components Description Optimal column size Suggested maximum crude mixture concentration Suggested working crude mixture concentration Capability 4.6-mm ID analytical columns for method development; 10-mm ID preparative columns for purification 2 mg/injection 0.01 to 1.00 mg/injection Additional capabilities: Triggering fraction collection from a Waters 2998 photodiode array, and from a Waters mass spectrometer or ELS detector Automating sample injection Collecting fractions in an open bed See also: Review component descriptions View components: UV-directed system View flow diagram: UV-directed system View components: mass-directed system View flow diagram: mass-directed system View components: UV-directed system The table below lists components in the UV-directed version of the Prep 15 SFC. UV-directed system components: Abbreviation FDM HE1 GLS Component Fluid delivery module Post-ABPR heat exchanger Gas-liquid separator 22 March 14, 2014, Rev. A

23 View components: mass-directed system Abbreviation ABPR SM HPLC 515 PDA N/A N/A N/A N/A Component Automatic back-pressure regulator 2767 sample manager Collection makeup pump 2998 photodiode array detector Chiller unit Column oven Stacked injection module (optional) Solvent reservoirs See also: Review Prep 15 SFC capabilities Review component descriptions View flow diagram: UV-directed system View flow diagram: mass-directed system View components: mass-directed system The table below lists components in the mass-directed version of the Prep 15 SFC. Mass-directed system components: Abbreviation MS Splitter PDA ABPR HE1 SM HPLC 515 Component Mass spectrometer SFC flow splitter 2998 photodiode array detector Automatic back-pressure regulator Post-ABPR heat exchanger 2767 sample manager Collection makeup pump March 14, 2014, Rev. A 23

24 2 Reviewing System Components Abbreviation HPLC 515 GLS FDM N/A N/A N/A N/A Component MS makeup pump Gas-liquid separator Fluid delivery module Chiller unit Column oven Stacked injection module (optional) Solvent reservoirs See also: Review Prep 15 SFC capabilities Review component descriptions View flow diagram: UV-directed system View flow diagram: mass-directed system Review component descriptions The following two tables describe Prep 15 SFC components: Components included in both UV-directed and mass-directed systems Components included in mass-directed systems only 24 March 14, 2014, Rev. A

25 Review component descriptions Components included in both UV-directed and mass-directed systems: Component Fluid delivery module Chiller unit Post-ABPR heat exchanger Description The fluid delivery module contains two pumps. One delivers high-pressure, supercritical CO 2 to the Prep 15 SFC system. The second pumps co-solvent to the 2767 sample manager. Maintains steady flow for liquid CO 2, and for co-solvents. Total flow = 15 ml/min (CO 2 and cosolvent). Co-solvent flow rate is 5% to 55% of total flow. Pump head is cooled to maintain liquid CO 2. CO 2 at the FDM is liquid, and not yet supercritical. Solvent selector for up to six cosolvents. The chiller unit chills a water and ethylene glycol mixture (anti-freeze) in the circulating bath. The circulating bath cools and liquefies the CO 2 before it enters the fluid delivery module's CO 2 pump. Circulating coolant also removes heat of compression from CO 2 pump heads. Post-ABPR heat exchanger reduces the effects of CO 2 expansion, such as freezing, dry ice formation, and sample precipitation. 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 temperature controller regulates the temperature set point for the heat exchanger. March 14, 2014, Rev. A 25

26 2 Reviewing System Components Component Description 2767 sample manager Manages sample injection and fraction collection. The sample manager has separate sampling and fraction dispensing probes. The dual-probe robotic arm has access to all positions on the worktable. Column oven Under control of MassLynx and FractionLynx software. Self-venting probe that performs accurate sample injections from tightly covered containers. High velocity wash pump with a flow rate greater than 30 ml/min, to flush the probe and tubing, and maintain high sample throughput. Fume hood, for ventilation of hazardous vapors. (Optional) Stacked injection module manages stacked injections for increased throughput. It supplants the injection mechanism in the 2767 sample manager. Column oven's primary purpose is to maintain column temperature for separation. Note: The critical temperature for CO 2 is 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 configuration of the racks inside the oven. 26 March 14, 2014, Rev. A

27 Review component descriptions Component Description 2998 photodiode array detector Integrated software and optics deliver chromatographic and spectral sensitivity: Automatic back-pressure regulator Collection makeup pump Gas-liquid separator Maximum noise less than 10 µau. Quantifies high-level and low-level components within a single chromatographic separation. Flexible sampling rates for both normal and fast SFC separation. Definitive compound identification and co-elution detection. Optimized signal strength across the full range of wavelengths. Electronically controlled needle valve in the ABPR maintains system pressure. Note: The critical pressure for CO 2 is bar (1070 psi). System pressure is typically set between 100 bar and 200 bar (1450 psi and 2900 psi). 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. Separates CO 2 gas from the liquid portion of the mobile phase near the end of the purification process. March 14, 2014, Rev. A 27

28 2 Reviewing System Components Components included in mass-directed systems only: Component Mass spectrometer Mass spectrometer (MS) makeup pump SFC flow splitter Description 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 splitter is an essential component in its implementation. 515 HPLC pump adds conditioning solvent to the mobile phase as it flows through SFC flow splitter to the mass spectrometer. The conditioning solvent helps to maintain peak shape, promotes solubility, 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. Splits a small portion of the mobile phase from the main preparative flow, and redirects it to the mass spectrometer. After the split, the MS makeup pump adds conditioning 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: Review Prep 15 SFC capabilities View components: UV-directed system View flow diagram: UV-directed system View components: mass-directed system View flow diagram: mass-directed system 28 March 14, 2014, Rev. A

29 View flow diagram: UV-directed system View flow diagram: UV-directed system The diagram below illustrates components and direction of flow for the UVdirected version of the Prep 15 SFC. Prep 15 SFC UV-directed system: See also: Review Prep 15 SFC capabilities View components: UV-directed system March 14, 2014, Rev. A 29

30 2 Reviewing System Components View components: mass-directed system Review component descriptions View flow diagram: mass-directed system The diagram below illustrates components and direction of flow for the mass-directed version of the Prep 15 SFC. Prep 15 SFC mass-directed system: 30 March 14, 2014, Rev. A

31 Gas-liquid separator See also: Review Prep 15 SFC capabilities View components: UV-directed system View components: mass-directed system Review component descriptions Gas-liquid separator The 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 CO 2 gas is safely vented. In the post-abpr section of the collection path, a phase change occurs as the liquid CO 2 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 CO 2 gas flows through the collection path. As the compressed CO 2 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 CO 2 portion of the mobile phase. Fraction 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: Description of operation Specifying the GLS MBPR pressure March 14, 2014, Rev. A 31

32 2 Reviewing System Components Troubleshooting Description of operation The 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: ABPR Input tee for the GLS makeup solvent flow (with associated GLS makeup pump) Post-ABPR heater GLS with manual back-pressure regulator (MBPR) Collection valve 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: Initiator (or dripper) tube Glass, V-bottomed cyclone housing Exit port 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. 32 March 14, 2014, Rev. A

33 Gas-liquid separator 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 CO 2 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 accumulated liquid in this area will not affect system performance, and is generally 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 UVdirected 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. March 14, 2014, Rev. A 33

34 2 Reviewing System Components 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 cosolvent percentage. During this gradient reset, you can sometimes observe an accumulation of liquid in the GLS that remains until the increased CO 2 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 pressure The 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: 15 ml/min total flow 5% co-solvent GLS makeup flow of 4 ml/min 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. 34 March 14, 2014, Rev. A

35 Gas-liquid separator 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 fractions that contain as much as 55% co-solvent, then specify a 55% method. Download the high co-solvent percent method. While the system equilibrates, 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 accumulated 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 accumulate, download again the 5% co-solvent method. Allow the system to reequilibrate, and then record the MBPR s pressure. This setting is the startup 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 collection 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 startup conditions. As the co-solvent percentage increases, and therefore CO 2 flow rate decreases, the reading on the GLS s MBPR decreases consistent with the decreased CO 2 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. Troubleshooting The 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 approximately 20 psi above the nominal pressure is needed to prevent accumulation of liquid in the GLS, the collection path between the GLS and liquid waste container may be blocked. March 14, 2014, Rev. A 35

36 2 Reviewing System Components 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 operator 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 downward 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. Nevertheless, enough drops to cause an accumulation of solvent above the initiator 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. 36 March 14, 2014, Rev. A

37 Gas-liquid separator If the flow does not appear satisfactory, contact Waters' Technical Service department. If the GLS performs as expected at 5% co-solvent (a maximum CO 2 flow rate of 95%), it will perform properly as co-solvent flow is increased and CO 2 decreased, so no further evaluation beyond 5% co-solvent is necessary. See also: Specify GLS vent-outlet pressure March 14, 2014, Rev. A 37

38 2 Reviewing System Components 38 March 14, 2014, Rev. A

39 3 Getting Started with System Setup Contents: Configure the inlet 39 Specify column names and solvent names 40 Configure system components 41 Software requirements 42 Configure the inlet 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 > Instrument 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. March 14, 2014, Rev. A 39

40 3 Getting Started with System Setup 10. On the Finished Configuring page, click Finish, to exit the Inlet Configuration 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 spectrometer, 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: Configure major components Configure SFC components Specify column names and solvent names 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 cosolvents, one at a time. 40 March 14, 2014, Rev. A

41 Configure system components 4. After you enter all column names and co-solvent names for your current purification runs, click OK. See also: Configure the inlet Configure SFC components Specify settings for the FDM Specify pressure settings and column oven settings Configure system components 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: Columns & Solvents General Defaults ABPR Thar Oven 3. Click OK to close the TharSFC Component Configuration pane. See also: Configure the inlet Configure major components Review equipment control settings March 14, 2014, Rev. A 41

42 3 Getting Started with System Setup Software requirements 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: Tour MassLynx software interface Start and exit MassLynx software 42 March 14, 2014, Rev. A

43 4 Getting Started with MassLynx Software Contents: Review MassLynx software workflow 43 Review online Help for control software 46 Start and exit MassLynx software 46 Tour the MassLynx software interface 47 Work with MassLynx software projects and sample lists 48 Activate FractionLynx software 51 Review MassLynx software workflow MassLynx software controls system instruments, devices, and their automation. 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: Selecting a column in the column oven. Specifying co-solvent gradients. Creating, editing, and storing inlet methods and sample lists. Configuring and controlling system components. Executing and monitoring purification runs. Tracking fractions and maintaining fraction data. Managing and analyzing separation data. Setting chromatogram offsets. Setting delay for the FractionLynx software method. For general information about MassLynx software, from the application's top menu, select Help. March 14, 2014, Rev. A 43

44 4 Getting Started with MassLynx Software The diagram below illustrates a typical MassLynx software workflow. 44 March 14, 2014, Rev. A

45 Review MassLynx software workflow MassLynx software flow diagram: March 14, 2014, Rev. A 45

46 4 Getting Started with MassLynx Software See also: Start and exit MassLynx software View the results of a purification run Review online Help for control software Online Help for MassLynx software and FractionLynx software contain detailed instructions about routine operations: MassLynx software online Help explains how to set up your mass spectrometer, and how to use MassLynx control software. 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: See also: Getting Started with FractionLynx software Configuring FractionLynx software Creating Methods Running Samples Viewing Results Review MassLynx software workflow Work with MassLynx software projects and sample lists Start and exit MassLynx software If the workstation requires you to log in to MassLynx software, follow registration prompts to establish your user name and password. If your installation 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. 46 March 14, 2014, Rev. A

47 Tour the MassLynx software interface 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: See also: On the MassLynx main page, from the top menu, select File > Exit. Review MassLynx software workflow Tour the MassLynx software interface MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor After you launch MassLynx software, you configure instruments and operate 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. March 14, 2014, Rev. A 47

48 4 Getting Started with MassLynx Software Inlet Method editor: Use the tools available in this editor to set up, execute, and monitor purification runs. For example, after you specify settings for an inlet method, click Load Method (the right-most button in the toolbar) to set the components to the values specified in the method. See also: MassLynx Getting Started Guide See also: Review MassLynx software workflow Work with MassLynx software projects and sample lists Work with MassLynx software projects and sample lists MassLynx main page Before using MassLynx software to control the Prep 15 SFC, you should become familiar with project files and sample lists. 48 March 14, 2014, Rev. A

49 Work with MassLynx software projects and sample lists Project files MassLynx software stores sample lists, inlet methods, instrument configuration 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: Sample lists 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. Control 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 appropriate 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. March 14, 2014, Rev. A 49

50 4 Getting Started with MassLynx Software 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: Inlet, to configure the fluid delivery module, the ABPR, or the column oven. Autosampler, to configure the 2767 sample manager. 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 File Name File Text Description Name of the data file associated with the run. Brief description of the run. 50 March 14, 2014, Rev. A

51 Activate FractionLynx software Sample list column Tray:Vial Inject Volume Inlet File Description Tray and vial the sample manager injector will use for the sample injection. The vial address contains three numbers: The first number designates the rack. The second number designates the tray or plate within the rack. The third number designates the vial in the tray. Indicates the volume to be injected from the sample vial, in microliters. File name of the inlet method used for the run. Recommendations: Monitor progress of a run at the sample list, as you check other system status indicators available in MassLynx software. Return to the sample list to queue more runs. Sample list lines already in the queue run automatically. 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: Review MassLynx software workflow Tour the MassLynx software interface Specify injection vial in a sample list Activate FractionLynx software MassLynx main page March 14, 2014, Rev. A 51

52 4 Getting Started with MassLynx 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 indicator 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: Activating FractionLynx software loads the collection bed layout and sets up communication between MassLynx software and the fraction collector in the sample manager. When FractionLynx software is activated, a blue, double-arrow icon appears in the system tray. When FractionLynx software is not activated, the same icon appears with color faded. See also: Activate the Collection Control application in FractionLynx 52 March 14, 2014, Rev. A

53 5 Managing Inlet Methods Contents: Create, edit, and load an inlet method 53 Specify settings for the fluid delivery module 56 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 63 Review terms and settings for purification runs 69 Create, edit, and load an inlet method 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 software: Create a new inlet method Edit an existing inlet method 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 information, refer to topics that explain settings for the fluid delivery module, the PDA detector, and the sample manager. March 14, 2014, Rev. A 53

54 5 Managing Inlet Methods 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 injection. For the Prep 15 SFC system, select the left sample loop and injection 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: Modify Instrument Method for CO 2 and co-solvent pumps Modify Instrument Method for PDA detector 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 CO 2 pump. Important: Specify all mobile phase parameters for isocratic or gradient runs in the Modify Thar Mobile Phase Method dialog box. Tips: A typical setting for the ABPR is 120 bar. 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. 54 March 14, 2014, Rev. A

55 Create, edit, and load an inlet method 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 CO 2 pump. By means of test runs, determine the best settings and gradients for cosolvent concentration and the corresponding CO 2 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. See also: Alternative: In the Inlet Method editor, from the top menu, select LC > Load Method. Specify settings for the FDM Specify ABPR and column oven settings Configure the photodiode array detector Specify settings for the sample manager Review terms and settings for purification runs Create a cleaning method March 14, 2014, Rev. A 55

56 5 Managing Inlet Methods Specify settings for the fluid delivery module 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: Split ratio Isocratic parameters for isocratic runs Co-solvent Gradient parameters for gradient runs 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. 56 March 14, 2014, Rev. A

57 Specify settings for the fluid delivery module Pumping System tab of the Modify Thar Mobile Phase Method dialog box: Split ratio Select 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 spectrometer. 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 percentage for the run. March 14, 2014, Rev. A 57

58 5 Managing Inlet Methods Solvent selection Select 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 rate The 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 gradient If 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 Gradient Parameters table. To set up the Gradient Parameters table: Click Add Gradient, to add a new row to the table. Click Edit Gradient, to modify an existing row. Click Remove Gradient, to delete a row from the table. The figures in the Start Perc and End Perc columns of the Gradient Parameters table refer to the proportion of co-solvent in the total flow. Subtract these figures from 100 to determine the proportion of CO 2 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 Gradient Parameters table are in milliliters per minute. 58 March 14, 2014, Rev. A

59 Configure the photodiode array detector Run time Specify 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 delivery module, navigate to the appropriate panel to specify the same run time for these two instruments: PDA detector Mass spectrometer See also: Monitor system status Prime the FDM Configure the photodiode array detector 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. March 14, 2014, Rev. A 59

60 5 Managing Inlet Methods See also: Configure SFC components Review equipment control settings Specify pressure settings and column oven settings 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 Additional 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 Additional Options tab. 60 March 14, 2014, Rev. A

61 Specify pressure settings and column oven settings Additional Options tab of the Modify Thar Mobile Phase Method dialog box: Specify pressure settings on the Additional Options tab: Settings System back-pressure, in bar System back-pressure alarm, in bar External heater temperature, in C External heater temperature alarm, in C Description Specify the appropriate back-pressure setting, typically 120 bar. Specify the pressure that triggers the system's back-pressure alarm. Not applicable to the Prep 15 SFC system. Not applicable to the Prep 15 SFC system. March 14, 2014, Rev. A 61

62 5 Managing Inlet Methods Specify column oven settings on the Additional Options tab: Setting Column oven temperature, in C Post-BPR temperature, in C Column Description Specify the appropriate column oven temperature setting, typically 40 C. Specify the appropriate post-abpr temperature setting, typically 25 C. 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: Review the operation of the column oven Maintain ABPR and post-abpr heat exchanger Specify settings for the sample manager 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 μl capacity, and to center the sample in the loop. Caution: For mass-directed systems, you must choose the lefthand 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. 62 March 14, 2014, Rev. A

63 Set FractionLynx method timing parameters See also: Waters 2767 Sample Manager, Injector, and Collector Installation and Maintenance Guide, Revision B, and Using the SFC Stacked Injection Module. See also: Load samples Create, edit, and load an inlet method Set FractionLynx method timing parameters MassLynx main page > Chromatogram window MassLynx main page > FractionLynx tab > FractionLynx Method editor When you collect fractions, you must set the time delay between peak detections 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 PDA DAD ELS detector MS EIC Split/Collector Delay MS/Analog Delay MS/DAD Delay Definition Photodiode array detector Diode array detector (Alternate name for photodiode array detector) Evaporative light scattering detector Mass spectrometer Extracted ion chromatogram The time between peak detection and fraction collection 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 Time offset between the mass spectrometer and the PDA s 3D channels March 14, 2014, Rev. A 63

64 5 Managing Inlet Methods In the FractionLynx Method editor, click the Timing tab. Three parameters appear in the Peak Timing section: Split/Collector Delay (secs) MS/Analog Delay (secs) 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: 64 March 14, 2014, Rev. A

65 Set FractionLynx method timing parameters 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 detectors, for those systems that integrate multiple detectors. Do not specify these two parameters for systems that use only a PDA detector. Suggested timing parameters The table below lists delay reference values and peak timing parameters for various detector configurations and flow rates. Notes: The suggested collection delay timing values are appropriate for most applications, but they can vary with different splitter configurations. March 14, 2014, Rev. A 65

66 5 Managing Inlet Methods Depending on the detectors incorporated in a given system, the splitter can affect collection delays. For some atypical applications, additional offset determinations or delay testing may be necessary. 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 configurations and flow rates PDA only PDA-to-collector delay reference value Split/Collector Delay MS/Analog Delay MS/DAD Delay 15 ml/min 9 sec 9 sec 0 sec 0 sec 10 ml/min 10 sec 10 sec 0 sec 0 sec 5 ml/min 11 sec 11 sec 0 sec 0 sec PDA with mass detector 15 ml/min 9 sec 9 sec 0 sec 0 sec 10 ml/min 10 sec 10 sec 0 sec 0 sec 5 ml/min 15 sec 15 sec 0 sec 0 sec PDA with ELS detector 15 ml/min 9 sec 10 sec -1 sec N/A 10 ml/min 10 sec 11 sec -1 sec N/A 5 ml/min 16 sec 17 sec -1 sec N/A Detector configurations and delay parameters To 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 configuration, 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 March 14, 2014, Rev. A

67 Set FractionLynx method timing parameters 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-tocollector 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 detectors You can determine the offset between two detectors in the MassLynx software 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 injections yield the best data for determining these offsets. High-intensity, offscale 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. March 14, 2014, Rev. A 67

68 5 Managing Inlet Methods Requirement: Once you determine the MS/DAD offset via overlaid chromatograms, 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 collection delay decreases. The determined detector offset equals: The MS/DAD Delay for systems with a PDA and a mass spectrometer. 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 positive 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: The MS/DAD Delay indicates the time offset between the mass spectrometer and the PDA s 3D channels. The MS/Analog Delay indicates the 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. 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. 68 March 14, 2014, Rev. A

69 Review terms and settings for purification runs See also: Create, edit, and load an inlet method Review terms and settings for purification runs The following table lists default settings for the Prep 15 SFC system. Default settings: Setting Total flow Makeup flow setting Back pressure Value 15 ml/min Oven temperature 40 C Post-ABPR temperature 4 ml/min (minimum liquid flow through the gasliquid separator) 120 bar (1740 psi) 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 Co-solvent Co-solvent flow or percent CO 2 flow or percent Test tube designated to collect the fraction, if any, that results from the run. Solvent mixed with CO 2 before mobile phase enters the column. Enter co-solvent names in the TharSFC Component Configuration pane. Co-solvent flow rate, in ml/min, or as a percent of total flow. CO 2 flow rate in ml/min, or as a percent of total flow. March 14, 2014, Rev. A 69

70 5 Managing Inlet Methods Term Oven temperature System pressure Makeup flows Definition The temperature set point for the column oven, in C. Range: Ambient to 90 C. The "Off" setting affords no temperature control. System pressure, specified at the automatic backpressure regulator (ABPR). Flow rates, in ml/min, for the system's makeup pumps: Collection makeup pump: UV and mass-directed systems MS makeup pump: mass-directed systems only See also: Review MassLynx software workflow Create, edit, and load an inlet method Specify settings for the FDM 70 March 14, 2014, Rev. A

71 6 Preparing the System for Use Contents: Prepare the system: checks and procedures 71 Supply CO2 and nitrogen 81 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 software 90 Specify GLS vent-outlet pressure 91 Prepare the system: checks and procedures After all electrical, communication, and plumbing connections are in place, you can operate the system. Refer to the following procedures when conducting purification runs using the Prep 15 SFC system. To prepare the system for operation, you must perform these preliminary tasks: Power-on the CO 2 chiller unit. Open the valve to the CO 2 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 CO 2 inlet pressure is 55 to 76 bar (798 to 1100 psi). For each system component that requires electrical power, move the power switch to the On position. Power-on the computer workstation. Launch MassLynx software. MassLynx software opens to the last project and sample list in use. Inspect solvent vessels and supply tubes, to ensure they are ready for use. March 14, 2014, Rev. A 71

72 6 Preparing the System for Use Prime the co-solvent pump in the fluid delivery module. Prime the collection makeup pump, if necessary. Open and inspect the column oven, to ensure that columns are connected properly. 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 FractionLynx software. The preparation procedures in the following table assume a cold start. Observe them when starting a system that has been shut down, or restoring one after a loss of electrical power. 72 March 14, 2014, Rev. A

73 Prepare the system: checks and procedures 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: 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. Make sure the status indicators on the column oven, the fluid delivery module, and other components do not show an error. 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 interface opens. 3. Find the appropriate MassLynx project. March 14, 2014, Rev. A 73

74 6 Preparing the System for Use Preparation Procedure Prepare the mass spectrometer. 1. From the MS Tune panel in MassLynx software, start the API gas. Confirm that the correct column is installed. 2. Put the mass spectrometer in Operate 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 properly, you must perform those tasks. When doing so, refer to the operator's guide for the mass spectrometer in your system. 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. 74 March 14, 2014, Rev. A

75 Prepare the system: checks and procedures Preparation 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). Determine solvent levels. Procedure 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 You must confirm all the containers contain sufficient solvent before you start a run. Refill vessels as required. If a vessel empties during system operation, pause the run immediately, and refill the vessel. March 14, 2014, Rev. A 75

76 6 Preparing the System for Use Preparation Procedure Start the CO 2 flow. 1. Ensure that the CO 2 supply is sufficient for the run. 2. Open the tank or bulkhead valve, allowing CO 2 to flow to the FDM's CO 2 pump inlet. 3. After the pump is running, when the CO 2 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 CO 2 pump pressure,which depends on flow rate, co-solvent, and column pressure, is properly higher than the ABPR setting. 76 March 14, 2014, Rev. A

77 Prepare the system: checks and procedures Preparation Procedure 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 priming 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 pressure 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: Conditioning makeup pump for the mass spectrometer Collection makeup pump for the gas-liquid separator Both pumps are Waters 515 HPLC pumps. Prime and purge these pumps of unwanted gas when the system has been idle for an extended period. March 14, 2014, Rev. A 77

78 6 Preparing the System for Use Preparation Load the inlet method for your run. Procedure 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 gradients and other key parameters for your run. To do so, refer to the settings for the FDM's CO 2 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. 78 March 14, 2014, Rev. A

79 Prepare the system: checks and procedures Preparation 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. Procedure 1. From the MassLynx main page, select Inlet Method in the lefthand pane. 2. From the Inlet Method editor, from the top menu, select Waters 2767 > Prime System. Result: The system primes the sample manager's wash pump and injector syringe. 3. From the Inlet Method editor, from the top menu, select Waters 2767 > Wash Probe. Result: The system washes contaminants from the injector needle and valve, the sample loop, and all related tubing. See also: Priming the 2767 sample manager in Waters 2767 Sample Manager, Injector, and Collector Installation and Maintenance Guide, Revision C, pages 3-9 to March 14, 2014, Rev. A 79

80 6 Preparing the System for Use Preparation Prepare sample injection vials and fraction collection tubes. When you initialize a run, the sample manager's operations proceed entirely under system control. To prevent damage to equipment and glassware, set up the sample manager with care. Procedure To prepare the sample vials, collection tubes, and racks in the sample manager: 1. Prepare the sample to be injected during the purification run. 2. Place the sample in one or more injection vials. 3. Place the vial or vials in the sample rack. Requirement: Verify the vials' rack positions correspond to the positions specified in the sample list. 4. Place the sample rack's plate over the sample vials, and carefully secure the plate to the rack. 5. Place clean fraction collection tubes in appropriate positions in the sample manager's fraction collection rack. Requirement: Verify that the collection tubes' size and rack positions correspond to the size and positions specified in MassLynx software and FractionLynx software. Important: Prepare the sample manager racks carefully. To avoid mistakes, set up the actual layout in the sample manager first, and then open the bed layout in MassLynx software and FractionLynx software to check for discrepancies. Make necessary adjustments to make sure the layout specified in the control system matches the actual layout. 80 March 14, 2014, Rev. A

81 Supply CO2 and nitrogen Preparation Procedure See also: Setting up the worktable in Waters 2767 Sample Manager, Injector, and Collector Installation and Maintenance Guide, Revision C, pages 2-31 to 2-50, and Collecting fractions, pages 3-11 to When you complete these procedures and checks, the system is ready for routine operations. Let the system equilibrate, and verify that all components have attained normal operating conditions before you perform purification tasks. See also: Review purification procedures Review Prep 15 SFC capabilities Specify settings for the sample manager Replace a column in the column oven Prepare solvent vessels for use Fill solvent vessels Start and exit MassLynx software Prime the FDM Create, edit, and load an inlet method Load samples Prepare the fraction collection bed Supply CO 2 and nitrogen Supercritical CO 2 is the principal solvent for the Prep 15 SFC. Mass-directed systems also require nitrogen gas. March 14, 2014, Rev. A 81

82 6 Preparing the System for Use Carbon dioxide Nitrogen The Prep 15 SFC requires a suitable supply of liquid carbon dioxide. Carbon dioxide must be industrial grade or better. Supply CO 2 from a dip tube cylinder or via a house supply. Supply pressure must be 55 to 76 bar (798 to 1100 psi). Maximum inlet temperature is 25 C. Warning: To avoid injury or death from suffocation, ensure that CO 2 is efficiently vented from the laboratory environment. The mass spectrometer in mass-directed systems requires a supply of dry, oil-free nitrogen, with a purity of at least 95%. Nitrogen must be regulated at 7 bar (100 psi). Refer to the mass spectrometer site preparation guide for additional nitrogen gas supply requirements. See also: Monitor CO 2 pressure and temperature Prepare solvent vessels for use Vessels and solvents in use depend on your industry-specific applications and standard practice in your laboratory. These instructions assume a standard glass laboratory bottle. The mass-directed system typically requires these solvent vessels to supply four inlets: Co-solvent for the FDM Collection solvent for the GLS makeup flow Conditioning solvent for the mass spectrometer Wash for the sample manager 82 March 14, 2014, Rev. A

83 Prepare solvent vessels for use Solvent vessel contents: Vessel contents Description Co-solvent The FDM pumps co-solvent to mix with CO 2, thus composing the system's main flow. The FDM pumps as many as six co-solvents, one at a time. Collection solvent Conditioning solvent Wash solvent This vessel supplies the collection makeup pump, to assure sufficient flow for the gasliquid separator. This vessel supplies the conditioning makeup pump. Conditioning solvent mixes with the sample, immediately before it enters the mass spectrometer, and serves as an additive, to enhance ionization and the mass spectrometer's signal during analysis. The sample manager uses wash solvent to rinse the injection needle, the injector, and the sample loop. The UV-directed system requires these solvent vessels to supply three inlets: Co-solvent for the FDM Collection solvent for the GLS makeup flow Wash for the sample manager Note: The FDM pumps as many as six co-solvents. Supply one vessel each for co-solvent. To prepare a solvent vessel for use: 1. Label the vessel. Requirement: Follow your laboratory's labeling requirements. Include the type of co-solvent the vessel contains as well as the pump the vessel supplies. 2. Connect a supply tube to the inlet of the correct pump. 3. Place a filter on the vessel end of the supply tube. March 14, 2014, Rev. A 83

84 6 Preparing the System for Use 4. Feed the tube and filter through the mouth of the vessel until the filter rests on the bottom of the vessel. 5. Fill the vessel with the correct co-solvent. Recommendation: Wear surgical gloves to improve your grip on heavy glass bottles, and to protect your hands from chemicals. See also: Fill solvent vessels Review requirements for CO 2 and co-solvents Fill solvent vessels Regularly monitor the level of solvent in the solvent vessels, and refill the vessels when necessary. If the solvent level is low, refill the vessel before it empties. Caution: To avoid damaging pumps, confirm that solvent vessels contain enough solvent to supply upcoming runs. If a vessel empties during a run, pause the run immediately. To fill a co-solvent vessel: 1. Refer to the label on the bottle, to verify the contents of the vessel you want to refill. 2. Obtain a supply bottle of the correct solvent. 3. Remove the supply tube and filter from the solvent vessel. 4. Remove the vessel from the rack. 5. Pour solvent from the supply bottle into the solvent vessel. 6. Return the solvent vessel to the rack. 7. Re-insert the supply tube and filter through the solvent vessel's mouth. Recommendation: Wear surgical gloves to improve your grip on heavy glass bottles, and to protect your hands from chemicals. 84 March 14, 2014, Rev. A

85 Determine fluid delivery module status See also: Prepare solvent vessels for use Review requirements for CO 2 and co-solvents Determine fluid delivery module status MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor The FDM comprises the CO 2 and co-solvent pumps, the 6-position co-solvent selector, the cooling heat exchanger, and the solvent bottle tray. Status indicators appearing on the module's front panel, and in the MassLynx software, provide access to information about the FDM's startup and operating status. Operate the FDM using the MassLynx software controls. Determining FDM status: Status Power For CO 2 and co-solvent pumps Pump On Indicator location The "Fluid Module: OK" status indicator appears on the front panel of the FDM when the module is powered on. When the module is powered off, the OK indicator is dark (unlit). The "CO 2 : ml/min" and "Mod: ml/min" flow rate indicators on the front panel of the FDM show a flow reading when the pumps are operating. Also, for the CO 2 and co-solvent pumps, the Pump On indicator in the Inlet Status tab shows whether the pump is switched on or off. The Pump On indicator is green when the pump is powered-on, and gray when the pump is powered-off. March 14, 2014, Rev. A 85

86 6 Preparing the System for Use Status For CO 2 and co-solvent pumps current outflow Co-solvent selector bottle (1 6) For CO 2 pump inlet pressure Indicator location The "CO 2 : ml/min" and "Mod: ml/min" flow rate indicators on the FDM's front panel show the current outflows for CO 2 and co-solvent. The Inlet Method editor's Status tab shows the total flow in ml/min. It also shows the proportion of CO 2 (percent A) and the proportion of cosolvent (percent B) in the mobile phase. The "Solvent Pos:" indicator on the FDM's front panel shows which of six solvent bottles, numbered 1 to 6, is selected. See also the current solvent on the Inlet Method editor's Thar Inlet Additional Status tab. The inlet pressure status indicator in the Thar Inlet Additional Status tab shows the pressure in bar, as measured at the inlet of the CO 2 pump, after the cooling bath. Tip: When the CO 2 supply tank is full, the inlet pressure should read between 55 and 60 bar. A lower than normal pressure reading can possibly indicate a low volume of CO 2 in the supply tank or a plugged CO 2 inlet filter that needs replacement. 86 March 14, 2014, Rev. A

87 Determine fluid delivery module status Status Indicator location For CO 2 pump inlet temperature The inlet temperature status indicator in the Thar Inlet Additional Status tab shows the temperature of the CO 2 entering the pump after passing through the cooling heat exchanger. For that reason, if you run the chiller unit at a temperature set point below ambient temperature, the inlet temperature reads lower whenever the CO 2 pump is operating. Tip: You can use the inlet temperature reading to diagnose whether the pump's check valves are operating properly. A higher-than-normal temperature reading usually indicates one or more of the CO 2 pump's check valves failed. Thermoelectric chiller operation The chiller unit's operating status and cooling temperature set point are displayed on the chiller unit's front panel display. The chiller unit supplies coolant to the cooling heat exchanger and circulating bath to cool and liquefy the supply CO 2 before it enters the CO 2 pump. The circulating coolant also removes the heat of compression from the pump heads. Requirement: The CO 2 pump requires cooling to maintain the inlet temperature of approximately 4 C. To support this requirement, you must ensure the chiller unit is plumbed and configured to maintain an inlet temperature of 4 C within the fluid delivery module. For additional operation details and instructions, see the documentation that accompanies your chiller unit. March 14, 2014, Rev. A 87

88 6 Preparing the System for Use See also: Monitor system status in the Inlet Method editor Maintain the fluid delivery module Troubleshoot fluid delivery module Prime the FDM To prepare the FDM for use, prime each line in use. To prime the FDM: 1. Open the front of the FDM. 2. Open the black priming valve by turning it counterclockwise. Note: Do not open the priming valve with a syringe attached. 3. Insert a syringe in the black priming 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. See also: Load samples Prepare the system: checks and procedures Specify settings for the FDM Before you conduct a purification run, load your samples in the correct locations in the sample manager. 88 March 14, 2014, Rev. A

89 Load samples To load samples: 1. Remove the injector rack from the sample manager. Injector rack: 2. Remove the top metal plate from the injector rack to expose the trays that hold the sample vials. 3. Obtain enough clean and dry 4 ml vials to hold the volume of sample you intend to prepare. 4. Carefully mix the sample according to your laboratory's procedures. 5. Fill each vial with the sample mixture, and do not overfill. 6. Place each sample vial in the injector rack. Recommendation: For better sample tracking, fill each row before you begin a new one. 7. Replace the injector rack's top metal plate to cover the vials. Requirement: Confirm that you have fastened the plate securely. 8. Place the injector rack in the sample manager. Requirement: Confirm that the rack is seated properly. Warning: To avoid crush injury, do not place your hands inside the sample manager while the equipment is in operation. See also: Waters 2767 Sample Manager, Injector, and Collector Installation and Maintenance Guide See also: Specify settings for the sample manager Specify injection vial in a sample list Select or modify the bed layout March 14, 2014, Rev. A 89

90 6 Preparing the System for Use Prepare the fraction collection bed Prepare the fraction collection bed in the sample manager as part of your general preparations to run the Prep 15 SFC system. Requirement: Before you start a run from the sample list, open the Collection Control pane in FractionLynx software. To prepare the fraction collection bed: 1. Insert 25-mL test tubes in the fraction collection rack on the lefthand side of the sample manager. 2. Configure the fraction collection bed in MassLynx software. Requirement: Specify the number of collection tubes, their location, and their length. 3. Test the fraction collection arm in the sample manager. See also: Activate the Collection Control application in FractionLynx Prepare the system: checks and procedures Specify settings for the sample manager Select or modify the bed layout Activate the Collection Control application in FractionLynx software MassLynx main page After you set up a sample list and load the inlet method in MassLynx software, activate the Collection Control application in FractionLynx software before you initiate the run. To activate the Collection Control application: 1. On the MassLynx software main page, click the FractionLynx tab. 2. Click Collection Control. 3. In the Collection Control application, click Activate in the toolbar. 90 March 14, 2014, Rev. A

91 Specify GLS vent-outlet pressure Requirements: Do not activate the Collection Control application until the inlet completely initializes. Otherwise, a system error will occur. Assess the collection bed in the sample manager to ensure enough collection tubes are available for the run. If sufficient tubes are not available, click Control > Reset Beds in the Collection Control application. Result: When you reset the collection bed, the sample manager dispenses fraction into the tube located in the first position during the next run. See also: Activate FractionLynx software Prepare the fraction collection bed Prepare the system: checks and procedures Specify settings for the sample manager Select or modify the bed layout Specify GLS vent-outlet pressure To set the vent-outlet pressure for the gas-liquid separator (GLS), you first determine the outlet pressure, and then adjust as necessary. Note: Normally a field service engineer performs these procedures during system installation. You need not reset the vent-outlet pressure unless you modify the system. To determine the vent-outlet pressure: 1. Create a method that runs two co-solvents, each at 5%: Methanol at 120 bar (1740 psi) system back-pressure Isopropanol at 100 bar (1450 psi) system back-pressure Rationale: System back-pressure and vent-outlet pressure depend on the co-solvent in use. Specify the system back-pressure at the ABPR. Specify the vent-outlet pressure at the GLS's manual back-pressure regulator (MBPR). March 14, 2014, Rev. A 91

92 6 Preparing the System for Use 2. Load and run the inlet method created in step 1. Requirement: Allow the system to equilibrate for about five minutes. 3. View the inlet editor, and verify that the system back-pressure is steady, at 120 bar (1740 psi) for methanol and 100 bar (1450 psi) for isopropanol. 4. Note the vent-outlet pressure on the gauge next to the MBPR. Recommendation: Recommended vent-outlet pressures are 4.13 bar (60 psi) for methanol, and 5.52 bar (80 psi) for isopropanol. If necessary, adjust the manual back-pressure regulator to bring the outlet pressure for each co-solvent to its recommended level. To adjust the vent-outlet pressure: 1. In MassLynx software, note the system back-pressure to confirm that system pressure is steady at 120 bar (1740 psi) for methanol and 100 bar (1450 psi) for isopropanol. 2. To adjust the vent-outlet pressure, carefully turn the MBPR adjustment knob: Clockwise, to increase the outlet pressure Counterclockwise, to reduce the outlet pressure Requirement: Turn the knob in the appropriate direction, until the vent-outlet pressure gauge reads 4.13 bar (60 psi) for methanol, or 5.52 bar (80 psi) for isopropanol. Important: Monitor system back-pressure as you adjust the vent-outlet pressure. If system pressure fluctuates more than one bar (14.5 psi) from 120 bar (1740 psi) for methanol, or from 100 bar (1450 psi) for isopropanol, stop and wait for the system to equilibrate before you proceed. Vent outlet pressure normally decreases as the percent co-solvent increases. After initial adjustment, further adjustment of the vent-outlet pressure is not necessary as the percent co-solvent changes. See also: Setting the Gas-Liquid Separator (GLS) Vent Outlet Pressure on the Prep 15 SFC System (TUP-102, Revision 1.0) Gas-liquid separator (GLS), "Specifying the GLS MBPR pressure" 92 March 14, 2014, Rev. A

93 Specify GLS vent-outlet pressure See also: Gas-liquid separator (GLS) March 14, 2014, Rev. A 93

94 6 Preparing the System for Use 94 March 14, 2014, Rev. A

95 7 Executing Purification Runs Contents: 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 102 Determine status of the PDA detector 106 Verify system status during purification runs 107 Monitor CO2 pressure and temperature 110 View the results of a purification run 111 Review purification procedures Follow these steps to conduct purification runs with the Prep 15 SFC. The steps shown in the table illustrate a general workflow for a purification run. Purification procedures: Step Task 1 Confirm that all system components that require power are on. 2 Confirm that solvent vessels contain sufficient solvent. 3 Start the pumps, and wait approximately five minutes for the system to equilibrate. 4 Open your sample list in MassLynx software. 5 Verify the sample list includes appropriate samples and inlet methods. March 14, 2014, Rev. A 95

96 7 Executing Purification Runs Step Task 6 Load sample vials and fraction collection tubes in the sample manager, consistent with the bed layout specified in MassLynx software and FractionLynx software. 7 Run the appropriate lines in the sample list. 8 During the run, do as follows: Ensure equipment is operating properly. Monitor system status and data acquisition in MassLynx software. Monitor fraction collection at the sample manager. 9 Verify that run results meet expectations, given the samples and inlet methods used. 10 Flush the system, to prepare it for the next run. 11 Shut down the system pumps. 12 Confirm that the system is in the proper state, considering the amount of time during which it will remain idle. See also: Prepare the system: checks and procedures Conduct a typical purification run Fill solvent vessels Load samples Specify injection vial in a sample list Select or modify the bed layout Prepare the fraction collection bed Monitor system status Determine status of major components Verify system status during purification runs Monitor CO 2 pressure and temperature View the results of a purification run 96 March 14, 2014, Rev. A

97 Conduct a typical purification run Conduct a typical purification run To conduct a typical run, follow the steps below. The workflow assumes: All Prep 15 SFC components are operating. MassLynx software is open on the control workstation. The Collection Control application of FractionLynx software is open on the workstation. Recommendation: Consult the MassLynx software and FractionLynx software online Help for instructions about how to accomplish specific tasks in the software interface, such as how to enter data in your sample list, and how to set up a fraction file. To prepare for the run: 1. Prepare the sample or samples. 2. Place the samples in the correct location in the sample manager. 3. Verify that the actual bed layout in the sample manager matches the bed layout specified in MassLynx software. 4. Verify that the correct project is open in MassLynx software. 5. Open the appropriate sample list on your desktop. 6. Specify the information required to run the appropriate lines in the sample list. Tip: This information includes the inlet method, collection method, fraction trigger, mass spectrometer instrument method, if necessary, as well as injection volume and vial location. 7. Save the modified sample list. 8. Open the inlet method required for the run. Important: The inlet method for the run is as specified in the sample list, not the method last opened in the Inlet Method editor. To verify your inlet method, open the inlet file listed in the sample list in your Inlet Method editor, load the method, and verify settings are as expected. The same applies to the fraction file. The current run uses the fraction file specified in the sample list, not the last one opened. March 14, 2014, Rev. A 97

98 7 Executing Purification Runs 9. From the Inlet Method editor, from the top menu, select Waters 2767 > Prime System. 10. From the Inlet Method editor, select Waters 2767 > Probe/Port Wash. Requirement: Wash the probe and one or both injector ports, depending on which ports are in use. 11. Prime the FDM. To conduct the run: 1. In the sample list, verify that the inlet file you select contains the appropriate run settings. Tip: The inlet file contains all the settings for your inlet method. The method loads automatically at the beginning of a run. 2. Open the Collection Control application in FractionLynx software. 3. Select the sample line or lines that you want to run. 4. From the sample list, click the run arrow in the top toolbar. 5. In the dialog box, verify that you are about to run the correct line from the sample list. 6. Click OK to start the run. 7. Wait for the system to equilibrate. Note: When all components reach their set points, the System Ready light on the MassLynx main page, displays a green icon, and the run begins. 8. Monitor the pumps, sample manager, and status indicators in MassLynx software during the run. 9. Click Chromatogram above the sample list to view results. See also: Tip: To view results in real time, from the top menu, select Display > Real-Time Update. Review purification procedures Review MassLynx software workflow Verify system status during purification runs View the results of a purification run 98 March 14, 2014, Rev. A

99 Specify injection vial in a sample list Prime the FDM Activate the Collection Control application in FractionLynx Specify injection vial in a sample list Sample list You must specify, in the sample list, which injection vial the sample manager uses for a particular run. The Tray:Vial column in the sample list specifies the injection vial for a run. To change the vial, select the appropriate cell in the Tray:Vial column, and enter a new vial location. The vial location in the sample list is keyed to the bed layout. If the bed layout does not change, you can edit the vial location within the sample list. To change the vial location in the sample list: 1. Open the sample list that contains your run. 2. Double-click the cell in the Tray:Vial column that you want to edit. 3. Specify the coordinates for the correct vial location. Tip: Often you need only change the location of the vial in the tray, the numeral after the colon. If you want to view the bed layout to verify that the sample manager will inject sample from the appropriate vial, open the bed layout editor. To open the bed layout editor: 1. Right-click the appropriate cell in the sample list s Tray:Vial column. 2. From the menu that appears, select AutoSampler Bed Layout. March 14, 2014, Rev. A 99

100 7 Executing Purification Runs AutoSampler bed layout: In the bed layout above, rack 1 appears on the left-hand side. Rack 2, on the right-hand side, is subdivided into three 24-vial trays. Selected and shown below the bed layout is rack 2, tray 1, designated as Plate: 2,1. To select a vial location in the bed layout editor: 1. Select the appropriate tray in rack (or plate) In the selected tray, click the black circle that corresponds to the appropriate vial location. Result: The vial location you select turns green. 3. Click the middle button at the top, Replace Samples. 4. Click Close, to close the bed layout editor. 100 March 14, 2014, Rev. A

101 Select or modify the bed layout Result: The new vial location appears in the Tray:Vial cell you originally selected when you opened the bed layout editor. Bed locations: The fraction collection rack on the left-hand side is rack 1. The sample injection rack on the right-hand side is rack 2. The sample injection rack contains three trays, and each tray contains twenty-four vials. To specify a vial location, use a three-number address: Rack, Tray: Vial. Thus the address of the vial in the upper left-hand location of rack 2 is 2,1:1. To specify the vial next to it, in position two, enter 2,1:2. See also: Work with MassLynx software projects and sample lists Select or modify the bed layout Select or modify the bed layout MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor To select the bed layout: 1. In the Inlet Method editor, from the top menu, select Waters 2767 > Bed Layout. 2. In the Current Bed Selection drop-down list, select a bed layout that corresponds to the size and arrangement of your sample injection vials and fraction collection tubes. 3. Click OK, to close the Bed Layouts list. To modify the bed layout: 1. In the Inlet Method editor, from the top menu, select Waters 2767 > Bed Layout. 2. In the Bed Layouts list, select the bed layout to be modified. 3. Use the controls in the Bed Layouts list to add or delete racks and plates from the bed layout. March 14, 2014, Rev. A 101

102 7 Executing Purification Runs Tip: Click the Modify tab to select the plate types in the layout. 4. Click OK, to close the Bed Layouts list. See also: Specify injection vial in a sample list Load samples Prepare the fraction collection bed Monitor system status in the Inlet Method editor MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor Status tab You must monitor system status during purification runs. To do so, open the Inlet Method editor's Status tab. Find additional information in the Thar Inlet Additional Status tab. To open the Inlet Method editor, select Inlet Method in the MassLynx main page. Status indicators on the left-hand side of the Status tab are green during a run. The Pump On, Ready, and OK indicators are red when the system is not ready, for example, during equilibration or when a component is in an error condition. If an indicator displays a red icon, note the status message in the lower left corner of the MassLynx software main page. 102 March 14, 2014, Rev. A

103 Monitor system status in the Inlet Method editor Status tab of the Inlet Method editor: Status indicators in the Inlet Method editor's Status tab: Status indicator Running Pump On Inject Cycle Ready OK Description Displays a green icon when all pumps are on and a run is in progress. Appears gray when a run is not in progress. Displays a green icon when the FDM pumps are on. Appears gray when the FDM pumps are off. Displays a yellow icon when the sample manager executes a sample injection. Displays a green icon to indicate system has equilibrated and is ready to conduct a run. Appears red when the system is not ready to operate. Displays a green icon when no system errors are present. If red, click to see error message. March 14, 2014, Rev. A 103

104 7 Executing Purification Runs Status indicator Flow (ml/min) Pressure (bar) Description Total flow from the two pumps in the FDM. This flow is typically 15 ml/min. System pressure, read at the inlet of the ABPR. Default is 120 bar (1740 psi). To summarize, all status indicators except Inject Cycle appear green during a run. Select indicators appear red when the system is not ready, such as during equilibration, or when a component is in an error condition. If an indicator displays a red icon, click the indicator, or note the status message in the lower left-hand corner of the MassLynx software main page. Vessels A and B on the right-hand side of the Status tab show the current percentages of CO 2 and co-solvent in the main flow: Vessel A represents the percent flow of CO 2 in the mobile phase from the CO 2 pump in the FDM. Vessel B represents the percent flow of co-solvent in the mobile phase from the co-solvent pump in the FDM. Thar Inlet Additional Status tab To view additional system status information, click the Thar Inlet Additional Status tab in the Inlet Method editor. 104 March 14, 2014, Rev. A

105 Monitor system status in the Inlet Method editor Thar Inlet Additional Status tab of the Inlet Method editor: The following table lists system variables in the Thar Inlet Additional Status tab: System variables in the Thar Inlet Additional Status tab: System variable Oven temperature, in C CO 2 pressure, in bar Co-solvent pressure, in bar Pressure drop Description Interior oven temperature, typically 40 C. Pressure at the outlet of the CO 2 pump. Pressure at the outlet of the co-solvent pump. Difference between the FDM outlet pressure (pump pressure) and the ABPR set pressure. This value represents the pressure drop across the column. March 14, 2014, Rev. A 105

106 7 Executing Purification Runs System variable Current column in use Current solvent in use Inlet temperature, in C Inlet pressure, in bar ABPR's needle position Splitter status Description Column selected for current purification runs, selected in the Additional Options tab of the Modify Thar Mobile Phase Method dialog box. Co-solvent selected for current purification runs, selected in the Pumping System tab of the Modify Thar Mobile Phase Method dialog box. Temperature of the inlet CO 2. Pressure of the inlet CO 2. Position of the ABPR pressure regulating needle. Shows these status messages: "Off" if not running "Ready" if running at set point "Not Ready" if running, but not at set point See also: Determine status of major components Verify system status during purification runs Specify settings for the FDM Correct CO 2 flow problems Determine status of the PDA detector MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor Windows Start menu > Waters Instruments > Waters Instrument Console To determine the status of the PDA detector, refer to the Detector section at the bottom of the Inlet Method editor's Status tab. 106 March 14, 2014, Rev. A

107 Verify system status during purification runs You can also determine the status of the PDA detector from the Windows Start menu. Click Start > Waters Instruments > Waters Instrument Console. Tip: If your system has a mass spectrometer, you can view the PDA detector's status in the Mass Spectrometer Console. See also: Monitor system status in the Inlet Method editor Verify system status during purification runs Verify system status during purification runs MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor Verify system status frequently during purification runs, to monitor progress through these four phases of system operation: System equilibrating System ready System running System idle To view general information about system status, click the System Status indicator on the MassLynx software main page, at the bottom of the lefthand pane. The following table describes the information displayed when you click the System Status indicator. Status messages in the System Status indicator: Status message Not Ready Ready Description During equilibration, the status pane displays the term "Not Ready". The message also lists the system components that have not reached their set points. The list shortens as the system equilibrates, until all components are ready. System has equilibrated, and is ready to inject a sample. March 14, 2014, Rev. A 107

108 7 Executing Purification Runs Status message Running Ready to Trigger Description When all set points are reached, the system runs the first line in the queue. During a run, system status at the top of the pane indicates "Running". As the system prepares to run the next line in the queue, the system status indicates "Ready To Trigger". To view system status information in the Inlet Method editor, select Inlet Method in the left-hand pane of the MassLynx software main page. System status information is available in the Inlet Method editor's Status tab, and in the Thar Inlet Additional Status tab. System equilibrating During equilibration, the System Status indicator displays the message, "Not Ready". The message changes to "Ready" when all components are ready for the purification run, and the system overall maintains the correct temperature, pressure, and flow rate for the mobile phase. System ready As you prepare to initiate a run, verify that your system status indicators show ready. System status indicators appear in these places: System Status indicator: lower left-hand side of the MassLynx main page Inlet Method editor: Status tab The first system status indicator appears on the MassLynx software main page, at the bottom of the left-hand pane. 108 March 14, 2014, Rev. A

109 Verify system status during purification runs System Status indicator: A "Ready" message indicates the system is equilibrated, and all components have reached their specified set points. The Status tab of the Inlet Method editor contains more information. To open the Inlet Method editor, select Inlet Method on the MassLynx main page. When the system is ready, the indicators Pump On, Ready, and OK show a green icon. Verify that the information and settings in the Inlet Method editor's Thar Inlet Additional Status tab are correct. To summarize, the system is ready to inject when these conditions are met: System running All primary pumps and makeup pumps are pumping. Heat exchangers have reached their temperature set points. Automatic back-pressure regulator has reached its pressure set point. Keep the Status tab of the Inlet Method editor open during a purification run: The Status tab displays four green icons during most of a run: Running, Pump On, Ready, and OK. During the sample manager's injection cycle, the Inject Cycle icon is yellow. Click the Thar Inlet Additional Status tab for information about system temperatures and pressures. To monitor progress for a series of runs, open the sample list on the MassLynx software main page. A green pointer on the left-hand side indicates which line is currently running. The sample list heading describes the March 14, 2014, Rev. A 109

110 7 Executing Purification Runs current operation. For example, "Samples 7 to 10: Sample 8 Acquiring" indicates the system is processing four samples from sample list lines 7 through 10, and that it is acquiring detector data for sample 8. System idle When a run ends, the system enters an idle state: The Running indicator in the Inlet Method editor's Status tab turns from green to gray. The Pump On indicator in the Status tab remains green, indicating that the system's pumps continue to run when the Prep 15 SFC is idle. To determine whether the PDA detector is idle, view the detector's Mode field at the bottom of the Inlet Method editor's status tab. See also: Monitor system status in the Inlet Method editor Determine status of major components Monitor CO 2 pressure and temperature Monitor CO 2 pressure and temperature MassLynx main page > Instrument tab > Inlet Method > Inlet Method editor You must monitor CO 2 pressure and temperature, to ensure proper flow of the mobile phase throughout a purification run. Monitor CO 2 pressure and temperature in the Thar Inlet Additional Status tab. To open the Thar Inlet Additional Status tab, click the tab in the Inlet Method editor. The Thar Inlet Additional Status tab contains the following information: CO 2 pressure at the pump outlet ABPR needle position Inlet temperature Inlet pressure 110 March 14, 2014, Rev. A

111 View the results of a purification run The inlet pressure at the FDM is 55 to 60 bar (797 to 870 psi). A lower than normal pressure at the pump inlet can indicate a low-level volume of CO 2 in the supply tank, or a clogged CO 2 inlet filter that needs replacement. The ABPR, located after the column oven, maintains mobile phase pressure until the CO 2 passes through the ABPR. Use the inlet temperature at the FDM to ascertain whether the CO 2 pump's check valves are operating properly. The operating range for the CO 2 inlet temperature is 5 to 16 C. A higher-than-normal temperature at the pump's inlet often indicates that one or more of the pump's check valves has failed. See also: Monitor system status in the Inlet Method editor Determine status of major components View the results of a purification run MassLynx main page To view run data, open chromatograms based on results from the mass spectrometer and the PDA detector. To open a chromatogram for a completed run: 1. In the MassLynx software main page, open the sample list that contains the completed run. 2. In the sample list, select the row for the completed run. 3. Click Chromatogram. Result: The set of chromatograms specified in the default chromatograms list appears. To set default chromatograms to be displayed: 1. In the MassLynx main page, click the Tools button on the left side of the page. 2. Click Options in the left-hand pane. 3. Click the Chromatograms to Display tab. March 14, 2014, Rev. A 111

112 7 Executing Purification Runs 4. In the Chromatograms list on the left, select the type of chromatogram to be added or removed from the default view. 5. Click the + button to add the selected chromatogram to the default list, or click the - button to remove the selected chromatogram from the default list. Result: The Selected Chromatograms list on the right shows the chromatograms displayed by default. Tip: Click the up or down arrow to modify the order the default chromatograms are displayed. 6. Click OK to save your changes and exit the Options dialog box. The chromatogram's vertical axis measures absorbance units (AU) for PDA data, and percent maximum signal for mass spectrometry data. The horizontal axis measures retention time, in minutes. A description of the run and the data file associated with the run appear above the vertical axis. Tip: To view a chromatogram as it forms during a run, click Chromatogram above the sample list. Then select Display > Real-Time Update in the top menu. MassLynx software displays the chromatogram as it receives data from the PDA detector. 112 March 14, 2014, Rev. A

113 8 Maintaining the System Contents: Create a cleaning method 113 Shut down the system 113 Clean flow lines 114 Replace a flow line filter 115 Maintain the FDM 116 Replace CO2 cylinder 137 Clean fraction collection tubes 139 Replace component fuses 140 Maintain the ABPR and heat exchanger module 141 Create a cleaning method You can create a special inlet method to flush the system after a series of purification runs. To do so, set conditions to clean co-solvent and fraction collection lines, as well as pump and valve connections that comprise the system's flow paths. Before you shut down the system, run this method to flush co-solvents, sample compounds, or any substances that could contaminate the next run or damage the system. You can define and save different cleaning methods to prepare the system for different run conditions or maintenance needs. See also: Create, edit, and load an inlet method Shut down the system Shut down the system MassLynx main page March 14, 2014, Rev. A 113

114 8 Maintaining the System You must shut down the Prep 15 SFC properly, to ensure safe and effective operation the next time you use the system. Configure the control system to shut the system down in these circumstances: Routine shutdown at the end of a work session. Automatic shutdown if the system is paused due to an error state. Specify your preferences for shutting the system down in the ShutDown Pumps dialog box. To open the ShutDown Pumps dialog box, click the currently enabled shutdown routine listed in the lower right-hand corner of the MassLynx software main page. A typical shutdown routine is Batch & Error Enabled. In this routine, the pumps shut down in these circumstances: One minute after a batch completes. If an error or other interruption pauses the queue. To set Batch & Error Enabled in the ShutDown Pumps dialog box: 1. Under Batch Control, select "Enable shutdown after batch". 2. Click Browse, and select the appropriate shutdown method. Recommendation: Develop a shutdown method that cleans the system, and then shuts the system down automatically. 3. Under Batch Control, select "Shutdown if queue is in pause". 4. Under Shutdown on Error, click "Configure error shutdown" for more options. 5. Under Shutdown Time, enter a time. See also: Requirement: Specify the number of minutes the pumps should run after a batch completes or an error pauses the queue. Create a cleaning method Clean flow lines Clean flow lines At the end of each run, or at the end of a work session, flush the fraction collection lines with your solvent of choice. 114 March 14, 2014, Rev. A

115 Replace a flow line filter Important: Do not flush flow lines when the system is idle and not under pressure. Doing so floods the GLS. To flush fraction collection lines automatically: Develop an inlet method with a high percentage of co-solvent specially designed to clean lines. Run the method whenever necessary. Include a cleaning method at the end of a sample list, to flush the lines after a series of runs. Include steps in your inlet methods to clean lines at the end of each collection run. Include steps in your shutdown method to clean the lines. See also: Clean fraction collection tubes Replace a flow line filter Replace the frit inside each flow line filter once a month, or as needed to maintain proper CO 2 and co-solvent flow rates. Flow line filter in use: To replace a filter frit: 1. Remove the filter case from the flow line attached to each side of the filter. March 14, 2014, Rev. A 115

116 8 Maintaining the System 2. Use two open-end wrenches, one on each side of the filter case to loosen the two halves of the case. 3. Use your hands to turn the top half of the case counterclockwise until the two halves separate. 4. Remove the frit seated in the lower half of the case. 5. Place a clean frit inside the lower half of the filter case. 6. Use your hands to turn the upper and lower halves of the filter case together. 7. Tighten carefully with the two open-end wrenches. 8. Reattach the filter to the flow lines, one on the inlet and one on the outlet side of the case. 9. Inspect the fittings on the inlet and outlet sides of the filter to ensure they are secure, with no leaks. See also: Clean flow lines Correct CO 2 flow problems Maintain the FDM The FDM is shipped fully assembled and ready to use. During initial system setup, the system installer: Connects the FDM's CO 2 and co-solvent pumps to their supply sources. Plumbs the cooling lines. Connects the co-solvent pump outlet to the inlet of the sample injector. Connects the CO 2 pump outlet to the T connection on the outlet side of the sample injector. The result of this setup is modifier stream injection, where the CO 2 combines with the co-solvent and sample after the sample is injected. This topic, which conveys important safety information, product details, and instructions for operating and maintaining the fluid delivery module and its components, includes these sections: 116 March 14, 2014, Rev. A

117 Maintain the FDM Reviewing fluid delivery module operation Maintaining the CO 2 pump Maintaining the co-solvent pump Requirement: The FDM contains CO 2 and co-solvent pump assemblies that require servicing on a regular basis, to maintain optimum pumping performance. In addition, the CO 2 pump requires a coolant supply and chiller unit to support its operation with liquid carbon dioxide. To service and maintain the coolant supply and chiller unit, refer to the documentation that accompanies the chiller unit. Reviewing FDM operation Before operating or maintaining the FDM, review this important safety information and operation details. You must follow the information and warnings contained here to properly operate the fluid delivery module in a safe and successful manner. Operating considerations Observe safe laboratory practice when using the fluid delivery module. Always keep in mind the following safety practices: Familiarize yourself with proper handling, storage, and disposal of all chemicals used with your system. Refer to the Material Safety Data Sheet (MSDS) for each solvent you use, and know its chemical properties. Wear the appropriate personal protective equipment (PPE) when working with any type of hazardous chemical. Wear eye protection when the oven door is open, to protect eyes from possible failure of column seals or fittings. Ensure that the system is depressurized before loosening any fittings in the CO 2 and co-solvent flow path. Electrical considerations The operating voltage of your FDM was preset at the factory to comply with the electrical requirements specified when the device was ordered. Power conversion in the field is not recommended. The module operates at 50/60 Hz. Before powering-on the module, ensure that the power cord is connected March 14, 2014, Rev. A 117

118 8 Maintaining the System to an appropriate source of power. Plug the power cord only into an outlet equipped with a protective ground (earth) contact. Do not defeat the protective action of this connection by using an extension cord without a ground conductor. Any interruption of the protective grounding conductor capable of causing personal injury. Important: Waters assumes no responsibility for improperly grounded receptacles or power cords that it does not provide. All wiring to the supply outlet must meet local electrical codes. Warning: To avoid a fault condition that can result in personal injury or death from electric shock, do not disconnect or otherwise interrupt the continuity of an electrical socket's protective grounding conductor or the power cord's grounding conductor. Warning: To avoid a fault condition that can result in personal injury or death from electric shock, do not remove the device's protective panels unless directed to in these instructions. The panels often do not conceal user-serviceable parts, and their removal is properly the function only of technicians authorized by Waters. Caution: To avoid electrical noise from affecting signals, and reduce electromagnetic radiation capable of interfering with the performance of other devices or instruments, use shielded cables and connectors for all remote connections. Chemical considerations Always practice good chemical hygiene. Familiarize yourself with the proper handling, storage, and disposal of all chemicals used with your system. Refer to the Material Safety Data Sheet (MSDS) for each solvent you use, and know its chemical properties. The components in the FDM flow path are constructed of 316 and 17-4 stainless steel, Nitronic 60, Teflon (PTFE), sapphire, ruby, and UHMWPE (optional). Solvents used in the mobile phase must be compatible with these materials. Acceptable fluids include liquids and liquefied gasses. Do not introduce particulate matter into the pump. Damage to the check valve's sealing surface can result. Place a filter directly upstream of the pump to capture any solids that have formed. 118 March 14, 2014, Rev. A

119 Maintain the FDM Flammability warning Leaks of flammable mobile phase into the column oven could cause flammable or explosive mixtures to form. The oven components can achieve temperatures as high as 90 C. These temperature levels exceed the flash point of many flammable vapors. Avoid using any solvent for which the ignition temperature is in this range. Always inspect the system for leaks before operating it unattended. Warning: To avoid explosion, do not use a highly flammable solvent for any component of the mobile phase. FDM description The FDM's CO 2 pump is a high pressure pump that incorporates a lowdead-volume head and check valves, which provide efficient pumping of liquid CO 2. As the pump's piston retracts, the lower check valve opens to allow fluid into the chamber. Concurrently, the upper check valve closes, to prevent the system fluid from entering the chamber. When the piston completes its fill cycle, it begins to move forward, causing the lower check valve to close. When the pressure in the piston chamber exceeds the system pressure, the upper check valve opens and allows the fluid to flow into the system. The pump consists of three pistons that provide pulse-free solvent delivery. The FDM's co-solvent pump is a dual piston, reciprocating pump used to drive co-solvent into the system. Requirement: To service the fluid delivery module, you need the FDM service kit, part number Maintaining the CO 2 pump You maintain these component parts for the CO 2 pump: CO 2 inlet filter Inlet and outlet check valves Piston seals Cooling O-ring March 14, 2014, Rev. A 119

120 8 Maintaining the System Replacing the CO 2 inlet filter The CO 2 inlet line contains an inline filter, to help prevent particles from entering the system and damaging components such as check valves. When the filter becomes clogged, it restricts flow to the pump and causes poor pump performance. Replace the CO 2 inlet filter according to your maintenance schedule or whenever the CO 2 flow is restricted owing to a clogged filter. To replace the inlet filter: 1. Shut the valve off to the CO 2 supply tank, and then open the bleeder valve to completely depressurize the pump. 2. Remove the four upper thumbscrews securing the module's left-hand side panel. 3. Loosen the two lower thumbscrews securing the panel, and then lift and remove it. 4. Using a 7/8-inch wrench, loosen and remove the inlet filter cover and its internal cover spring. 120 March 14, 2014, Rev. A

121 Maintain the FDM CO 2 inlet filter assembly: 5. Remove the inlet filter from the inlet filter housing. 6. Insert a new inlet filter with the solid end facing outward into the housing. 7. Replace the seal in the inlet filter cover with the seal supplied with the filter, and then reinstall the internal cover spring. 8. Replace and tighten the inlet cover. 9. Open the valve to the CO 2 supply tank, and inspect for leaks. 10. Reinstall the FDM's side panel. Replacing the CO 2 pump's inlet and outlet check valves The inlet check valves, on the bottom of the pump heads, allow flow into the pump heads as the piston retracts. As the piston advances, and pressure in March 14, 2014, Rev. A 121

122 8 Maintaining the System the chamber begins to build, the inlet check valves close and prevent flow back into the source. The outlet check valves, on the top of the pump heads, allow flow out of the pump heads as the piston advances. As the piston retracts the outlet check valves prevent system pressure from entering the pump heads. When there is a bad check valve, the best indicator is the FDM pump temperature. It will be higher than normal, or greater than 14 C. To determine which check valve is bad, leave some pressure on the system (50 to 60 bar). Remove the fittings (tubing) from the bottom check valve assemblies on the pump heads, and bubble check the valves with soapy water. The CO 2 leaks back through the check valve if the valve is bad. From the check valve assemblies at the bottom of the pump heads, work your way up to test the valves at the top of the pump heads. To replace the check valve: 1. Shut off the valve to the CO 2 supply tank, and then open the bleeder valve to completely depressurize the pump. 2. For the check valve you are replacing, use a 1/4-inch wrench and a 1/2-inch backup wrench to disconnect the inlet or outlet line from the pump check valve nut and the manifold. 3. Remove the tubing. 4. Remove the check-valve nut. Tip: If the check valve is an outlet valve, use needle-nose pliers to remove the valve from the head. 122 March 14, 2014, Rev. A

123 Maintain the FDM Outlet check valve with arrow up: 5. Clean the retainer-nut threads, and spray them with a PTFE dry lubricant. 6. Install the new check valve with the arrow pointing up. 7. Finger tighten the nut into the head until the nut contacts the check valve. 8. Using a 1/2-inch wrench, tighten the check-valve nut approximately Reinstall the tubing. 10. Open the valve to the CO 2 supply tank, and inspect for leaks. Replacing the CO 2 pump's piston seals The CO 2 pump incorporates low friction sapphire for the pistons. The sapphire passes through a GFPM spring-loaded seal. After some time, the seal begins to wear and requires replacement. Typical symptoms of a leaking seal are decreased pump efficiency, excessive bubbles from behind the head, and a build-up of frost from leaking CO 2. Tip: Upon a cold start, the CO 2 pump seals normally leak briefly until the pump head warms during operation. March 14, 2014, Rev. A 123

124 8 Maintaining the System Recommendation: Replace both piston seals at the same time. Requirement: Always replace the backup seal when replacing the piston seal. To replace the piston seals: 1. Shut the valve off to the CO 2 supply tank, and then open the bleeder valve to completely depressurize the pump. 2. Remove the cooling lines attached to the front of the pump heads. 3. Using a 1/4-inch wrench and a 1/2-inch backup wrench, disconnect the inlet and outlet lines from the pump's check-valve nuts and the manifold. Removing a pump head: 4. Using a permanent marker, place a small mark on the top of the heads. Tip: Be sure to mark the top of each pump head so that you can reinstall the heads in the correct orientation. 5. Using a 3/8-inch wrench, remove the four head nuts and washers securing the head to the front of the pump. 124 March 14, 2014, Rev. A

125 Maintain the FDM 6. Carefully pull the head assembly straight out from the front of the pump. Pump head assembly removed: 7. Clean the piston sapphire using acetone and a lint-free cloth. 8. Using the plastic hooked tool from the service kit, remove the tancolored backup bearing and piston seal from the pump head. 9. Using a cotton swab, clean the seal cavity using acetone. 10. Slide the new seal into the pump head with the spring facing toward the front of the head. Requirement: When installed, the spring must not be visible. March 14, 2014, Rev. A 125

126 8 Maintaining the System Pump head, piston spring seal and backup seal: 11. Slide the backup bearing into the pump head until it is flush with the back of the pump head. 126 March 14, 2014, Rev. A

127 Maintain the FDM Pump head with installed spring seal: 12. With the mark made in step 4 at the top, align the pump head with the head bolts, and slide the pump head back into place. 13. Loosely install the 4 head nuts between the head and the pump chassis. 14. Begin tightening the nuts approximately one or two turns each, in a clockwise pattern, until all are tight. 15. Reinstall the tubing to the check-valve nuts and the manifold. 16. Reinstall the cooling lines attached to the front of the pump heads. 17. Open the valve to the CO 2 supply tank, and inspect for leaks. Replacing the CO 2 pump head cooling O-ring The CO 2 pump incorporates a cooling cavity on the pump heads to increase the CO 2 pumping efficiency. This cavity contains an O-ring seal. Evidence of a coolant flow coming from the seam around the front of the pump head indicates a seal failure. To replace the cooling O-ring: 1. Turn off the flow of coolant to the pump heads. March 14, 2014, Rev. A 127

128 8 Maintaining the System Requirement: It is not necessary to remove the tubing from the pump heads; however, ensure you clamp off the tubing to prevent siphoning of the coolant. 2. Using a 3/8-inch wrench, remove the four head nuts and washers securing the head to the front of the pump. 3. Holding the pump head in place with one hand, use the other hand to pull the pump head face plate out of the pump head. Important: Do not remove the pump head from the front of the unit. Pump head cooling cavity seal: 4. Locate the O-ring seal inside the pump head and remove it. 5. Clean any remaining pieces of O-ring from the head cavity. 6. Install the replacement O-ring seal in the cavity, and then press the face plate into the pump head. 7. Loosely install the four head nuts between the head and the pump chassis. 8. Begin tightening the nuts approximately one or two turns each, in a clockwise pattern, until all are tight. 128 March 14, 2014, Rev. A

129 Maintain the FDM Maintaining the co-solvent pump You must maintain these items for the co-solvent pump: Piston seals Inlet and outlet check valves Prime purge valve seal Co-solvent isolation check valve Refer to the following figure when servicing the co-solvent pump head: Stainless steel self-flushing pump head assembly: Replacing the piston seals Lower-than-normal pressure, pressure variations, and leaks in the pumping system all indicate possible problems with the piston seal. Depending on which solvents you use, the need to replace piston seals can manifest itself as frequently as every 1,000 hours of running time. Each replacement seal kit contains one seal, one backup washer, one selfflush seal, one non-flush guide bushing, two seal insertion/removal tools, and a pad to clean the piston when changing the seal. March 14, 2014, Rev. A 129

130 8 Maintaining the System To remove the existing seals: 1. Remove the inlet line from the inlet check valve. 2. Remove the outlet line from the outlet check valve. 3. Remove inlet and outlet self-flush check valves. Caution: To avoid unintentional dismantling of assemblies and consequent performance problems, when removing the check valves, keep them in this orientation entire time they are not installed in the pump head. the 4. Carefully remove the two knurled nuts at the front of the pump head. Caution: To avoid damaging the piston, when removing the pump head, use care not twist it. 5. Carefully separate the pump head from the pump by pulling the pump head straight out from the pump until it is free from the piston. Requirement: Remove the seal and seal backup washer from the piston, if it failed to stay in the pump head. 6. Carefully separate the flush housing from the pump by pulling the flush housing straight out from the pump until it is free from the piston. Requirement: Remove the self-flush seal from the piston, if it failed to stay in the flush housing. 7. Insert the flanged end of the seal insertion/removal tool into the seal cavity on the pump head, tilting it slightly so that flange is under the seal, and then pull out the seal. Caution: To avoid scratching the smooth surface of the pump head, when removing the seal, use only the seal insertion/removal tool from the seal replacement kit. Do not use another tool. 8. Repeat the procedure for the low-pressure seal in the flush housing. 9. Continue with the next section, to clean the piston. 130 March 14, 2014, Rev. A

131 Maintain the FDM To clean the piston: 1. After removing the pump head and self-flush housing, gently remove the seal back-up plate using a toothpick or small screwdriver in the slot on top of the pump housing. 2. Grasp the metal base of the piston assembly, so that you avoid exerting any side load on the sapphire rod, and then remove the piston from the slot in the carrier by sliding it up and out. 3. Use the scouring pad included in the seal-replacement kit to clean the piston, gently squeezing the piston within a folded section of the pad and rubbing the pad along the length of the piston. Requirement: Rotate the piston frequently, to ensure the entire surface is scrubbed. Do not exert pressure perpendicular to the length of the piston, as doing so can cause the piston to break. 4. Use a lint-free cloth, dampened with alcohol, to wipe the piston clean. 5. Grasp the metal base of the piston assembly, and insert it into the slot in the piston carrier until it bottoms. 6. Continue with the next section, to replace the seals and check valves. To replace the seals and check valves: 1. Place a high-pressure replacement seal on the rod-shaped end of the seal insertion/removal tool so that the spring is visible when the seal is fully seated on the tool. 2. Insert the tool into the pump head so that the open side of the seal enters first, facing the high-pressure cavity of the pump head. Requirement: Take care to align the seal with the cavity while performing the insertion. 3. Withdraw the tool, leaving the seal in the pump head. Tip: When you look into the pump head cavity, only the polymer portion of the seal remains visible. 4. Place a self-flush replacement seal on the seal insertion/removal tool so that the spring in the seal is visible when the seal is on the tool. 5. Insert the tool and seal into the seal cavity on the flushing housing, taking care to align the seal with the cavity, and then withdraw the tool. March 14, 2014, Rev. A 131

132 8 Maintaining the System Tip: When you look into the pump head cavity, only the polymer portion of the seal remains visible. 6. Place the seal back-up washer over the high-pressure seal. Place the seal back-up plate back into pump housing if it was removed. Tip: Orientation is unimportant in these cases. 7. Install the inlet valve on the bottom of the pump head, and the outlet valve, at the top of the pump head. Caution: To avoid unintentional dismantling of assemblies and consequent performance problems, when removing the check valves, keep them in this orientation entire time they are not installed in the pump head. the 8. Carefully align the self-flush housing and gently slide it into place on the pump. If misalignment with the piston occurs, gently push up on the piston holder. 9. Line up the pump head and carefully slide it into place. Requirement: Be sure that the inlet valve is on the bottom and the outlet valve is on the top. Do not force the pump head into place. 10. Finger tighten both knurled nuts into place. To tighten firmly, alternately turn nuts 1/4-turn while gently wiggling the pump head, to center it. 11. Re-attach the inlet and outlet lines. Reconnect the self-flush lines and fittings to the self-flush check valves. 12. Continue with the next section to condition the new seals. To condition new seals: Requirement: Use only organic solvents to acclimatize new seals. Avoid buffer solutions and salt solutions. 1. Using a union in place of the extraction vessel, run the pump using a 50:50 solution isopropanol (or methanol) / water for 60 minutes at the backpressure and flow rate listed below. 132 March 14, 2014, Rev. A

133 Maintain the FDM Acclimatizing conditions for pump head seals: Pump head type Pressure and flow rate 5.0 ml SS/Nitronic-60 ~60 bar < 3 ml/min 2. Periodically inspect the co-solvent pump heads for leaks, and monitor the run conditions, to ensure the pressure and flow rate are stable. 3. Flush the flow path with 100% isopropanol (or methanol) for 10 minutes, to remove all traces of water from the flow path. Cleaning or replacing the co-solvent pump's inlet and outlet check valves Many check valve problems are the result of small particles interfering with the operation of the check valve. You can solve most of these problems by removing the check valve and sonicating it in methanol or your solvent of choice. If after cleaning, the check valve is still not functioning properly, replace it. Replacing the prime purge valve seal Refer to the following figure when servicing the prime purge valve. March 14, 2014, Rev. A 133

134 8 Maintaining the System Prime/purge valve rebuilding kit: To replace the prime/purge valve seal: 1. Using the plastic knurled cap, open the priming valve completely. 2. Unscrew the hex nut, and remove the valve stem assembly from the valve. 3. Using the supplied Allen wrench, remove the seal retainer, taking care not to scratch or otherwise damage the internal parts of the valve. 134 March 14, 2014, Rev. A

135 Maintain the FDM 4. Remove the Teflon seal ring from the valve body, taking care not to scratch the sealing surface around the seal. 5. Using tweezers or small pliers, pull the plastic tip from the stem. 6. Insert a new tip from the kit, blunt end first, into the stem assembly, and seat the tip firmly, applying light pressure against a clean, hard surface. 7. Insert a new seal ring fully into the valve body until the ring rests flat against the shoulder, beyond the threads. 8. Replace the seal retainer, and tighten firmly. 9. Refit the stem assembly into the valve body, and rotate it one or two turns past the point where you feel the initial resistance. 10. Close the valve firmly, to seat the tip seal. Replacing the co-solvent isolation check valve To replace the co-solvent isolation check valve: 1. Remove the four upper screws securing the right-hand, side panel of the unit. 2. Loosen the two lower screws securing the right-hand, side panel of the unit, and then lift and remove it. 3. Shut the valve off to the CO 2 supply tank, and then open the bleeder valve to completely depressurize the pump. March 14, 2014, Rev. A 135

136 8 Maintaining the System Isolation check valve assembly: 136 March 14, 2014, Rev. A

137 Replace CO2 cylinder 4. Using 1/4-inch and 1/2-inch wrenches, remove the inlet line outlet line from the isolation check valve. 5. Using two, 1/2-inch wrenches, open the check-valve holder. and 6. Remove the check valve, and install the replacement check valve, making sure the arrow on the check valve matches the arrow on the holder. 7. Tighten the two halves of the check-valve holder together. 8. Connect the inlet of the check valve holder to the line coming from the prime/purge valve. 9. Connect the outlet of the check valve to the line leading to the mixer. 10. Open the valve to the CO 2 supply tank, and inspect for leaks. 11. Reinstall the FDM's side panel. See also: Determine fluid delivery module status Troubleshoot fluid delivery module Replace CO 2 cylinder These instructions apply if you do not have a bulkhead CO 2 supply in your laboratory, and you must therefore replace the CO 2 cylinder connected to your system. External CO 2 supply connections If you are using liquid carbon dioxide cylinders to supply CO 2 to your system, ensure that they meet these requirements. CO 2 supply requirements: Component Requirement CO 2 cylinder High-pressure cylinder, with dip tube, no regulator Pressure Approximately 60 bar (870 psi) March 14, 2014, Rev. A 137

138 8 Maintaining the System Component Requirement CO 2 grade Normal grade, greater than % purity, less than 50 ppm impurities CO 2 cylinder: Connecting the CO 2 supply cylinder to the CO 2 inlet fitting Requirement: The system is shipped with a CGA 320 fitting for connecting to the CO 2 supply cylinder. Consult with your gas supplier to ensure this type of connection is acceptable in your locale. Before connecting the CGA fitting to a new CO 2 cylinder, you must vent the cylinder for 2 to 3 seconds, until liquid escapes. To vent the cylinder, open and close the valve in a fast, smooth motion until liquid escapes. Wipe the 138 March 14, 2014, Rev. A

139 Clean fraction collection tubes valve clean, and dry it with a lint-free cloth before connecting the CGA fitting. To connect a new CO 2 cylinder: 1. Secure the CO 2 supply cylinder using safety strapping. 2. Remove the cap and protective wrap from the tank valve. 3. While directing the valve outlet away from all personnel, vent the cylinder for 2 to 3 seconds until liquid escapes. 4. Wipe the valve clean, and dry it with a lint-free cloth. 5. Insert the Teflon disc seal in the CGA fitting, attach the inlet tubing assembly to the cylinder nozzle, and tighten the fitting using a 1 1/8- inch wrench. 6. Uncoil the tubing from the cylinder nozzle. 7. Locate the CO 2 inlet fitting on the CO 2 pump, and connect the tubing to the inlet fitting. See also: Tip: CGA fittings and tubing are in the system startup kit shipped with the system. Supply CO 2 and nitrogen gas Correct CO 2 flow problems Clean fraction collection tubes After a run ends, replace the fraction collection tubes in the 2767 sample manager with clean ones. To prepare fraction collection tubes for reuse, clean them of solvent and sample residue. Do not allow compounds collected during one run to contaminate the purity of fractions collected during future runs. To clean a fraction collection tube: 1. Remove the tube from the open bed fraction collection rack in the sample manager. 2. Assure the fraction is removed from the tube before you clean it. March 14, 2014, Rev. A 139

140 8 Maintaining the System 3. Use methanol or another cleaning solvent to clean the inside of the tube. 4. Let the fraction collection tube dry before you reuse the tube. See also: Prepare the fraction collection bed Clean flow lines Replace component fuses System components use fuses to interrupt electrical power when they fail, or some other problem occurs. Fuses protect both the operator and the equipment. Under normal operation, fuses rarely fail. Multiple failures likely indicate an electrical fault in a component. Contact Waters customer support for troubleshooting assistance, if necessary. Locate a component's fuse holder above the power switch on the rear panel. Always replace a fuse with one of equal rating. The component's rear panel contains specifications for replacement fuses. To replace a fuse that serves a system component: 1. Move the component's power switch to the off (0) position, and disconnect the power cord from the back of the unit. 2. Open the fuse-holder door, above the power switch. 3. Remove the old fuse, and replace it with one of the same type and rating. 4. Close the fuse-holder door. 5. Reconnect the unit's power cord, and move the power switch to the on (1) position. Tip: Before you replace a fuse, determine whether an underlying problem caused the fuse to fail. Warning: To prevent electrical shock, disconnect the component from its power source before you replace its fuse. Warning: To prevent electrical fires, use fuses that comply with the ratings affixed to the component's rear panel. 140 March 14, 2014, Rev. A

141 Maintain the ABPR and heat exchanger module Contact Waters customer support for assistance with all electrical problems that prove difficult to resolve. See also: Review power requirements Maintain ABPR and post-abpr heat exchanger Maintain the ABPR and heat exchanger module The ABPR and the post-abpr heat exchanger do not have customer-serviceable parts. To replace the needle inside the ABPR, or to calibrate the backpressure regulator valve, contact your Waters field representative. Exceptions: The electrical fuse at the rear of the ABPR is user-replaceable. If your heat exchanger has an externally mounted fuse on the rear panel, the fuse is user-replaceable. See also: Replace component fuses Review component descriptions March 14, 2014, Rev. A 141

142 8 Maintaining the System 142 March 14, 2014, Rev. A

143 9 Troubleshooting the System Contents: 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 Correct CO 2 flow problems The troubleshooting table below lists symptoms, problems, and corrective actions related to CO 2 flow in the Prep 15 SFC system: March 14, 2014, Rev. A 143

144 9 Troubleshooting the System Troubleshooting CO 2 flow problems: Symptom Problem Corrective Action Unable to achieve pressure, or unable to achieve flow rate. Low CO 2 supply Inadequate cooling CO 2 supply does not contain a dip tube Defective check valve Leaking piston seal Leaks in system System pressure is incorrect. closed Restriction in lines valves Defective pressure transducer Miscalibrated pressure transducer Replace CO 2 supply tank. Ensure bath temperature is between 5 and +5 C. Ensure that cooling lines to pump heads and heat exchangers are intact and properly secured, ensure that the fluid level is adequate. Contact CO 2 supplier; replace tank. Replace check valve. Replace piston seal. Correct leaks. Ensure that valves are properly positioned and that tubing is correctly sized and properly cut. Replace and recalibrate pressure transducer. Note: To replace or recalibrate the pressure transducer, contact your Waters field service representative. Recalibrate unit. 144 March 14, 2014, Rev. A

145 Resolve system problems Symptom Problem Corrective Action Repeated failures of check valves. See also: Contamination in CO 2 PTFE particles in lines Dirt particles in lines Troubleshoot fluid delivery module Replace CO 2 cylinder Resolve system problems Contact CO 2 supplier. Ensure that PTFE tape is properly applied to fittings. Inspect the flow meter seals and the seal between the tank and supply-line fitting. Clean lines after cutting. Three types of problems can affect the Prep 15 SFC system: Temporary software alarm Communication failure System alarm To address each type: When you install a new CO 2 cylinder, vent it for 2 to 3 seconds until liquid escapes. Then connect the cylinder to the supply line. 1. Open and view the most recent events recorded in the LogLynx audit log to determine the cause or location of the problem. 2. Exit MassLynx software, cycle power to failed components, and then reboot workstation or execute the StopMassLynxProcesses routine. 3. Reset MassLynx software. March 14, 2014, Rev. A 145

146 9 Troubleshooting the System Tip: If the system re-equilibrates, you have cleared the problem or alarm. 4. If the problem persists, report all relevant information to your Waters service representative. System problems and solutions: Problem Temporary software alarm A temporary condition can trigger a software alarm. A system overpressure condition caused by a pressure spike is a typical trigger. Communication failure System communication with a component can fail, triggering a software alarm. System alarm A system-level problem can trigger an internal hardware or software alarm, causing a component or the system to shut down. See also: Review audit log Troubleshoot fluid delivery module Solution See standard four-step procedure, above. See standard four-step procedure, above. Inspect the failed component's cables and connections, to ensure they are tight. Refer to the operator's guide for the failed component. It might contain additional troubleshooting information. See standard four-step procedure, above. If necessary, shut down the entire system from MassLynx software, and restart. If you suspect a problem involving delivery of CO 2 or co-solvent from the FDM, verify the flow rate at the pump outlet. The following table lists 146 March 14, 2014, Rev. A

147 Troubleshoot fluid delivery module potential problems related to the FDM's two pumps, possible causes, and solutions: FDM troubleshooting: Problem Possible Causes Solution Unable to achieve correct CO 2 flow rate. Repeated failures of CO 2 pump check valves. Low CO 2 supply Replace CO 2 supply tank. Inadequate cooling At the chiller unit, make sure bath temperature is between 5 and +5 C. Inspect cooling lines to and from the fluid delivery module, and ensure that the coolant fluid level is adequate. CO 2 supply does not contain a dip tube Defective check valve Leaking piston seal Restriction in lines Closed valves Contaminated CO 2 PTFE particles in lines Dirt particles in lines Contact CO 2 supplier; switch tank. Replace the CO 2 pump's check valve. Replace the CO 2 pump's piston seal. Ensure that tubing is correctly sized and properly cut. Ensure that valves are properly positioned. Contact CO 2 supplier. Ensure that PTFE tape is properly applied to fittings. Inspect the seal between the tank and supply line fitting and the flow meter seals. Clean lines after cutting; add filter in supply line. March 14, 2014, Rev. A 147

148 9 Troubleshooting the System Problem Possible Causes Solution Unable to achieve correct co-solvent flow rate. See also: Low co-solvent supply Defective check valve on co-solvent pump Leaking piston seal Clogged co-solvent filter Selector valve out of alignment Pressure test the system Refill co-solvent reservoir. Replace check valve. Replace piston seal. Replace co-solvent filter. Align valve. Determine fluid delivery module status Maintain the fluid delivery module Pressure test the system Pressure test the system when any of these events occur: The system or one of its components is newly installed. The system is recommissioned after being shut down for a week or more. Evidence of freezing or solvent leakage is visible at tube connections or fittings. The system is unable to maintain its specified, pressure set point. To pressure test the system and diagnose results: 1. Open the CO 2 supply valve, and do as follows: Inspect all fittings and connectors for leaks. Look for dry ice at the fitting, and listen for the sound of escaping CO March 14, 2014, Rev. A

149 Troubleshoot the column oven Tighten fittings and connectors where you discover a leak. 2. Note the CO 2 supply gauge to determine whether the supply of CO 2 is low. Requirement: If the CO 2 supply is low, refill or replace the CO 2 source. 3. Start an inlet method specifying a CO 2 flow rate of 5 ml/min and an ABPR pressure of 100 bar (1450 psi). Tip: Doing so confirms whether the system can achieve and maintain a pressure set point of 100 bar (1450 psi). Recommendation: Allow four minutes for the system to reach 100 bar (1450 psi). If it does not reach and maintain that pressure, reinspect the system fittings and connections for leaks (see step 1). 4. Specify the CO 2 flow rate at 2 ml/min and the ABPR pressure at 100 bar (1450 psi). See also: Tip: Doing so confirms the proper operation of the CO 2 pump. Important: Ascertain the CO 2 pump's inlet temperature and pressure. A higher-than-normal temperature at the pump inlet usually indicates that one or more of the CO 2 pump's check valves failed. Low pressure or pressure fluctuations greater than 0.4 can indicate CO 2 supply problems. Troubleshoot fluid delivery module Specify pressure settings and column oven settings Troubleshoot the column oven Troubleshoot the column oven using information in the errors display on the oven's front panel. To open the errors display: 1. Press the F1 key under the oven's touch sensitive screen. 2. In the main menu, press Errors. March 14, 2014, Rev. A 149

150 9 Troubleshooting the System The errors display either displays no errors, or it displays one or more of the following error codes. Contact your Waters service representative if corrective action does not resolve the problem. Oven error codes: Error code Description Corrective action 1 Oven temperature alarm. Verify the oven temperature set point. 2 Heater temperature above the maximum allowed temperature. 3 An external zone temperature above the maximum allowed temperature. Verify the alarm temperature set point. Contact Waters. Verify the zone temperature set point. Verify the alarm temperature set point. 4 Gas sensor alarm. Inspect the oven for solvent leaks. 5 Inflow and outflow oven valves are in different positions. Verify valve positions. Set both valves for the same column. 6 Open thermocouple circuit. Confirm extra channels without a thermocouple connected are unused. 7 Gas sensor burnout. Contact Waters. 8 Oven door open longer than time specified for the door open interval. Close the oven door and clear the alarm. 9 Oven heats too slowly. Confirm oven is connected to the correct power source. 150 March 14, 2014, Rev. A

151 Review audit log Error code Description Corrective action 10 Heater and oven temperatures too far apart. 11 Oven measured and set point temperatures too far apart. Confirm door seal is properly in place. Ensure air flows into and out of heater vents and within oven chamber are unrestricted. Allow oven to cool for several minutes, and then reheat oven. Confirm door seal is properly in place. Ensure air flows into and out of heater vents and within oven chamber are unrestricted. Allow the oven to cool for several minutes, and then reheat oven. See also: Specify pressure settings and column oven settings Review the operation of the column oven Control the column oven from the front panel Review audit log The LogLynx audit log stores data about system events. You can filter the events in the audit log by date and other criteria. When you suspect that a problem occurred with the system, open and view the most recent system events to diagnose the problem and determine corrective action. You can use this approach to solve most problems that occur during a run. Refer to the MassLynx software online Help for more information about LogLynx audit logs. See also: Resolve system problems March 14, 2014, Rev. A 151

152 9 Troubleshooting the System 152 March 14, 2014, Rev. A

153 10 Reviewing Column Oven Procedures Contents: Review the operation of the column oven 153 Review safety information for the column oven 154 Control the column oven from the front panel 157 Replace a column in the column oven 158 Clean oven surfaces 160 Review the operation of the column oven Separations take place in one or more preparative SFC columns located in the column oven. Mobile phase and injected samples enter the oven inlet, and then pass through the column at the correct pressure and temperature range, ambient +5 degrees to 90 C. The eluent emerges from the oven as the automatic back-pressure regulator maintains correct pressure. The column oven is shipped from Waters fully assembled. To use the oven, verify that set points are properly specified, and install the column or columns you need for your runs. The oven's component parts include these: Two switching valves, to select the column for a particular run. Connections for multiple columns, 4.6 mm or 10 mm internal diameter. Controllers and connections for four external heating zones. See also: Waters SFC Column Oven Operator's Guide The oven's integral controller monitors and controls the oven's selector valves, reads its sensors, and controls its heaters. The heat source comprises an enclosed heater with a forced air blower. Connections in the rear of the oven provide for controlling devices in four external zones. Use March 14, 2014, Rev. A 153

154 10 Reviewing Column Oven Procedures these zones to monitor and control heat exchangers and other thermal devices via a type J thermocouple. The oven door is hinged at the bottom and held shut by means of a magnetic gasket at the top. To open the door, pull the handle toward you. The rack allows use of 4.6 mm and 10 mm internal diameter columns. Move the racks inside the oven to accommodate different column configurations. See also: Warning: To avoid burn injuries, do not handle columns or other components heated to high temperature in the column oven. Wait until the oven and the columns inside it have cooled. Review safety information for the column oven Control the column oven from the front panel Review safety information for the column oven Before you use the column oven, review these safety information and operating instructions: General safety Observe safe laboratory practice when using the oven. Always adhere to these safety practices: Familiarize yourself with proper handling, storage, and disposal of all chemicals used with your system. Refer to the Material Safety Data Sheet for each solvent you use, and know its chemical properties. Wear the appropriate personal protective equipment when working with any type of hazardous chemical. Wear eye protection when the oven door is open to protect eyes from possible failure of column seals or fittings. Depressurize the system before you loosen fittings in the mobile phase flow path. Exercise caution whenever the oven door is open. Internal surfaces of the components can become very hot during normal operation. Direct contact could cause injury. The internal oven compartment is capable of reaching temperatures high enough to cause burns and the internal 154 March 14, 2014, Rev. A

155 Review safety information for the column oven surfaces do not cool instantly upon opening the door. Internal surfaces such as heat sinks, valves, tubing, and fittings may become very hot. Warning: To avoid burn injuries, take care when reaching for internal components. Internal surfaces of the oven components can become extremely hot during normal operation. Electrical safety The operating voltage of the column oven was preset at the factory, to comply with the electrical requirements specified when the device was ordered. Power conversion in the field is not recommended. The oven operates at 50/60 Hz. Before powering-on the oven, ensure that the power cord is connected to an appropriate source of power. Plug the power cord into a properly grounded outlet. Do not defeat the protective action of this connection by using an extension cord without a ground conductor. Interruption of the protective grounding conductor can cause personal injury. Important: Waters assumes no responsibility or legal liability for improperly grounded receptacles or power cords that it does not provide. All wiring to the supply outlet must meet local electrical codes. Warning: To avoid injury or death from electric shock, do not disconnect or otherwise interrupt the continuity of an electrical socket's protective grounding conductor or the power cord's grounding conductor; do not remove the oven's protective panels. Instead contact your Waters field service representative. Note: Electromagnetic radiation can affect performance of other instruments. Use shielded cables and connectors for all remote connections. Chemical safety Familiarize yourself with the proper handling, storage, and disposal of all chemicals that you use in your system. Refer to the Material Safety Data Sheet for each solvent you use, and know its chemical properties. March 14, 2014, Rev. A 155

156 10 Reviewing Column Oven Procedures Warning: To avoid the harmful effects of personal contact (including inhalation) with compounds that are toxic or corrosive, observe Good Laboratory Practice when handling all solvents. For information about the physical properties and safe handling of the particular solvents that you use, consult their Material Safety Data Sheets. Flow path materials Components in the oven flow path are constructed of stainless steel and other durable materials. Solvents in the mobile phase must be compatible with these components. Flammable materials Leaks of flammable mobile phase into the extraction oven could cause flammable or explosive mixtures to form. The oven contents can achieve temperatures as high as 90 C. These temperatures exceed the flash point of many flammable vapors. Warning: To avoid an explosion, do not use a highly flammable solvent for any component of the mobile phase. Warning: To avoid danger of fire, always inspect the oven and related components for leaks before you operate the system. Warning: To avoid eye injury or other injury caused by the explosive force of a high-pressure release of carbon dioxide, sample, and cosolvent, use eye protection when opening the oven door. Integrated safety features The column oven heating element is enclosed and inaccessible. Tubing and other materials cannot touch the heating element. When an unsafe condition occurs inside the oven, the oven's control system generates an error message. The oven and external zones shut down when these conditions occur: The oven's vapor sensor detects a buildup of hydrocarbon gases. An interior heat sensor detects that the oven's element has overheated. 156 March 14, 2014, Rev. A

157 Control the column oven from the front panel The safety thermostat detects an interior temperature greater than 120 degrees centigrade. See also: Observe Good Laboratory Practice Review the operation of the column oven Control the column oven from the front panel To monitor and control the column oven from the front panel, use the touch sensitive display, and the five function keys under the display. Oven touch panel: The function keys to display these screens: Key F1 F2 F3 F4 F5 Display Main menu Valve screen System status screen External zones screen Previous screen March 14, 2014, Rev. A 157