Waters Gel Permeation Chromatography Cleanup System

Transcription

1 Waters Gel Permeation Chromatography Cleanup System Operator s Guide 34 Maple Street Milford, MA WAT022516TP, Revision 1

2 NOTICE The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, the use of this manual WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED. THIS BOOK OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER. Waters is a registered trademark of Waters Corporation. Envirogel is a trademark of Waters Corporation. Acrodisc and Gelman are registered trademarks of Pall Corporation. Fluoropore, Milli-Q, and Millipore are registered trademarks of Millipore Corporation. Teflon, Tefzel, and Viton are registered trademarks of the E.I. du Pont de Nemours Company. PreCleaned Certified is a trademark of Environmental Sampling Supply Inc. All other trademarks are the sole property of their respective owners. The quality management system of Waters chromatography applications software design and manufacturing facility, Milford, Massachusetts, complies with the International Standard ISO 9001 Quality Management and Quality Assurance Standards. Waters quality management system is periodically audited by the registering body to ensure compliance.

3 STOP Attention: This is a highly sensitive instrument. Read the accompanying user's manual before using. When you use the instrument, follow generally accepted procedures for quality control and methods development. If you observe a change in the retention of a particular compound, in the resolution between two compounds, or in peak shape, immediately take steps to determine the reason for the changes. Until you determine the cause of a change, do not rely upon the results of the separations. Caution: Use caution when working with any polymer tubing under pressure. Always wear eye protection when in proximity to pressurized polymer tubing. Extinguish all nearby flames. Do not use Tefzel tubing that has been severely stressed or kinked. Do not use Tefzel tubing with tetrahydrofuran (THF) or concentrated nitric or sulfuric acids. Be aware that methylene chloride and dimethyl sulfoxide cause Tefzel tubing to swell and greatly reduce the rupture pressure of the tubing. Caution: For continued protection against fire hazard, replace fuses with those of the same type and rating. STOP Attention: Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment. STOP Attention: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy, and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference, in which case you must correct the interference at your own expense. Shielded cables must be used with this unit to ensure compliance with Class A FCC limits.

4 Canadian Emissions Notice This digital apparatus does not exceed the Class A limits for radio noise emissions from digital apparatus set forth in the Radio Interference Regulations of the Canadian Department of Communications. Le présent appareil numérique n émet pas de bruits radioélectriques dépassant les limites applicables aux appareils numériques de la classe A prescrites dans les règlements sur le brouillage radioélectrique édictés par le Ministère des Communications du Canada. Symbols Used on the Waters Gel Permeation Chromatography Cleanup System Direct current Alternating current Protective conductor terminal Frame or chassis terminal Caution, risk of electric shock (high voltage) Caution or refer to manual Caution, hot surface or high temperature

5 Table of Contents How to Use This Guide Chapter 1 Introduction GPC Overview GPC Cleanup GPC System Components Waters GPC Cleanup System Waters 515 HPLC Pump Waters 717plus Autosampler Waters Envirogel GPC Cleanup Columns Waters 2487 Dual l Absorbance Detector Waters Fraction Collector II Chapter 2 Installing and Preparing the System Installation Site Requirements Solvent Preparation Installation Changing the 717plus Autosampler Syringe Columns Making Fluidic Connections Connecting Solvent Reservoir to Pump Connecting Pump to Autosampler Table of Contents 5

6 2.6.3 Connecting Autosampler to Detector Connecting Detector to Fraction Collector II Flushing the System Installing the Columns Column Connections and Equilibration Chapter 3 Calibrating the System Precalibration System Check Preparing Calibration Solution Calibrating Columns and Setting Fraction Collection Times Performing a 5-mL Injection Determining Peak Resolution Programming Collection Times Adjusting Collection Times Sample Cleanup Guidelines Sample Preparation Sample Volume Sample Load Equipment Considerations Recalibration Table of Contents

7 Chapter 4 Troubleshooting and Maintenance Troubleshooting Maintenance and Storage Short-Term Storage Procedures Long-Term Storage Procedures Appendix A Warranty Information and Spare Parts A.1 Warranty Information A.2 Spare Parts Index Table of Contents 7

8 List of Figures 1-1 Components of a GPC System Sample Chromatogram Waters 515 HPLC Pump Waters 717plus Autosampler Waters Envirogel GPC Cleanup Columns Waters 2487 Dual l Absorbance Detector Waters Fraction Collector II Suggested System Setup Removing the Knurled Nut Installing the Syringe Column Installation Solvent-Reservoir-to-Pump Connection HPLC Pump to 717plus Autosampler Connection plus Autosampler to 2487 Detector Connection Detector to Fraction Collector II Connection plus Autosampler to Column Connection Sample Calibration Chromatogram Peak Resolution Table of Contents 8

9 List of Tables 2-1 Component Installation References Required Concentrations of Calibration Solution Compounds Troubleshooting A-1 System Parts A HPLC Pump Parts A-3 717plus Autosampler Parts A-4 Envirogel Columns A Dual l Absorbance Detector Parts A-6 Fraction Collector II Parts A-7 Disposables from Waters A-8 Disposables from ESS Table of Contents 9

10 How to Use This Guide Purpose of This Guide The Waters Gel Permeation Chromatography Cleanup System Operator s Guide describes how to unpack, install, operate, maintain, and troubleshoot the Waters Gel Permeation Chromatography (GPC) Cleanup System. It also includes appendixes for spare parts, warranty and service information, and care and use of Envirogel columns. Audience This guide is intended for individuals who need to install, operate, maintain, and/or troubleshoot the Waters GPC Cleanup System. Structure of This Guide The Waters Gel Permeation Chromatography Cleanup System Operator s Guide is divided into four chapters and an appendix. Each chapter and appendix page is marked with a tab and a footer to help you access information within the chapter. The table below describes the material covered in each chapter and appendix of this guide. Chapter/Appendix Chapter 1, Introduction Chapter 2, Installing and Preparing the System Chapter 3, Calibrating the System Chapter 4, Troubleshooting and Maintenance Appendix A, Warranty Information and Spare Parts Content Describes the Waters GPC Cleanup System, including features and options. Describes how to unpack and install the Waters GPC Cleanup System. Describes how to calibrate the Waters GPC Cleanup System. Describes troubleshooting and maintenance procedures for the Waters GPC Cleanup System. Provides warranty and service information, as well as a list of recommended and optional spare parts. 10 How to Use This Guide

11 Related Documentation The following table lists related documentation. Title Waters 515 HPLC Pump Operator s Guide Waters 717plus Autosampler Operator s Manual Waters 2487 Dual λ Absorbance Detector Operator s Guide Part Number WAT068980TP WAT173-02TP WAT048740TP Waters Fraction Collector II Operator s Guide Related Adobe Acrobat Reader Documentation For detailed information about using the Adobe Acrobat Reader, refer to the Adobe Acrobat Reader Online Guide. This Online Guide covers procedures such as viewing, navigating and printing electronic documentation from Adobe Acrobat Reader. Printing From This Electronic Document Adobe Acrobat Reader lets you easily print pages, pages ranges, or the entire electronic document by selecting Print from the File menu. For optimum print quantity, Waters recommends that you specify a Postscript printer driver for your printer. Ideally, use a printer that supports 600 dpi print resolution. How To Use This Guide 11

12 Conventions Used in This Guide This guide uses the following conventions to make text easier to understand. Purple Text indicates user action. For example: Press 0, then press Enter for the remaining fields. Italic text denotes new or important words, and is also used for emphasis. For example: An instrument method tells the software how to acquire data. Underlined, Blue Color text indicates hypertext cross-references to a specific chapter, section, subsection, or sidehead. Clicking this topic using the hand symbol automatically brings you to this topic within the electronic document. Right-clicking and selecting Go Back from the popup context menu brings you back to the originating topic. For example: Replace the standard 250-µL syringe in the 717plus Autosampler with this larger volume syringe, as described in Section 2.4, Changing the 717plus Autosampler Syringe. Notes, Attentions, and Cautions Notes call out information that is important to the operator. For example: Note: Record your results before you proceed to the next step. Attentions provide information about preventing possible damage to the system or equipment. For example: STOP Attention: To avoid damaging the detector flow cell, do not touch the flow cell window. Cautions provide information essential to the safety of the operator. For example: Caution: To avoid chemical or electrical hazards, always observe safe laboratory practices when operating the system. Caution: To avoid the possibility of electrical shock and possible injury, always turn off the detector and unplug the power cord before performing maintenance procedures. Caution: To avoid the possibility of burns, turn off the lamp at least 30 minutes before removing it for replacement or adjustment. 12 How to Use This Guide

13 1 Introduction GPC Overview The Waters Gel Permeation Chromatography (GPC) Cleanup System is a high-performance liquid chromatography (HPLC) system designed to perform cleanup of environmental samples as required by EPA Method 3640A, GPC Cleanup, and the U.S. EPA Contract Laboratory Program Statement of Work for Organic Analysis, Document OLMO.O GPC Cleanup Theory Gel permeation chromatography (GPC) separates molecules according to their size in solution. A GPC column contains a highly-porous, polymeric gel packing material. The size of the pores and the pore size distribution determine the molecular range of separation. Because small molecules enter and reside in a larger percentage of the gel pores than do large molecules, the small molecules take proportionally longer to elute from the gel. The resulting column eluent contains the component molecules sorted by size, with largest molecules eluting first, medium-sized molecules slightly later, and the smallest molecules eluting last. This sorting mechanism makes GPC an ideal method for removing undesired components in a sample, while retaining and collecting desired components. GPC Cleanup Process Sample cleanup by GPC removes non-volatile, high-molecular-weight materials, such as lipids, proteins, polymers, and natural resins from environmental samples. These materials foul the injection port area and columns of the gas chromatographs used to detect priority pollutants and pesticides in environmental samples. EPA Method 3640A, GPC Cleanup, and the Contract Laboratory Program Statement of Work for Organic Analysis require that all environmental samples be processed by gel permeation chromatography before analysis by gas chromatography. GPC Overview 13

14 1.1.2 GPC System Components 1 The basic components of a GPC system (Figure 1-1) are: System pump Injector or autosampler Column Detector Fraction collector Data collection device Waters 717plus Autosampler Data Collection 515 HPLC Pump Solvent Reservoir To Waste or Fraction Collector TP01622A Figure 1-1 Components of a GPC System 14 Introduction

15 Pump The system pump delivers the mobile phase from the solvent reservoir to the system at a constant flow rate against backpressures of up to 6000 psi. Injector Sample is introduced into the system through the injector. The injector contains a small length of tubing, called a sample loop. The sample loop holds a fixed or variable volume of solution for delivery to the column. The injection volume is determined by the sample loop size or by the volume loaded into the loop. 1 When the injector is in the LOAD position, the sample loop is disconnected from the flow path, so that mobile phase bypasses the sample loop and flows directly to the column. Sample is drawn into the sample loop through a port in the syringe needle. When the injector valve is in the inject position, mobile phase flows through the sample loop and washes the sample loop contents to the column. Column When sample reaches the column, the sample molecules flow around the packing material. Smaller sample molecules diffuse in and out of the pores in the packing material. Larger molecules may not enter the pores at all and elute from the column first. Detector Column eluent is monitored by a detector. The detector identifies eluent components by monitoring changes in concentration of solute in the eluent. The type of detector to use depends upon the nature and amount of the compound being detected. For example, refractive index (RI) detectors monitor the difference in refractive index of the mobile phase versus the refractive index of the mobile phase and solute. Ultraviolet (UV) wavelength detectors monitor the variation in UV absorbance caused by the presence of a solute. GPC Overview 15

16 Data Collection 1 The output signal from the detector is connected to a data collection device, such as a strip chart recorder, data integrator, or other data system. The data collection device creates a graphic recording of the output signal, called a chromatogram (see Figure 1-2). Detector Response Peak 1 Peak 2 Baseline Time Figure 1-2 Sample Chromatogram Each compound in the chromatogram is represented by a peak. The amount or concentration of each compound can be calculated from the area or height of its peak. Because molecules of the same size elute at the same time, you can compare chromatograms of compounds of known molecular weights (standards) to unknown compounds to identify the unknown components. Fraction Collector The fraction collector captures portions of the column eluent needed for further analysis. The fractions are fed to collection vessels, while the undesired components are diverted to waste. 16 Introduction

17 1.2 Waters GPC Cleanup System The Waters GPC Cleanup System consists of: Waters 515 HPLC Pump Waters 717plus Autosampler Two Waters Envirogel GPC Cleanup Columns (19mm 150mm and 19mm 300mm) Waters 2487 Dual λ Absorbance Detector Waters Fraction Collector II Waters 515 HPLC Pump The Waters 515 HPLC Pump (Figure 1-3) delivers mobile phase at precise flow rates from to 10.0 ml/min in increments of ml/min at backpressures of up to 6000 psig. TP01407 Figure 1-3 Waters 515 HPLC Pump Waters GPC Cleanup System 17

18 Enter Clear Releas e Engag e Waters 717plus Autosampler 1 The Waters 717plus Autosampler (Figure 1-4) is an automated, multiple-sample, injection device. Waters 717plus Autosampler Hom e CAROUSE L 0 I 0 Figure 1-4 Waters 717plus Autosampler The 717plus Autosampler features: Injection volumes from 0.1-µL to 5000-µL (with auxilary 5-ml sample loop and 2500-µL syringe installed), in 0.1-µL increments. Two carousel sizes (48-vial and 96-vial), as well as a variety of sample vials. Multi-method automatic sampling, allowing you to program up to 96 steps, with each step containing a different sampling method. Priority (stat) sampling, which interrupts a programmed sampling method, performs the priority injections, and then continues autosampling from the interruption point. Automated options that allow you to inject a common solution with each sample, run standards automatically during a run, or transfer, mix, and inject solutions from multiple vials. Interaction with other LC Modules, allowing the Waters 717plus to control external devices during a run. IEE-488 and RS-232 Communication. Purge and compression testing capability. Needle Wash System, which prevents cross-contamination of samples and extends seal life. 18 Introduction

19 Lot No. Mxxxxxxx Lot No. Mxxxxxxx Semiprep Chromatography support, using an optional 2000-µL sample loop and 2500-µL syringe. Microbore Chromatography support, using an optional 25-µL syringe and low volume sample vials. Adjustable syringe speed to adjust for sample viscosity. Sample Heater/Cooler to maintain a constant sample compartment temperature from 4 to 40 C. For more information on the features of the Waters 717plus Autosampler, refer to the Waters 717plus Autosampler Operator s Manual Waters Envirogel GPC Cleanup Columns The cleanup system uses two Waters Envirogel GPC Cleanup Columns: 19 mm 150 mm, and 19 mm 300 mm (Figure 1-5). The Envirogel columns are high-efficiency, high-resolution columns that contain 100-Å-pore-size material, with a nominal particle size of 10 µm. The columns are shipped with methylene chloride as the mobile phase. Envirogel GPC Clean-Up column 19x150mm Part No FLOW Waters Envirogel GPC Clean-Up column 19x300mm Part No FLOW Waters Figure 1-5 Waters Envirogel GPC Cleanup Columns Waters GPC Cleanup System 19

20 1.2.4 Waters 2487 Dual λ Absorbance Detector 1 The Waters 2487 Dual λ Absorbance Detector (Figure 1-6) monitors the ultraviolet absorbance of the column eluent stream to detect the presence of sample molecules. The 2487 detector operates at wavelengths from 190 nm to 700 nm. For the GPC Cleanup operation, the detector uses a 3-mm path length (semiprep) flow cell. Waters 2487 Dual l Absorbance Detector TP01497 Figure 1-6 Waters 2487 Dual λ Absorbance Detector 20 Introduction

21 1.2.5 Waters Fraction Collector II The Waters Fraction Collector II (Figure 1-7) can collect fractions ranging in volume from 1 µl to more than 1 L. The fraction collector can be programmed to collect specific peaks or portions of peaks, and to divert unwanted peaks to waste. 1 TP01620 Figure 1-7 Waters Fraction Collector II Waters GPC Cleanup System 21

22 2 Installing and Preparing the System 2.1 Installation Site Requirements This chapter describes how to install your Waters GPC Cleanup System for operation. Each component is shipped with its own manual. Refer to these manuals for unpacking instructions and installation requirements. 2 Environmental Requirements The GPC Cleanup System requires: Approximately 6 linear feet ( 1.8 m) of bench space A shelf or rack above the system capable of holding 4 L of solvent Constant laboratory temperatures (72 F, 22 C) to prevent shifting of retention times and outgassing of methylene chloride. Caution: Be sure that the chosen area is well ventilated and complies with applicable safety codes. Electrical Requirements The GPC Cleanup System requires a minimum of five grounded receptacles on a 115 V, 20 A, dedicated line. Installation Site Requirements 22

23 2.2 Solvent Preparation The cleanup procedure requires a supply of HPLC-grade methylene chloride. STOP Attention: Always observe safe laboratory practices when handling solvents. Know the chemical and physical properties of solvents. Refer to the Materials Safety Data Sheets for the solvents in use. 2 STOP Attention: Envirogel columns can be permanently damaged if they contact unsuitable solvents. The only mobile phases suitable for use with these columns are methylene chloride or a 50:50 mixture of methylene chloride and cyclohexane. All residues of other solvents must be thoroughly washed from the system prior to installation of the columns. To check the acid level of the methylene chloride: 1. Shake together equal portions of methylene chloride and Milli-Q grade (18 Megohm) water. 2. Check the ph of the water layer using ph paper or other suitable methods. 3. If the ph of the water layer is 5, filter the entire supply of methylene chloride through a 2-inch 15-inch glass column containing activated basic alumina. Be sure that an adequate volume of methylene chloride is available for calibration and sample cleanup operations. At a flow rate of 5.0 ml/min, the GPC Cleanup System uses 0.30 L of methylene chloride per hour. Two cleanup or calibration procedures can be performed in one hour. 23 Installing and Preparing the System

24 2.3 Installation Unpack and install the system components as described in the individual component operators manuals. Included in your Waters GPC Cleanup System are: Viton tubing Use this tubing for the drain tube on the fraction collector. Viton tubing resists solvent degradation by methylene chloride µL syringe Replace the standard 250-µL syringe in the 717plus Autosampler with this larger volume syringe, as described in Section 2.4, Changing the 717plus Autosampler Syringe. Installation of the cleanup system involves: Positioning the components (Figure 2-1) Making power and signal connections Making fluidic connections Figure 2-1 shows the placement of components on a bench. This configuration minimizes tubing lengths between the components and, therefore, decreases bandspreading. 2 Waters 717plus Autosampler Waters 2487 Dual l Absorbance Detector 515 HPLC Pump TP01623 Figure 2-1 Suggested System Setup Installation 24

25 Installation Guidelines Make electrical connections as specified in the component manuals, but do not connect the fluidic lines. After you make the required electrical connections, power on each module for at least 30 minutes to stabilize all electronics before continuing with the fluidic connections. 2 Observe the following guidelines when setting up the system: The Waters HPLC modules described in this guide are strongly recommended for this application. If you use alternative modules, set them up in a similar way using their operator s manuals. Check that your autosampler includes the optional 2500-µL syringe and the fraction collector has the optional 3-way (diverter) valve. Check that the draw rate on the injector is set to the minimum rate to prevent cavitation of the methylene chloride. Make sure the autosampler has a sample loop of adequate size. A second, large-volume loop is required if 5-mL samples are to be injected onto the large columns used in the EPA methods. Remove this second loop when injection volumes of no greater than 2 ml are used. Use 0.23-mm (0.009-inch) tubing between the injector and the detector inlet. Waters recommends 0.23-mm tubing because high backpressure will not cause flow rate problems in this system, and because loss of resolution can occur with larger diameter tubing. In Waters absorbance detectors, use a semi-preparative (3-mm path length) flow cell to reduce the magnitude of the detector signal. With an analytical (10-mm) flow cell, detector signals can result in readings above 2.0AU when used for the GPC cleanup method. Note: Refer to Table 2-1 for references about and modifications to the installation procedures for each system component. Refer to the How to Use This Guide section at the beginning of this manual for editions of the specified manuals. 25 Installing and Preparing the System

26 Table 2-1 Component Installation References Component Reference Modifications Waters 515 HPLC Pump Waters 515 HPLC Pump Operator s Guide (WAT068980TP), Chapter 2, Installing the Waters 515 Pump Do not prime the pump with methanol. Waters 717plus Autosampler Waters 717plus Autosampler Operator s Manual (WAT173-02TP), Chapter 2, Installation Install the large sample loop in the autosampler as described in Section 2.6.3, Installing the Large Sample Loop. 2 Do not connect the autosampler to the column. Install the 48-vial carriage. Waters 2487 Dual λ Absorbance Detector Waters 2487 Dual λ Absorbance Detector Operator s Guide (WAT048740TP), Chapter 2, Installing the 2487 Detector In Section 2.4, Making Fluidic Connections, assemble the tubing and fittings for the column, but do not connect tubing to the column. Install the 3-mm (semiprep) cell. Waters Fraction Collector II Waters Fraction Collector II Operator s Guide ( ), Chapter 2, Installing the Waters Fraction Collector II In Section 2.5.2, Connecting the Drain Tubing, install the Viton tubing at the drain joint. Follow Section 2.8.1, Installing an Optional 3-Way Valve, to install the diverter valve. Select BOTTLE POSITION mode to collect large-volume fractions. The collection time for peaks is set during column calibration. Installation 26

27 For data collection, refer to the specific installation instructions for the chosen analog or digital data collection device, and also refer to: The Waters 2487 Dual λ Absorbance Detector Operator s Manual, Chapter 2, Installing the 2487 Detector, for connections to the detector. The Waters 717plus Autosampler Operator s Manual, Chapter 2, Installation, for connections to the autosampler. 2.4 Changing the 717plus Autosampler Syringe 2 The 717plus Autosampler is shipped with a 250-µL syringe, which injects sample volumes of 1 µl to 150 µl. To inject larger sample volumes, replace the syringe with the 2500-µL syringe. The 2500-µL syringe injects sample volumes of 150 µl to 2.5 ml. With the 2500-µµL syringe and the Auto Addition routine in the 717plus software, you can inject a sample volume of up to 5.0 ml. STOP Attention: Use only Waters-approved syringes in the autosampler (see Appendix A, Warranty Information and Spare Parts, for ordering information). Unapproved syringes may cause erroneous results and/or may not meet required safety standards. Syringe replacement involves: Removing the installed syringe Installing the new syringe Purging the syringe Modifying configuration parameters Removing the Syringe To remove the syringe: 1. Set pump flow to 0 ml/min. 2. Remove the syringe compartment plastic cover by lifting up. 3. Remove the knurled nut that holds the plunger to the syringe guide mounting bracket (Figure 2-2). 4. From the Main Page on the 717plus keypad, press Diag Page. The Diagnostic Menu appears. 5. Press UserMaint Page. The User Maintenance Menu appears. 6. Press ValveMot Page. The Valve and Motor Operations page appears. 7. Select the Open entry in the Valve 3 field, then press Enter. 27 Installing and Preparing the System

28 8. Select the Fill entry in the Syringe field, then press Enter. The mounting bracket is lowered while the plunger tip remains up. 9. When the mounting bracket is fully lowered, remove the syringe by grasping the syringe barrel near the mounting block and turning the syringe counterclockwise until it is released from the mounting block. 2 Figure 2-2 Removing the Knurled Nut Changing the 717plus Autosampler Syringe 28

29 Installing the New Syringe To install the new syringe: 1. Screw the new syringe assembly partially into the mounting block (Figure 2-3) Do not tighten. 2 Figure 2-3 Installing the Syringe 2. While maintaining the alignment of the plunger with the syringe, slide the plunger down so that the threaded end slides through the syringe guide mounting bracket. 3. Tighten the syringe hand-tight. 4. From the 717plus keypad, select Empty in the Syringe field, then press Enter. The mounting block moves up. 5. Install the knurled nut. 29 Installing and Preparing the System

30 6. Move the screen arrow to the To Execute Normal State Press Enter field, then press Enter. This resets the Valve and Motor Operations screen to the original state. 7. Return to the Main menu by pressing UserMaint Page, Diag Page, then Main Page. Purging the Syringe To purge the syringe. Note: Purge the autosampler a minimum of three times to ensure that no bubbles remain in the syringe. 1. Set the pump flow to 1 ml/min. 2. From the 717plus Main Menu, press Purge Page. 3. Press Start Purge. 4. Repeat the purge at least two additional times. 5. If bubbles are present in the syringe after three purges, manually purge the syringe. See the Waters 717 Plus Autosampler Operator s Manual, Chapter 6, Troubleshooting, for more information. 2 Modifying Configuration Parameters If the size of the new syringe is different from that of the syringe you are replacing, you must change the syringe volume on Configuration Page 3. To modify the syringe volume parameters: 1. From the 717plus Main menu, press Config Page. The Configuration Page 1 menu appears. 2. Press NextCfg Page. The Configuration Page 2 menu appears. 3. Press NextCfg Page. The Configuration Page 3 menu appears. 4. Select the appropriate volume in the Syringe Volume field, then press Enter. Note: Compressibility test results (system messages 6300 and 6400) are not valid when performed with a 2500-µL syringe. Perform this test only with the standard 250-µL syringe. Changing the 717plus Autosampler Syringe 30

31 2.5 Columns The GPC Cleanup System uses two Envirogel columns, 19 mm 150 mm and 19 mm 300 mm, connected in series. The two columns provide the range of molecular separation required for the cleanup procedure. Column Care and Use 2 STOP Refer to the Waters Envirogel GPC Cleanup Column Care and Use Manual for column care and use guidelines. Connecting the Columns Attention: Incorrect installation can permanently impair the performance of Envirogel columns. Read Section 2.6.5, Flushing the System, and Section 2.6.6, Installing the Columns, before you connect the columns. To connect the columns in series: 1. Remove the end plugs from the columns and save them for use when you store the columns. 2. Ensure that solvent flows in the direction shown by the arrows on the column labels. The direction of flow is from the smaller column to the larger column. 3. Place a ring stand or other suitable column-mounting device between the 717plus Autosampler and the detector. Mount the columns so that sample flows into the columns, as shown in Figure Installing and Preparing the System

32 Ring Stand Envirogel CPC Clean-Up column 19x150mm Part No Lot No. Mxxxxxxx FLOW Waters Division of Millipore Envirogel CPC Clean-Up column 19x300mm Part No Lot No. Mxxxxxxx FLOW Waters Division of Millipore 2 Figure 2-4 Column Installation 4. Connect the outlet of the smaller column to the inlet of the larger column, using the inch ID, U-shaped, column-joining tube. Note: At this time, do not connect the columns to the autosampler or the detector. Make these connections after you purge the system. See Section 2.6.5, Flushing the System. 5. Proceed to Section 2.6, Making Fluidic Connections. Columns 32

33 2.6 Making Fluidic Connections 2 Preparing the fluidic path involves: Connecting the solvent reservoir to the pump (Section 2.6.1) Connecting the pump to the 717plus autosampler (Section 2.6.2) Connecting the 717plus autosampler to the detector (Section 2.6.3) Connecting the detector to the fraction collector (Section 2.6.4) Flushing the system (Section 2.6.5) Installing the columns (Section 2.6.6) Equilibrating the columns (Section 2.6.7) Note: Before continuing with this installation, power on each module for at least 30 minutes to stabilize all electronics. Required Materials 10-mL priming syringe (Startup Kit) 5/16-inch open-end wrench (Startup Kit) 1/8-inch Teflon tubing 1/16-inch Teflon tubing Waste container 33 Installing and Preparing the System

34 2.6.1 Connecting Solvent Reservoir to Pump To make the fluidic connection between the 515 HPLC pump and the 717plus Autosampler, use the 1/8-inch Teflon tubing to connect the solvent reservoir to the solvent inlet tube on the front of the pump (Figure 2-5). 1/8-inch Teflon Tubing 515 HPLC Pump Methanol 2 Solvent Inlet TP01624A Figure 2-5 Solvent-Reservoir-to-Pump Connection Making Fluidic Connections 34

35 2.6.2 Connecting Pump to Autosampler To connect the 515 HPLC Pump to the 717plus Autosampler: 1. Connect the solvent outlet connection on the front of the pump to the blue (0.040-inch ID) tubing from the 717plus Autosampler (Figure 2-6). Waters 717plus Autosampler HPLC Pump Blue (0.040-inch) Tubing Red (0.009-inch) Tubing Waste Flask Solvent Outlet Figure HPLC Pump to 717plus Autosampler Connection TP Place the Red (0.009-inch ID) tubing from the 717plus Autosampler into a waste container. 3. Fill the solvent reservoir with methanol. 4. If the pump already holds solvent, press the Up, Down, or Menu key to display the Flow parameter, then set the pump Flow parameter to ml/min. If the pump is dry, however, start with a lower Flow rate such as ml/min. a. Press Edit/Enter until the blinking cursor is positioned over the digit that you need to edit. b. Use the Up or Down key to increase or decrease the value of the digit. c. If necessary, repeat steps a and b until the Flow parameter value is correct. d. Press Menu to store the parameter value. Note: Do not press Run/Stop to start the pump at this point in the procedure. 35 Installing and Preparing the System

36 5. Fully depress the priming syringe plunger to remove all air from the syringe, then insert the syringe into the Luer fitting at the center of the draw-off valve handle 6. Turn the draw-off valve handle counterclockwise about 1/2 turn to open the valve. 7. Withdraw several milliliters of solvent with the syringe, then close the valve handle and remove the syringe. Expel the contents of the syringe into a suitable waste container. 8. Press Run/Stop to start the pump. 9. Let the pump run for about a minute, then verify normal pump pressure for the selected solvent and flow rate. If the pump pressure is not correct, repeat steps 6 through 10 as necessary. 10. Once the correct pump pressure is achieved, allow the flow to continue for approximately 5 minutes to flush the flow paths in the pump and injector. 11. Stop the flow by pressing Run/Stop, then proceed with the connections between the autosampler and detector. 2 Making Fluidic Connections 36

37 2.6.3 Connecting Autosampler to Detector Note: This is a temporary connection used to purge the system. Install columns between the autosampler and the detector after you purge the system. To connect the 717plus autosampler to the 2487 detector: 1. Connect the Red (0.009-inch ID) tubing from the 717plus autosampler to the sample inlet tube on the detector (Figure 2-7) using a union connector (P/N 97332). Tighten the fitting with the wrench 1/4-turn beyond hand-tight. 2 Waters 717plus Autosampler Red (0.009-inch) Tubing Figure plus Autosampler to 2487 Detector Connection TP01621A 2. Place a waste container at the detector outlet tube. 3. Set the pump flow rate to 5.0 ml/min. 4. Flush the detector flow path for 5 minutes. 5. Stop the flow, then proceed with the connection to the fraction collector. 37 Installing and Preparing the System

38 2.6.4 Connecting Detector to Fraction Collector II 1 To connect the 2487 detector to the Waters Fraction Collector II: 1. Remove the tubing connected to the detector sample cell outlet port. 2. Cut off the end of the fraction collector inlet tubing. 3. Connect the 1/16-inch Teflon tubing from the 3-way (diverter) valve on the fraction collector to the sample cell outlet port on the detector using a stainless steel union and compression screw (Figure 2-8). Hand-tighten only to avoid crushing the tubing. Fraction Collector II 1/16-inch Teflon Tubing Detector 3-Way Valve Drain Tube Waste Container TP01626 Figure 2-8 Detector to Fraction Collector II Connection 4. Be sure that the drain tube on the 3-way valve is connected to a waste container. Set the flow rate to 5 ml/min, then flush the line for 5 minutes. 5. Check all fluidic connections for leaks, then tighten the fittings as necessary. 38 Installing and Preparing the System

39 2.6.5 Flushing the System Envirogel columns are packed with particles of styrene divinyl benzene in methylene chloride. These particles can shrink or swell if solvents of significantly different polarity are passed through the column. This shrinking or swelling destroys the efficiency of the columns. 1 STOP The only solvents guaranteed to provide proper performance of Envirogel columns are methylene chloride or a 50:50 mixture of methylene chloride and cyclohexane, as specified in the AOAC and EPA methods. Water, alcohols, ketones, or alkanes must not be used as solvents. Because Waters HPLC systems are stored and shipped in methanol, you must flush the system with methylene chloride before the columns are installed, to remove the methanol from the system. Required Materials Methylene chloride Column union 0.23-mm stainless steel tubing. (startup kit) To flush the system: Attention: Ensure that the columns are not connected to the system before performing this procedure. 1. Join the inlet and outlet connectors for the columns with a union. 2. Prime the 717plus Autosampler needle-wash system with methylene chloride according to the directions in the Waters 717plus Autosampler Operator s Manual. 3. Prime the solvent delivery system with methanol and begin pumping methanol at 5 ml/min through the system (without the columns). 4. Set the 2500-µL syringe to its lowest draw rate and purge the system with methanol. Make sure the 717plus autosampler goes through at least one complete purge cycle. 5. During the purge procedure, check that the system plumbing (without the columns in place) provides enough backpressure to force sufficient flushing solvent through the lines. If the volume of flushing solvent is insufficient, you can add a 3-m (10-foot) length of 0.23-mm (0.009) ID stainless steel tubing to the system, in the location normally occupied by the columns. Making Fluidic Connections 39

40 1 6. When the methanol purge is finished, begin pumping methylene chloride through the system at 5 ml per minute. 7. Purge the 717plus autosampler with methylene chloride. Once this second purge is finished, the system is properly flushed with methylene chloride. Stop the flow and proceed with column installation Installing the Columns Waters strongly recommends installing a Waters Guard Column (Part No ) just before the first column. The Guard Column traps any particulate matter in the samples, preventing column contamination and the blocking of the inlet frit of the first column. Note: Use of the Guard Column is a precautionary measure and is not an alternative to proper sample filtration. Install the shorter (15-cm) column first (Figure 2-9). This provides some separation of analytes and also acts as a large guard column for the main (30-cm) column. Begin the initial washing of the columns as described in the Envirogel Care and Use Manual. The flow rate for Envirogel columns is 5.0 ml/minute of methylene chloride. Degas the mobile phase by the usual means, such as vacuum filtration, just before use and at least once per day during subsequent use. STOP Attention: Do not exceed a flow rate of 6.0 ml/minute. Make changes in the flow rate gradually. If you adjust the flow rate manually, increase or decrease the rate in steps of no more than 1 ml/min. Wait at least 10 seconds between each change in flow rate. 40 Installing and Preparing the System

41 2.6.7 Column Connections and Equilibration Column Connections To connect the columns to the Cleanup System: 1. Disconnect the Red tubing (between the autosampler and the detector) from the sample inlet of the detector. 2. Connect the free end of the Red tubing to the column inlet of the 15-cm column, or to the guard column (Figure 2-9). Then, using a wrench, tighten the connection 1/4 to 1/2-turn beyond hand-tight. 1 Autosampler Waters 717plus Autosampler Ring Stand Waste Container TP01627 Figure plus Autosampler to Column Connection 3. Cut a length of tubing to connect the outlet of the second column to a waste container. Connect one end of the tubing to the column outlet, and the other end to a waste collection container. 4. Set the pump flow rate to 1 ml/min, then collect column effluent for 1 minute. 5. Increase the flow rate in 1 ml/min increments for at least 1 minute at each flow rate until the flow rate is 5 ml/min. 6. When the flow rate is 5 ml/min, collect effluent for 20 minutes (100 ml of solvent). Making Fluidic Connections 41

42 1 7. Stop the flow and allow the system pressure to return to 0 psi (approximately 30 seconds). 8. Connect the outlet tubing of the second column to the inlet of the detector. Column Equilibration To equilibrate the columns: 1. Resume flow as in steps 4 and 5 above. 2. Adjust the absorbance level to zero by pressing Auto Zero on the detector. 3. Monitor the UV absorbance on the detector to ensure that the column is fully equilibrated. At a flow rate of 5 ml/min, full-column equilibration can take up to 20 minutes. During this time check that no large fluctuations occur in pump pressure (as indicated by the pump pressure meter). Typical pump pressures are approximately 200 psi at flow rates of 5 ml/min. 4. Continue the flow until you obtain a stable absorbance reading. The columns are equilibrated when the change in the absorbance reading is AU after 500 ml of solvent have passed through the column. 5. Press Auto Zero again, if necessary. The system is now fully equilibrated. Continue with Chapter 3, Calibrating the System. 42 Installing and Preparing the System

43 3 Calibrating the System 3.1 Precalibration System Check Prior to calibrating the GPC Cleanup System: Fill the solvent supply reservoir with methylene chloride. At a flow rate of 5 ml/min, the cleanup system uses 0.30 liters of solvent per hour of operation. Check the pressure reading on the pump pressure meter. At a flow rate of 5mL/min, a stable system with both columns installed has less than 200 psi. Record the normal operating pressure for future reference. Check the flow rate at the fraction collector outlet by collecting solvent in a graduated cylinder for a set amount of time. For example, after 10 minutes at a flow rate of 5 ml/min, verify that 50 ml of solvent was collected. Check the UV absorbance reading. Monitor the baseline on a data collection device for 10 to 20 minutes to observe for drift or component elution from the column. Check that the capacity of the waste container is sufficient to contain the anticipated solvent volume. Place collection tubes in the fraction collection rack and position the rack under the 3-way valve. When using Envirogel columns, use 28-mm scintillation vials, or 125-mL Erlenmeyer screw-top flasks in the appropriate rack. See Appendix A, Warranty Information and Spare Parts, for part numbers. 3 Precalibration System Check 43

44 3.2 Preparing Calibration Solution STOP A calibration solution consists of compounds of known amounts dissolved in a known total volume of solvent. Because the Waters GPC Cleanup System performs either 2-mL or 5-mL sample injections, the concentrations of compounds in the calibration standard must be adjusted accordingly. For example, the compounds in a calibration solution appropriate for a 2-mL injection must be 2.5 times the concentration of the same compounds in a calibration solution used for 5-mL injections. Prepare the calibration solution by mixing the calibration compounds into low-acid methylene chloride. Refer to Section 2.2, Solvent Preparation, for the procedures to determine and adjust the acid level of methylene chloride. Attention: Always observe safe laboratory practices when handling solvents. Know the chemical and physical properties of solvents. Refer to the Materials Safety Data Sheets for the solvents in use. 3 The concentration of each compound in the calibration solution depends on the volume of calibration solution you inject. Table 3-1 shows the concentrations of compounds required for 2-mL and 5-mL injection volumes. Table 3-1 Required Concentrations of Calibration Solution Compounds Compound Required Concentration (mg/l) For 2-mL Injection For 5-mL Injection Corn oil 62,500 25,000 Bis(2-ethylhexyl)phthalate Methoxychlor Perylene Sulfur Calibrating the System

45 To prepare the calibration solution: 1. Select a volumetric flask whose total volume is the volume of calibration solution you want to prepare. 2. Calculate the amount of each compound you need to dissolve in the total volume of the calibration solution (refer to Table 3-1). 3. Dissolve the calculated amount of elemental sulfer in the appropriate amount of corn oil. Warm the mixture until all the sulfur dissolves. 4. Add the remaining compounds listed in Table 3-1 to the volumetric flask. 5. Add the warm corn oil-sulfur mixture to the volumetric flask. 6. Add methylene chloride to bring the solution to full volume. 7. Store the calibration solution away from light, at 4 C in an amber bottle with a Teflon-lined screw-cap. The solution can be stored up to 6 months. After storage, allow the solution to stand at room temperature until components redissolve. Note: A small amount of sulfur may precipitate from the solution when the methylene chloride is added or after the solution is stored. Remove this precipitate by filtering the calibration solution through a 0.45-µm Gelman Acrodisc CR PTFE filter before injecting the calibration solution. (Refer to Appendix A, Warranty Information and Spare Parts, for filter part numbers.) 3 Preparing Calibration Solution 45

46 3.3 Calibrating Columns and Setting Fraction Collection Times Calibrating the columns and setting fraction collection times involves: Performing an injection (Section 3.3.1) Observing peak resolution (Section 3.3.2) Determining retention times (Section 3.3.3) Adjusting fraction collection times (Section 3.3.4) Performing a 5-mL Injection 3 Before you begin, be sure that: All components are powered on The columns are equilibrated as specified in Section 2.6.7, Column Connections and Equilibration The UV detector is set to 254 nm. The fraction collector is set to send the flow to waste. To set up the cleanup system for an injection: Configure the Autosampler Hardware 1. Install the 2500-µL syringe (WAT077342). Refer to the Waters 717plus Autosampler Operator s Guide, Chapter 5, Maintenance, for instructions. 2. Check that the draw rate on the injector is set is set to the minimum rate. 3. Install the 5-mL sample loop (WAT036212). Refer to the Waters 717plus Autosampler Operator s Guide, Chapter 2, Installation, for instructions. Prepare the Vials 1. Fill the 4-mL screw-top glass vials up to the neck of the vial. 2. Load the 4-mL screw-top glass vials, with caps and PTFE septa (WAT073018), into the 48-vial carousel. 3. Load the 48-vial carousel. 46 Calibrating the System

47 Configure the Autosampler Software 1. Select Config Page from the Main menu, then go to Page Move the cursor to Syringe Volume (µl) , then select Move the cursor to Loop Volume (µl), then select Return to the Main menu, then select Auto Page. 5. In the Edit Auto Run table, enter Step In the From Vial field, enter the starting vial number. 7. In the To Vial field, enter the same vial number (from step 10). 8. In the # Inj field, enter In the Inj Vol field, enter In the Run Time field, enter the desired run time. 11. Enter Step 2 in the Edit Auto Run table. 12. Use nonconsecutive vials (for example 1,3,5, etc.) for each step. 13. Repeat line information for each step entered in the Edit Auto Run table. 14. Press Step Function to display the Edit Step Function screen. 15. In the Editing Step field, enter Move the cursor to the Option? field, then select Addn. 17. Press Addn Page to display the Edit Auto Addition screen. 18. In the Run Addition field, select Yes. 19. In the Common Vial field, enter 2 (for step 1 in the Edit Auto Run table). 20. In the Common Vol field, enter Select Step Function to display the Editing Step Function screen. 22. In the Editing Step field, enter In the Option? field, select Addn. 24. Press Addn Page to display the Edit Auto Addition screen. 25. In the Run Addition field, select Yes. 26. In the Common Vial field, enter In the Common Vol field, enter Repeat the programming procedure for each step in the Edit Auto Run table by pressing Step Function and entering the Step number in the Editing Step field. 3 Calibrating Columns and Setting Fraction Collection Times 47