gel permeation cleanup system operator's guide€¦ · cleanup system direct current...

Waters Gel Permeation Chromatography Cleanup

SystemOperator’s Guide

34 Maple StreetMilford, MA 01757

WAT022516TP, Revision 1

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.

© 1999 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.

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.

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.

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.

STOP

STOP

STOP

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

Table of Contents

How to Use This Guide..................................................................... 10

Chapter 1 Introduction ...................................................................................... 13

1.1 GPC Overview ...................................................................... 131.1.1 GPC Cleanup............................................................. 131.1.2 GPC System Components......................................... 14

1.2 Waters GPC Cleanup System............................................... 171.2.1 Waters 515 HPLC Pump ........................................... 171.2.2 Waters 717plus Autosampler..................................... 181.2.3 Waters Envirogel GPC Cleanup Columns ................. 191.2.4 Waters 2487 Dual l Absorbance Detector ................. 201.2.5 Waters Fraction Collector II ....................................... 21

Chapter 2 Installing and Preparing the System ................................................ 22

2.1 Installation Site Requirements .............................................. 222.2 Solvent Preparation .............................................................. 232.3 Installation............................................................................. 242.4 Changing the 717plus Autosampler Syringe......................... 272.5 Columns................................................................................ 312.6 Making Fluidic Connections .................................................. 33

2.6.1 Connecting Solvent Reservoir to Pump..................... 342.6.2 Connecting Pump to Autosampler ............................. 35

Table of Contents 5

2.6.3 Connecting Autosampler to Detector......................... 372.6.4 Connecting Detector to Fraction Collector II.............. 382.6.5 Flushing the System .................................................. 392.6.6 Installing the Columns ............................................... 402.6.7 Column Connections and Equilibration...................... 41

Chapter 3 Calibrating the System .................................................................... 43

3.1 Precalibration System Check................................................ 433.2 Preparing Calibration Solution .............................................. 443.3 Calibrating Columns and Setting

Fraction Collection Times .................................................... 463.3.1 Performing a 5-mL Injection....................................... 463.3.2 Determining Peak Resolution .................................... 503.3.3 Programming Collection Times.................................. 513.3.4 Adjusting Collection Times ........................................ 51

3.4 Sample Cleanup Guidelines ................................................. 523.4.1 Sample Preparation ................................................... 523.4.2 Sample Volume.......................................................... 533.4.3 Sample Load.............................................................. 533.4.4 Equipment Considerations......................................... 543.4.5 Recalibration.............................................................. 54

6 Table of Contents

Chapter 4 Troubleshooting and Maintenance .................................................. 55

4.1 Troubleshooting .................................................................... 554.2 Maintenance and Storage ..................................................... 59

4.2.1 Short-Term Storage Procedures ................................ 594.2.2 Long-Term Storage Procedures................................. 60

Appendix A Warranty Information and Spare Parts ............................................. 61

A.1 Warranty Information ............................................................ 61A.2 Spare Parts........................................................................... 61

Index ............................................................................................ 66

Table of Contents 7

Table of Contents 8

1-1 Components of a GPC System...................................................... 141-2 Sample Chromatogram.................................................................. 161-3 Waters 515 HPLC Pump ............................................................... 171-4 Waters 717plus Autosampler......................................................... 181-5 Waters Envirogel GPC Cleanup Columns ..................................... 191-6 Waters 2487 Dual l Absorbance Detector ..................................... 201-7 Waters Fraction Collector II ........................................................... 21

2-1 Suggested System Setup .............................................................. 242-2 Removing the Knurled Nut............................................................. 282-3 Installing the Syringe ..................................................................... 292-4 Column Installation ........................................................................ 322-5 Solvent-Reservoir-to-Pump Connection ........................................ 342-6 515 HPLC Pump to 717plus Autosampler Connection.................. 352-7 717plus Autosampler to 2487 Detector Connection ...................... 372-8 Detector to Fraction Collector II Connection.................................. 382-9 717plus Autosampler to Column Connection ................................ 41

3-1 Sample Calibration Chromatogram ............................................... 493-2 Peak Resolution............................................................................. 50

List of Figures

Table of Contents 9

2-1 Component Installation References .............................................. 26

3-1 Required Concentrations of Calibration Solution Compounds ..... 44

4-1 Troubleshooting ............................................................................ 56

A-1 System Parts .......................................................................... 62A-2 515 HPLC Pump Parts ............................................................ 62A-3 717plus Autosampler Parts ..................................................... 63A-4 Envirogel Columns .................................................................. 63A-5 2487 Dual l Absorbance Detector Parts................................... 64A-6 Fraction Collector II Parts........................................................ 64A-7 Disposables from Waters ........................................................ 64A-8 Disposables from ESS ............................................................ 65

List of Tables

How to Use This GuidePurpose 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.

AudienceThis guide is intended for individuals who need to install, operate, maintain, and/or troubleshoot the Waters GPC Cleanup System.

Structure of This GuideThe 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 Content

Chapter 1, Introduction Describes the Waters GPC Cleanup System, including features and options.

Chapter 2, Installing and Preparing the System

Describes how to unpack and install the Waters GPC Cleanup System.

Chapter 3, Calibrating the System

Describes how to calibrate the Waters GPC Cleanup System.

Chapter 4, Troubleshooting and Maintenance

Describes troubleshooting and maintenance procedures for the Waters GPC Cleanup System.

Appendix A, Warranty Information and Spare Parts

Provides warranty and service information, as well as a list of recommended and optional spare parts.

10 How to Use This Guide

Related DocumentationThe following table lists related documentation.

Related Adobe™ Acrobat Reader DocumentationFor 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 DocumentAdobe 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.

Title Part Number

Waters 515 HPLC Pump Operator’s Guide WAT068980TP

Waters 717plus Autosampler Operator’s Manual WAT173-02TP

Waters 2487 Dual λ Absorbance Detector Operator’s Guide

WAT048740TP

Waters Fraction Collector II Operator’s Guide 71500011202

How To Use This Guide 11

Conventions Used in This GuideThis 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:

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.

STOP

12 How to Use This Guide

1
1
Introduction

1.1 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.

1.1.1 GPC Cleanup

TheoryGel 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 ProcessSample 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

1
1.1.2 GPC System Components
The basic components of a GPC system (Figure 1-1) are:

• System pump• Injector or autosampler

• Column

• Detector

• Fraction collector• Data collection device

Figure 1-1 Components of a GPC System

TP01622A

515 HPLC Pump

Waters 717plus Autosampler

Solvent Reservoir

To Waste orFraction Collector

DataCollection

14 Introduction

1

PumpThe 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.

InjectorSample 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.

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.

ColumnWhen 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.

DetectorColumn 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

1

Data CollectionThe 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).

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 CollectorThe 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

1
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

1.2.1 Waters 515 HPLC PumpThe Waters 515 HPLC Pump (Figure 1-3) delivers mobile phase at precise flow rates from 0.001 to 10.0 mL/min in increments of 0.001 mL/min at backpressures of up to 6000 psig.

Figure 1-3 Waters 515 HPLC Pump

TP01407

Waters GPC Cleanup System 17

1
1.2.2 Waters 717plus Autosampler
The Waters 717plus Autosampler (Figure 1-4) is an automated, multiple-sample, injection device.

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

1

• 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.

1.2.3 Waters Envirogel GPC Cleanup ColumnsThe 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.

Figure 1-5 Waters Envirogel GPC Cleanup Columns

��

�� !��

��" #$

$��

��

��%

��!��" #$

$��


1
1.2.4 Waters 2487 Dual λ Absorbance Detector
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.

Figure 1-6 Waters 2487 Dual λ Absorbance Detector

"#��

$��%&'�(��)*��*��

20 Introduction

1
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.

Figure 1-7 Waters Fraction Collector II

��


2

2Installing 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.

Environmental RequirementsThe 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 RequirementsThe GPC Cleanup System requires a minimum of five grounded receptacles on a 115 V, 20 A, dedicated line.

Installation Site Requirements 22

2

2.2 Solvent Preparation

The cleanup procedure requires a supply of HPLC-grade 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.

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.

STOP

STOP

23 Installing and Preparing the System

2

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.

• 2500-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.

Figure 2-1 Suggested System Setup

��

��

$��'�'�(�)(��

$��%&'�(��)*��*��

Installation 24

2

Installation GuidelinesMake 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.

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.


2

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 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.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 (71500011202), 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

2

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

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.

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 SyringeTo 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.

STOP


2

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.

Figure 2-2 Removing the Knurled Nut

Changing the 717plus Autosampler Syringe 28

2

Installing the New SyringeTo install the new syringe:

1. Screw the new syringe assembly partially into the mounting block (Figure 2-3) Do not tighten.

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.


2

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 SyringeTo 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.

Modifying Configuration ParametersIf 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

2

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 UseRefer 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 2-4.

STOP


2

Figure 2-4 Column Installation

4. Connect the outlet of the smaller column to the inlet of the larger column, using the 0.009-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

2

2.6 Making Fluidic Connections

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


2

2.6.1 Connecting Solvent Reservoir to PumpTo 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).

Figure 2-5 Solvent-Reservoir-to-Pump Connection

515 HPLC Pump

TP01624A

Methanol

1/8-inch Teflon Tubing

Solvent Inlet

Making Fluidic Connections 34

2

2.6.2 Connecting Pump to AutosamplerTo 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).

Figure 2-6 515 HPLC Pump to 717plus Autosampler Connection

2. 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 3.000 mL/min.

If the pump is dry, however, start with a lower Flow rate such as 1.000 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.

��

��

$��'�'�(�)(��

Waste Flask

Blue (0.040-inch) Tubing

Solvent Outlet

Red (0.009-inch)

Tubing


2

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

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.

Figure 2-7 717plus Autosampler to 2487 Detector Connection

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.


TP01621ARed

(0.009-inch) Tubing


1
2.6.4 Connecting Detector to Fraction Collector II
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.

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.

��Waste Container

Drain Tube

3-Way Valve

1/16-inch Teflon Tubing

Detector

Fraction Collector II


1
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.

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.

STOP


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.

2.6.6 Installing the ColumnsWaters strongly recommends installing a Waters Guard Column (Part No. 98801) 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.

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.

STOP


1
2.6.7 Column Connections and Equilibration
Column ConnectionsTo 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.

Figure 2-9 717plus 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).

��'

$��'�'�(�)(��

Waste ContainerRing Stand

Autosampler


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 EquilibrationTo 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 ≤ 0.002 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.


3

3Calibrating 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.

Precalibration System Check 43

3

3.2 Preparing Calibration Solution

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.

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

CompoundRequired Concentration (mg/L)

For 2-mL Injection For 5-mL Injection

Corn oil 62,500 25,000

Bis(2-ethylhexyl)phthalate 2500 1000

Methoxychlor 500 200

Perylene 50 20

Sulfur 200 80

STOP

44 Calibrating the System

3

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.)

Preparing Calibration Solution 45

3

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)

3.3.1 Performing a 5-mL InjectionBefore 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.


3

Configure the Autosampler Software

1. Select Config Page from the Main menu, then go to Page 3.

2. Move the cursor to Syringe Volume (µL) 25 250 2500, then select 250.3. Move the cursor to Loop Volume (µL), then select 2000.

4. Return to the Main menu, then select Auto Page.

5. In the Edit Auto Run table, enter Step 1.6. 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 1.

9. In the Inj Vol field, enter 250.10. 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 1.16. 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 250.

21. Select Step Function to display the Editing Step Function screen.22. In the Editing Step field, enter 2.

23. 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 4.

27. In the Common Vol field, enter 250.28. 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.

Calibrating Columns and Setting Fraction Collection Times 47

3

Make the 5-ml Injection

To make a 5-mL injection:

1. When programming is complete, press Step Function, then press Auto Page to return to the Edit Auto Run screen.

2. Press Start Auto. The Auto Addition routine runs as part of each programmed step. A message appears indicating that Auto Addition is running.

Turn Off Auto Addition

To turn off Auto Addition:

1. Access the Edit Auto Addition screen.2. In the Run Addition field, select No. The information programmed for Auto

Addition is saved, but Auto Addition does not run. For more information about Auto Addition, see the Waters 717plus Autosampler Operator’s Guide, Chapter 4, Operating the Autosampler.

Note: With the larger-volume syringe installed, the injection volume is 10 times the displayed programmed volume.


3

Injecting Calibration Solution

1. Filter approximately 10 mL of calibration solution through a 0.45-µm Gelman Acrodisc CR PTFE filter.

2. Fill two 717plus autosampler sample vials with 3 to 4 mL of calibration solution, and place the vials in carriage positions 1 and 2.

3. Press AUTO/START to inject the calibration solution.

The initial (corn oil) peak appears about 10 to 12 minutes after injection. The final (sulfur) peak appears about 25 minutes after injection. Figure 3-1 shows a sample calibration chromatogram.

Figure 3-1 Sample Calibration Chromatogram

2000.00

1500.00

1000.00

500.00

0.00

10.0

0

15.0

0

20.0

0

25.0

0

30.0

0

Abso

rban

ce U

nits

Minutes

Collect

1 2 3

4

5

1 Corn Oil2 Phthalate3 Methoxychlor4 Perylene5 Sulfur

5.00


3

3.3.2 Determining Peak Resolution

Calculating Peak ResolutionTo calculate the resolution between two peaks, measure the height of the valley between the peaks, and the height of the smaller peak (Figure 3-2).

Figure 3-2 Peak Resolution

Calculate the peak resolution using the following equation:

%R = 100 (1 − A/B)

Where:

R = resolution in percentA = height of the valley

B = height of smaller peak

Resolution RequirementsEPA Method 3640A requires that:

• All peaks are observed and symmetrical.

• Resolution is greater than 85 percent between the:– Corn oil and phthalate peaks

– Phthalate and methoxychlor peaks

– Methoxychlor and perylene peaks• The resolution between the perylene and sulfur peaks is greater than 90 percent.

A B


3

3.3.3 Programming Collection TimesDetermine the fraction collection time for each component by examining the chromatogram for retention times and compensating for any delay volume between the detector and the fraction collector. With 0.009-inch ID tubing, the delay is normally 1 to 2 seconds. Refer to the Waters Fraction Collector II Operator’s Guide, Chapter 4, Collecting Fractions Using Simple Modes, for more information.

After you determine the collection times for the peaks of interest, program the fraction collector to collect the samples.

SemivolatilesDetermine the retention times for the phthalate, methoxychlor, and perylene peaks. Set the fraction collector to collect those peaks; discard the corn oil and sulfur peaks.

Organochlorine Pesticides and PCBsSet the fraction collector to collect only the methoxychlor and perylene peaks. A correctly set collection time removes more than 85 percent of the phthalate, yet retains greater than 95 percent of the methoxychlor.

3.3.4 Adjusting Collection TimesTo adjust collection times:

1. Refill the sample vial with calibration solution and perform another calibration run. Collect the fractions as programmed.

2. Verify that the fraction collector captures the correct components by reinjecting each fraction. Examine the chromatograms, and, if necessary, adjust the collection times. Repeat steps 1 and 2.

3. When the proper collection times are established, inject a blank consisting of only methylene chloride, to be sure that all calibration components are washed from the column.

If any of the calibration peaks appear in the chromatogram, repeat step 3.

A stable baseline indicates that the system is ready to perform sample cleanup.


3

3.4 Sample Cleanup Guidelines

Observe the guidelines in this as you perform the sample cleanup described in the Contract Laboratory Program Statement of Work for Organic Analysis or the EPA Method 3640A, “GPC Cleanup.”

This section describes:

• Sample Preparation

• Sample Volume • Sample Load

• Equipment Considerations

• Recalibration

3.4.1 Sample PreparationPrepare sample extract according to the EPA Method 3640A or the Contract Laboratory Program Statement of Work for Organic Analysis.

Note: The most likely cause of fouling or blockage of Envirogel columns is solids precipitating from solution in the mobile phase. All samples must be dissolved in methylene chloride before filtration and injection.

If samples are dissolved and injected in solvents other than the mobile phase (100% methylene chloride or 50:50 methylene chloride:cyclohexane) it is highly likely that some components will drop out of solution and deposit on the column. Therefore, when samples are not dissolved in mobile phase, you need to evaporate off the solvent, redissolve the residue in mobile phase, and filter the solution. For more information on solvents, see Section 2.2, Solvent Preparation.

Extracts can be safely evaporated under a stream of nitrogen. The evaporation does not need to be exhaustive because small volumes of residual solvents will not harm the column. If the sample does not contain significant amounts of lipophilic substances, a small amount of the (clean) corn oil or some other nonvolatile lipophilic substance can be added as a “keeper”. (For more details see Section 3.4.2, Sample Volume.)

All samples should be filtered just prior to injection. The filters best suited to resist methylene chloride are Gelman 0.45-µm Acrodisc CR PTFE filters (Part Number WAT2000502).

High backpressure or loss of resolution may indicate the deposition of solids in the system. To check if solids are deposited in the system, check the in-line filter located just before the column.


3

To check the filter for blockage:

1. Turn off the solvent flow.

2. Disconnect the fitting on the exit side of the in-line filter.

3. Check the backpressure without the columns in line. This will indicate if the filter is blocked.

If the filter is blocked, reverse it and flush it at 9 mL/minute. (The columns should not be attached during the flushing process.) When the flush is complete, reverse the filter to its normal position. If the flush does not clear the filter, replace the filter unit inside the in-line filter (Part No. 84056).

Note: Some Waters Autosamplers have in-line filters near the injector assembly. These also should be checked if high backpressures or cavitation in the sampling syringe is noticed.

3.4.2 Sample VolumeThe recommended maximum sample volume for injection into Envirogel columns is 2 mL. Smaller sample volumes can be used and do give better resolution. However, small sample volumes should be used only in a controlled manner with samples known to be relatively clean, because the solubility of some analytes may be exceeded by excessive concentration, resulting in poor yields.

The volume of a sample can be increased to 5 mL if the autosampler is fitted with the programming software that allows two volumes of a sample from adjacent vials to be combined in one injection. The 717plus autosampler must be programmed for this operation and must be fitted with the large sample loop (as explained above) for this application.

Injecting 5 mL into an Envirogel column results in a significant loss of resolution and is not recommended. The 5-mL injection technique can be used for injecting samples onto the standard EPA columns (which may be desired if EPA-style columns are to be packed in solvents such as acetone or cyclohexane), for analyte extraction problems, or for non-EPA/AOAC methods.

3.4.3 Sample LoadAn excessive amount of soluble solids in the sample may overload the capacity of the column either by a mass overload or by being too viscous. As a guideline, load no more than 400 mg of triglyceride lipid onto the recommended set of one 15-cm and one 30-cm column in series. Otherwise, you risk having some carryover into other fractions.

Sample Cleanup Guidelines 53

3

Very viscous samples can cause increased retention, particularly of the higher-molecular-weight components.

Higher loadings can be used if extra columns are added, but this takes longer (and uses more solvent). It takes only slightly longer total time to dilute the sample and make two injections and fraction collections.

This is only a guideline, and proper studies should be carried out on difficult samples to gauge the effect of excessive sample loads or high viscosity. Such excessive sample loads are abnormal for the samples using GPC cleanup by the EPA or AOAC methods.

3.4.4 Equipment ConsiderationsTo run multiple cleanup samples, program the 717plus autosampler as described in Chapters 3 and 4 of the Waters 717plus Autosampler Operator’s Guide.

Be sure the sample reservoir contains sufficient methylene chloride to perform multiple cleanup operations, and that the waste receptacle can hold the anticipated volume of waste.

Typical collection volumes are less than 60 mL with the Envirogel columns. Use 28-mm scintillation vials in the fraction collector when using the Envirogel columns. Refer to Appendix A, Warranty Information and Spare Parts for part numbers.

3.4.5 RecalibrationPerform a system calibration at least once per week as recommended in the EPA method. Compare the retention times between calibration runs. If the retention times shift more than 5% between calibration runs, stabilize and recalibrate the system as described in Chapter 2, Installing and Preparing the System, and in this chapter.


4

4Troubleshooting and Maintenance

4.1 Troubleshooting

Most problems you may encounter while using your GPC Cleanup System are easily corrected. Use Table 4-1 as a guide to diagnosing and correcting possible problems. Refer to the troubleshooting and maintenance chapters in the manuals of the individual system components for information on possible problems and corrective action.

Contacting Waters Technical ServiceIf you encounter any problems when troubleshooting the system, contact Waters Technical Service at 1-800-252-4752, U.S. and Canadian customers only. Other customers, call your local Waters subsidiary or Technical Service Representative, or call Waters corporate headquarters for assistance at 1-508-478-2000 (U.S.).

When you call, have the following information available:

• Symptom

• Type of column• Operating pressure

• Flow rate

• Diagnostic error codes• System configuration (for example, GPC Cleanup System)

Troubleshooting 55

4

Table 4-1 Troubleshooting

Problem Possible Cause Corrective Action

High pressure shutdown

Clogged fluidic path Change all filters in fluidic path. (Refer to the Waters 717plus Autosampler Operator’s Guide for the location of filters.)Replace all tubing.Flush the column(s) at 2 ml/min by reversing the direction of flow through the column.Replace the column(s).

Low operating pressure

Leaks Tighten fluidic connections

Insufficient solvent Refill solvent reservoir

Air in line Purge the system

Retention times too long

Pump flow rate set too low

Check flow rate setting. Verify that flow rate set matches output at fraction collector.

Leaks Tighten fluidic connections

Wrong columns Verify column length and diameter.

Retention times too short

Pump flow rate set too fast

Check flow rate setting and verify output at fraction collector.

Data collection device improperly connected

Ensure data collection starts at inject signal.

Wrong columns Verify column length and diameter.

56 Troubleshooting and Maintenance

4

No peaks, or poor recovery

Solvent flow too low Check pump pressure.

Injector blocked or leaking

Check for leaks or blockage. Contact Waters Technical Service for back-flush procedure.

Detector malfunctioning

Check wavelength, sensitivity, and signal output cables.

Broad, smeared peaks, or peaks appear as doublets

Rearrangement of packed bed, or void in the column packing

Check system performance with each column separately.Reverse flow through affected column using 2-ml injections of toluene or xylene at 2 ml/min. If problem is not corrected after 10 injections, replace column.Recalibrate columns after any cleaning procedure.

Injector sample loop blocked or leaking; autosampler in-line or inlet filter partially clogged

Check for leaks or excessive pressure drop across sample loop. Back-flush loop.Reverse flow through column and inject 2 mL of tetrahydrofuran, butylchloride, or a mixture of 1:1 methylene chloride:cyclohexane. Change the inlet filters.Recalibrate columns after any cleaning procedure.

Cyclic pattern of positive and negative peaks instead of positive peaks

Air in pump Purge pump

Immiscible solvent trapped in flow path

Disconnect sample inlet line to detector. Attach a syringe containing alcohol or THF to inlet, then purge flow path with 5 to 10 mL of solvent.

Table 4-1 Troubleshooting (Continued)


Troubleshooting 57

4

Detector response decreases over time

Film on detector cell Remove column from flow path. Flush detector flow path with methanol. Refill and flush system with methylene chloride. Reinstall column.

Retention times shift >5%

Change in flow rate Check flow rate

Change in laboratory temperature

Stabilize laboratory temperature or column temperature.

Peaks on chromatogram appear flat on top

Detector sensitivity too high

Decrease sensitivity.

Solvent background absorbance too high

Replace mobile phase with fresh solvent.

Column efficiency higher than expected

This is not a problem if baseline resolution is within method requirements. To see the peak tops, reduce sample size by 10 to 15% or dilute sample.

Low or erratic injection volumes

Cavitation or outgassing of solvent

Stabilize or reduce solvent temperature.Sparge solvent with helium.

Table 4-1 Troubleshooting (Continued)



4

4.2 Maintenance and Storage

This section describes:• Short-term storage procedures

• Long-term storage procedures

Refer to the specific component manuals for equipment maintenance procedures.

4.2.1 Short-Term Storage ProceduresWhen you want to store the GPC Cleanup System for a few (2 to 3) hours to a few (2 to 3) days.

• Leave the system power on.• Reduce the flow rate to the column to 0.5 mL/min.

• Set the fraction collector to divert the flow to waste.

• Check that sufficient solvent is present in the reservoir.• Check that a waste container of sufficient volume is connected to the system.

Note: The mobile phase can be recirculated if there is no contamination that can leak from the columns.

Maintenance and Storage 59

4

4.2.2 Long-Term Storage Procedures

Storage PeriodWhen you want to store the system for several days to several weeks:

1. Set the flow rate to 0.0 mL/min, and set the fraction collector to divert the flow to waste.

2. Remove the cleanup columns from the system, and tightly cap both ends to prevent solvent evaporation. The columns need not be separated. Store the columns at room temperature.

3. Reconnect the autosampler to the detector (see Section 2.6.3, Connecting Autosampler to Detector).

4. Set the system flow rate to 5.0 mL/min.

5. Make several injections of solvent to clean out the lines between the sample injector and the fraction collector.

6. Store the system in methanol.7. Set the flow rate to 0.0 mL/min. Proceed with the power-off and long-term

storage instructions in the individual component manuals.

RestartTo restart the Waters GPC Cleanup System, perform a complete startup procedure as described in Chapter 2, Installing and Preparing the System, and Chapter 3, Calibrating the System.


A
Appendix AWarranty Information and Spare Parts
This appendix contains information on:

• Warranty Information • Spare Parts

A.1 Warranty Information

WarrantyRefer to the individual component manuals for service information and warranty periods for the Waters GPC Cleanup System.

Refer to the Envirogel Columns Care and Use Manual for column warranty periods.

A.2 Spare Parts

When ordering parts from Waters, please include the part number, description, and quantity of each item. To order the GPC Cleanup System and related items, contact Waters Technical Service as described in Section 4.1, Troubleshooting.

This section includes information on spare parts for:

• GPC Cleanup System

• 515 HPLC Pump• 717plus Autosampler

• Envirogel columns

• 2487 Dual λ Absorbance Detector• Fraction Collector II

• Optional components

• Disposables from Waters• Disposables from Fisher Scientific

Warranty Information and Spare Parts 61

A

GPC Cleanup System

515 HPLC Pump

Table A-1 System Parts

Part Description Part Number

515 HPLC Pump WAT020700

717plus Autosampler WAT078900

2487 Dual λ Absorbance Detector WAT081110

Fraction Collector II 725000126

Fraction Collector II 3-way valve (standard) 725000142

2487 semi-prep flow cell WAT081158

Waters GPC Cleanup System Operator’s Guide WAT022516

Table A-2 515 HPLC Pump Parts


Stainless steel tubing, 0.040 inch ID × 1/16 inch OD × 10 feet

WAT026805

Stainless steel tubing, 0.020 inch ID × 1/16inch OD × 10 feet

WAT026804

Waters 515 HPLC Pump Operator’s Guide WAT068980TP

62 Warranty Information and Spare Parts

A

717plus Autosampler

Envirogel Columns

Table A-3 717plus Autosampler Parts


Stainless steel tubing, 0.009 inch ID ×1/16 inch OD × 10 feet

WAT026973

Compression screws, 1/16 inch (10/pkg) WAT005070

Ferrules, 1/16 inch (10/pkg) WAT005063

In-line pre-column filter kit WAT084560

Replacement filter (5/pkg) WAT005139

Replacement gasket (10/pkg) WAT084567

2500-µL syringe WAT077342

5-ml sample loop WAT036212

Vial assembly, 4-mL (includes cap and PTFE septa) WAT073018

Waters 717plus Autosampler Operator’s Manual WAT173-02TP

Table A-4 Envirogel Columns


19 mm ×150 mm WAT036555

19 mm × 300 mm WAT036554

Waters Envirogel GPC Cleanup Column Care and Use Manual

WAT036556


A

2487 Dual λ Absorbance Detector

Fraction Collector II

Disposables from Waters

Table A-5 2487 Dual λ Absorbance Detector Parts


2487 Semi-prep flow cell WAT081158

2487 Dual λ Absorbance Detector Operator’s Guide

WAT048740TP

Table A-6 Fraction Collector II Parts


17-mm OD vial rack 725000146

28-mm OD vial rack 725000147

3-way valve 725000142

Viton tubing, 0.375 in. OD × 60 inches WAT037013

Waters Fraction Collector II Operator’s Guide 71500011202

Table A-7 Disposables from Waters


Priming syringe WAT027629

Gelman 0.45-µm Acrodisc CR PTFE filter WAT200502

64 Warranty Information and Spare Parts

A

Disposables from Environmental Sampling Supply (ESS)

Table A-8 Disposables from ESS


For Use with Waters Columns

40-mL PreCleaned Certified™ vials, 28 × 95-mm OD (72/case)

PC0040-0300


I

DEX

NIndex
Numerics2487 Dual Absorbance Detector 203-way valve 385 mL injections
compatibility with column types 53programming 47

515 HPLC pump 17515 HPLC pump, priming 35717plus autosampler 18

AAutosampler 18

connecting 37syringe, see Syringe

BBackpressure 39, 52Bench space 22Blockage, checking for 52

CCalibration chromatogram 49Calibration solution

filtering 45, 49injecting 49preparing 44storage 45

Chromatogram 16, 16Collection times

adjusting 51programming 51

Columns 15, 19

blockage 52connecting 31, 41equilibration 42installing 40theory 13, 15

Component installation 25Compressibility test results 30Configuring the 717plus syringe 30Connections, fluidic 33

DData collection 16Detector 15, 20

connecting 37, 38troubleshooting response 58

Diverter valve 38Draw rate 25

EElectrical requirements 22Envirogel columns 19, 39Environmental requirements 22Equipment considerations 54Extracts, evaporation of 52

FFilter

checking for blockage 52filtering samples 52

Flow rate 23, 40, 43Fluidic connections 33Flushing the system 39

Index 66

I

DEX

N
Fraction collection times
adjusting 51programming 51

Fraction collector 16, 21Fraction collector, connecting 38

GGPC

cleanup process 13equipment 14theory 13

Guard column 40

HHeater/cooler

compartment temperature 19

IInjection

procedure 46troubleshooting 58volume 27

Injector 15, 18Injector draw rate 25Installation 25

LLaboratory temperatures 22

MMethanol, flushing 39Methylene chloride, checking acid level

of 23

I

Microbore injectionssyringe and vials 19

Mobile phase 19, 39, 40, 52Mobile phase, recirculating 59Multiple cleanup operations 54

PPeak resolution

calculating 50requirements 50

Peaks, troubleshooting 57, 57Pressure

checking 43troubleshooting 56

Priming the pump 35Pump 15, 17Pump, priming 35

RRecalibration 54Refractive index 15Restarting the system 60Retention times 54

troubleshooting 56Retention times, troubleshooting 58

SSample

cleanup 13filtering 52load 53preparation 52volume 53

Sample loop 25Semiprep chromatography

sample loop and syringe 19

Index 67

I

DEX

N
Semi-preparative cell 20, 25Solvent considerations 23Solvent reservoir 34Spare parts
2487 dual absorbance detector 64515 HPLC pump 62717plus autosampler 63disposables 64envirogel columns 63fraction collector 64system 62

Specifications 61Storage

long-term 60short-term 59

Syringechanging 27purging 30safety standards 27

Systemcomponents 14pressure 43recalibration 54

TTroubleshooting 56, 56

detector response 58injection 58peaks 57pressure 56retention times 56, 58

Troubleshooting peaks 57Tubing 24, 25

UUltraviolet

absorbance 42, 43wavelength 15, 20

VViscosity, syringe speed 19

WWarranty 61Waters Technical Service 55

68 Index

gel permeation cleanup system operator's guide€¦ · cleanup system direct current...

Documents