PFC Analysis Kit for ACQUITY UPLC System Guide

Transcription

1 PFC Analysis Kit for ACQUITY UPLC System Guide /Revision A Copyright Waters Corporation 2009 All rights reserved

2 Copyright notice 2009 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA AND IRELAND. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER. The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with, or arising from, its use. Trademarks ACQUITY, ACQUITY UPLC, Oasis, UltraPerformance LC, UPLC, and Waters are registered trademarks of Waters Corporation, and IntelliStart, MassLynx, PFC Isolator, QuanLynx, Quattro micro, Quattro Premier, TargetLynx, and THE SCIENCE OF WHAT S POSSIBLE. are trademarks of Waters Corporation. PharMed and Tygon are registered trademarks of Saint-Gobain Ceramics & Plastics, Inc. Teflon is a registered trademark of E.I. du Pont de Nemours & Co., Inc. PEEK is a trademark of Victrex plc. Other registered trademarks or trademarks are the sole property of their owners. Customer comments Waters Technical Communications department invites you to tell us of any errors you encounter in this document or to suggest ideas for otherwise improving it. Please help us better understand what you expect from our documentation so that we can continuously improve its accuracy and usability. We seriously consider every customer comment we receive. You can reach us at tech_comm@waters.com. ii

3 Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use, transportation, removal, or disposal of any Waters product. You can reach us via the Internet, telephone, or conventional mail. Waters contact information Contacting medium Internet Telephone and fax Conventional mail Information The Waters Web site includes contact information for Waters locations worldwide. Visit and click Waters Division > Contact Waters. From the USA or Canada, phone HPLC, or fax For other locations worldwide, phone and fax numbers appear in the Waters Web site. Waters Corporation 34 Maple Street Milford, MA USA Safety considerations Some reagents and samples used with Waters instruments and devices can pose chemical, biological, and radiological hazards. You must know the potentially hazardous effects of all substances you work with. Always follow iii

4 Good Laboratory Practice, and consult your organization s safety representative for guidance. When you develop methods, follow the Protocol for the Adoption of Analytical Methods in the Clinical Chemistry Laboratory, American Journal of Medical Technology, 44, 1, pages (1978). This protocol addresses good operating procedures and the techniques necessary to validate system and method performance. Caution: Read the system guide thoroughly before using the product, and follow the instructions carefully. Waters cannot guarantee reliable assay results if you deviate from these instructions. Caution: Do not use the kit components if the kit s packaging is opened or damaged on arrival. Safety advisories Consult Appendix A for a comprehensive list of warning and caution advisories. Using this kit When using this kit, follow standard quality-control (QC) procedures and the guidelines presented in this section. Applicable symbols Symbol ABN Definition Confirms that a manufactured product complies with all applicable European Community directives Australia C-Tick EMC Compliant Confirms that a manufactured product complies with all applicable United States and Canadian safety requirements iv

5 Audience and purpose This document is for laboratory personnel. It includes procedures and information for installing, operating, and maintaining the PFC Analysis Kit for ACQUITY UPLC systems. Intended use of the PFC analysis kit Waters designed the PFC Analysis Kit for ACQUITY UPLC systems to analyze trace amounts of perfluorinated compounds in surface water samples. Calibrating To calibrate LC systems, follow acceptable calibration methods using at least five standards to generate a standard curve. The concentration range for standards must include the entire range of QC samples, typical specimens, and atypical specimens. When calibrating mass spectrometers, consult the calibration section of the operator s guide for the instrument you are calibrating. In cases where an overview and maintenance guide, not operator s guide, accompanies the instrument, consult the instrument s online Help system for calibration instructions. Quality-control Routinely run three QC samples that represent subnormal, normal, and above-normal levels of a compound. Ensure that QC sample results fall within an acceptable range, and evaluate precision from day to day and run to run. Data collected when QC samples are out of range might not be valid. Do not report these data until you are certain that the instrument performs satisfactorily. ISM classification ISM Classification: ISM Group 1 Class B This classification has been assigned in accordance with CISPR 11 Industrial Scientific and Medical (ISM) instruments requirements. Group 1 products apply to intentionally generated and/or used conductively coupled v

6 radio-frequency energy that is necessary for the internal functioning of the equipment. Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage, power-supply network. EC Authorized Representative Waters Corporation (Micromass UK Ltd.) Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Telephone: Fax: Contact: Quality manager vi

7 Table of Contents Copyright notice... ii Trademarks... ii Customer comments... ii Contacting Waters... iii Safety considerations... iii Safety advisories... iv Using this kit... iv Applicable symbols... iv Audience and purpose... v Intended use of the PFC analysis kit... v Calibrating... v Quality-control... v ISM classification... v ISM Classification: ISM Group 1 Class B... v EC Authorized Representative... vi 1 PFC analysis kit Kit contents ACQUITY UPLC retrofit kit for PFC analysis PFC standards Purpose Compatible ACQUITY UPLC system configurations Software control Table of Contents vii

8 2 Setting up the System Hardware Preparing an existing system Preparing the solvent reservoirs Installing the columns and tubing Removing the PFC analysis components Preparing the System for Analysis Flushing the system Preparing eluents and wash solvents Verifying System Performance Preparing the standards Performing the verification test Opening the project file Interpreting the test data Preparing Samples Typical sample preparation procedure Troubleshooting PFC analysis troubleshooting Thorough cleaning of solvent reservoirs A Safety Advisories... A-1 Warning symbols... A-1 Task-specific hazard warnings... A-1 Task-specific hazard warnings... A-2 Warnings that apply to particular instruments, instrument components, and sample types... A-3 viii Table of Contents

9 Caution symbol... A-5 Warnings that apply to all Waters instruments... A-5 Electrical and handling symbols... A-12 Electrical symbols... A-12 Handling symbols... A-13 Index... Index-1 Table of Contents ix

10 x Table of Contents

11 1 PFC analysis kit Use the kit to quantify trace levels of perfluorinated compounds (PFCs) in surface water samples. The kit minimizes contamination from system components and separates background contaminants from the analytes of interest. Contents Topic Page Kit contents 1-2 Purpose 1-6 Compatible ACQUITY UPLC system configurations 1-7 Software control

12 Kit contents Caution: To help minimize PFC contamination and bacterial growth in the system s solvent supply, follow the recommendations in this guide for appropriate glassware and bottle closures. The PFC analysis kit includes the elements described in the table, below. Some elements are described in subsequent sections of this guide. PFC analysis kit contents Part Description Analytical column ACQUITY UPLC BEH C mm analytical column ACQUITY UPLC retrofit kit for PFC analysis See table titled Retrofit kit for PFC analysis on page 1-3. PFC standards See PFC standards on page 1-3. Oasis HLB PLUS extraction cartridges, 225 mg/60 μm Vials, mm, 2-mL, polypropylene, screw-neck, with polyethylene, septumless caps Borosilicate glass solvent reservoirs High-density polyethylene solvent caps, with accessories Box of 50 Pack of 100 Clear, 500-mL Clear, 1000-mL (2) Amber, 500-mL (2) Amber, 1000-mL (2) Solvent-cap inner liners (7) Closed-thread solvent caps (7 colors) Open-thread solvent caps (7 colors) Solvent-cap-liner plugs (14) Filters, 10-μm (7) 1-2 PFC analysis kit

13 ACQUITY UPLC retrofit kit for PFC analysis Using the retrofit kit, you can install a complete flow path suitable for the trace analysis of perfluorinated compounds. Retrofit kit for PFC analysis Part PFC Isolator column assembly Tubing assembly, mixer to isolator column Tubing assembly, valve to isolator column Tubing assembly, column to detector Description mm Coiled stainless-steel Stainless-steel PEEK Tubing assemblies, solvent inlet PEEK (set of 7) Nut extender Flangeless, short Sample loop 10-μL PFC standards The standards are divided into two groups: analytical standards used for calibration, and preparation standards used for quality control. PFC calibration standards For analytical standards, the PFC kit supplies five PFC calibration standards, one PFC internal standard, and one PFC recovery standard. For quality control, five preparation standards are provided. Analytical standards for calibration Label Concentration Vial PFC Calibration Standard #1 PFC Calibration Standard #2 PFC Calibration Standard #3 5.0 ng/ml for each analyte 25 ng/ml for each analyte 50 ng/ml for each analyte 2-mL, flame-sealed ampule 2-mL, flame-sealed ampule 2-mL, flame-sealed ampule Kit contents 1-3

14 Analytical standards for calibration (Continued) Label Concentration Vial PFC Calibration Standard #4 PFC Calibration Standard #5 250 ng/ml for each analyte 500 ng/ml for each analyte 2-mL, flame-sealed ampule 2-mL, flame-sealed ampule PFC Calibration standards #1 through #5 contain the following analytes: Analytes in calibration standards Analyte Chemical formula CAS number 1 Perfluorobutyric acid CF 3 (CF 2 ) 2 CO 2 H Perfluoropentanoic acid CF 3 (CF 2 ) 3 CO 2 H Perfluorohexanoic acid CF 3 (CF 2 ) 4 CO 2 H Perfluoroheptanoic acid CF 3 (CF 2 ) 5 CO 2 H Perfluorooctanoic acid CF 3 (CF 2 ) 6 CO 2 H Perfluorononanoic acid CF 3 (CF 2 ) 7 CO 2 H Perfluorodecanoic acid CF 3 (CF 2 ) 8 CO 2 H Perfluoroundecanoic acid CF 3 (CF 2 ) 9 CO 2 H Perfluorododecanoic acid CF 3 (CF 2 ) 10 CO 2 H Perfluorotridecanoic acid CF 3 (CF 2 ) 11 CO 2 H Perfluorotetradecanoic acid CF 3 (CF 2 ) 12 CO 2 H Perfluorobutanesulfonic acid CF 3 (CF 2 ) 3 SO 3 H Perfluorohexanesulfonic acid CF 3 (CF 2 ) 5 SO 3 H Perfluoroheptanesulfonic acid CF 3 (CF 2 ) 6 SO 3 H Not available Perfluorooctanesulfonic acid CF 3 (CF 2 ) 7 SO 3 H Perfluorodecanesulfonic acid CF 3 (CF 2 ) 9 SO 3 H CAS (Chemical Abstracts Service, a division of the American Chemical Society) numbers are unique identifiers for chemical substances. 1-4 PFC analysis kit

15 Internal and recovery standards for calibration Label Analytes Concentration Vial PFC Internal Standard Perfluorooctanoic acid (1,2,3,4-13 C 4 ) Perfluorooctanesulfonic acid (1,2,3,4-13 C 4 ) 2500 ng/ml for each analyte 2-mL, flamesealed ampule PFC Recovery Standard Perfluorohexanoic acid (1,2-13 C 2 ) Perfluoro-n-[1,2-13 C 2 ]decanoic acid 2500 ng/ml for each analyte 2-mL, flamesealed ampule Perfluorohexanesulfonic acid ( 18 O 2 ) Preparation standards for quality control Label Analytes Concentration Vials PFC Quality Control Standards Perfluoropentanoic acid Perfluorohexanoic acid Perfluoroheptanoic acid Perfluorooctanoic acid Perfluorononanoic acid 5.0 to 500 ng/ml for each analyte Five, 2-mL, flamesealed ampules Perfluorodecanoic acid Perfluoroundecanoic acid Perfluorododecanoic acid Perfluorobutanesulfonic acid Perfluorohexanesulfonic acid Perfluorooctanesulfonic acid Perfluoroheptanesulfonic acid Perfluorodecanesulfonic acid Kit contents 1-5

16 Preparation standards for quality control (Continued) Label Analytes Concentration Vials PFC Recovery Standard Perfluorohexanoic acid (1,2-13 C 2 ) Perfluoro-n-[1,2-13 C 2 ]decanoic acid Perfluorohexanesulfonic acid ( 18 O 2 ) 2500 ng for each analyte 2-mL, flamesealed ampule Purpose The PFC Analysis Kit for ACQUITY UPLC enables the quantitative analysis of perfluorinated compounds in surface water samples. It is designed to minimize interference from PFCs found in many laboratory instrument components, common HPLC solvents, and laboratory water. The kit contains tubing, fittings, filters, and other components to replace those that can contribute to contamination. The kit also contains a PFC isolator column that helps isolate the background contaminants from the analytes in a sample. The isolator column is in the flow path between the mixer of the binary solvent manager and the injector of the sample manager. During analysis, the isolator column retains the background PFCs, separating them from the analytes of interest. The increased retention time of the background PFCs makes them easily distinguishable by mass analysis. For examples of TIC and MRM chromatograms from the analysis of standard PFC compounds using the PFC analysis kit and method, refer to the application note, ACQUITY UPLC System Solution for Quantifying Trace Levels of Perfluorinated Compounds with an ACQUITY PFC Analysis Kit, on the documentation CD. 1-6 PFC analysis kit

17 Compatible ACQUITY UPLC system configurations The PFC analysis kit is compatible with an ACQUITY UPLC system composed of the following components: Binary solvent manager, with or without i 2 Valves Sample manager Column heater or high temperature column heater Sample organizer (optional) Tandem quadrupole mass spectrometer (TQ detector, Quattro Premier XE, Quattro micro, or Xevo mass spectrometer) Caution: The kit does not support the ACQUITY UPLC column manager or detectors with Teflon flow cells (ACQUITY PDA and TUV, for example). Software control ACQUITY UPLC systems using the PFC analysis kit require MassLynx software version 4.1. Use the QuanLynx or TargetLynx applications to analyze data. The kit is shipped with a MassLynx project template for running standards and quality control samples to verify system operation. You can also use the project template to run analytical samples. (See To open the project file on page 4-3.) Compatible ACQUITY UPLC system configurations 1-7

18 1-8 PFC analysis kit

19 2 Setting up the System Hardware Before installing the PFC analysis kit on an existing system, you must first remove some components. Installing the kit establishes a complete flow path for analyzing PFC-containing samples while minimizing interference from background contamination. Caution: To avoid damaging the kit hardware, use the PFC analysis kit only for applications involving the analysis of perfluorinated compounds. Requirement: Verify that the standard ACQUITY UPLC installation procedure is completed before installing the kit on a new system. See also: The ACQUITY UPLC System Operator s Guide for basic ACQUITY hardware installation procedures. Contents Topic Page Preparing an existing system 2-3 Preparing the solvent reservoirs 2-5 Installing the columns and tubing 2-7 Removing the PFC analysis components

20 Required materials Warning: To prevent injury, always wear eye protection and particle-free, powder-free, non-latex gloves when handling tubing that can contain solvents. If possible, drain the tubing before handling. Locations of required materials Location PFC analysis kit ACQUITY UPLC startup kit PFC analysis kit or ACQUITY UPLC startup kit PFC retrofit kit ACQUITY UPLC bottle kit Material Flangeless nut extender Isolator column Stainless-steel tubing assemblies, labeled Mixer to PFC Isolator and Valve to isolator column Open-end wrenches, 5/8-inch and 1/4-inch ACQUITY UPLC BEH C μm 50 mm column 10-μL sample loop Bottle-cap inner liners Closed-thread bottle caps Open-thread bottle caps Bottle-cap-liner plugs Filters, 10-μm Clear or amber, glass, borosilicate bottles: 500-mL, 1000-mL See also: Controlling Contamination in UltraPerformance LC/MS and HPLC/MS Systems (part number ). 2-2 Setting up the System Hardware

21 Preparing an existing system If you are adding the PFC analysis kit to an existing ACQUITY UPLC system, perform the following procedure before installing the PFC analysis kit components. To prepare an existing system 1. Using the open-end wrenches, remove the stainless-steel tubing that extends from the mixer outlet of the binary solvent manager to port 5 of the sample manager injection valve. Tips: Store the tubing for future use. If you need more room to maneuver inside the binary solvent manager, you can remove the ends of the pre-installed Tygon tubing (running from the process waste port) and the PharMed tubing (running from the needle-clean system waste port) from the pass-through of the binary solvent manager drip tray. Mixer Tubing to port 5 of the sample manager injection valve Tygon and PharMed waste tubing Tubing from mixer outlet in binary solvent manager Port 5 of the sample manager injector valve Preparing an existing system 2-3

22 2. Using the flangeless-nut extender tool, remove the Teflon solvent tubing from the A1, B1, A2, B2, SNW, WNW, and seal wash (SW) ports of the binary solvent manager. 3. Remove the solvent tubing and solvent bottles from the system, and store them for future use. 4. Use the wrenches to remove the currently installed sample loop. For detailed instructions on how to remove a sample loop from an ACQUITY UPLC system, consult the ACQUITY UPLC System Operator s Guide. 5. Using the 1/4-inch wrench, remove the tubing that connects the analytical column to the existing detector. 6. Remove from the flow path any detectors employing Teflon flow cells (TUV or PDA, for example). To ensure adequate tubing lengths and distances for the PFC analysis kit components, physically remove these detectors from the ACQUITY stack. Requirements: Do not install any detectors where flow cells are made of Teflon. Do not place additional detectors in the ACQUITY stack. 2-4 Setting up the System Hardware

23 Preparing the solvent reservoirs Caution: To prevent contamination, heed these measures: Always wear particle-free, powder-free, non-latex gloves when handling system components. Select, prepare, and handle solvents and samples correctly. For best results, use polypropylene vials and caps. Avoid any vial with a Teflon-lined cap. Do not wash reservoirs in detergent, alongside other glassware, or in washing facilities that can retain detergent residue. Store glassware separately from common-use glassware. Use clean fittings, tubing, and columns. Keep laboratory air clean. To install solvent reservoirs, caps, and tubing 1. In the PFC analysis kit, unwrap the appropriate PEEK solvent tubing assemblies for the binary solvent manager s A1, B1, A2, B2, SNW, WNW, and seal-wash (SW) ports. Tip: Each tubing assembly is labeled with a specific port name. Solvent labels and colors Solvent type Label Color Strong needle wash SNW White Weak needle wash WNW Orange Solvent A1 A1 Yellow Solvent A2 A2 Blue Solvent B1 B1 Red Solvent B2 B2 Green Seal wash SW Brown Recommendation: Match the cap color to the color on the tubing labels and the degasser port. Preparing the solvent reservoirs 2-5

24 2. Install the bottle cap and solvent filter on one end of the PEEK tubing as follows: a. Unwind one end of the PEEK tubing, and then slide the outer cap onto it with the threads facing toward the end of the tubing. b. Slide the end of the tubing through an open hole in the inner liner (but not the center hole). Inner liner Center port with 10-μm filter attached Tubing port 5 c. Insert the solvent filter fitting and then the solvent filter onto the end of the tubing. Label PEEK tubing Inlet filter fitting Inlet (solvent) filter d. Push the tubing all the way into the solvent filter, and then finger tighten the fitting. e. If other solvent lines require the same solvent, repeat steps a through d. f. Insert plugs into the remaining open ports on the inner liner. 3. Install the tubing in the appropriate degasser port as follows: a. Install the compression screw on the degasser end of the tubing assembly. b. Slide the lock ring and ferrule onto the tubing, with the metal end nearest the compression screw. Compression screw Lock ring and ferrule Tip: Finger tighten the compression screw, and then use the flangeless-nut extender tool to tighten it an additional 1/4-turn. 4. If the reservoir has a cap and plastic ring around its neck, remove them. 2-6 Setting up the System Hardware

25 5. Rinse the reservoir with organic solvent, MS-grade water, and, finally, with the intended solvent. Tip: If more aggressive cleaning is required, see Thorough cleaning of solvent reservoirs on page Fill the reservoir with solvent (see Preparing eluents and wash solvents on page 3-3). 7. Insert the solvent filter and tubing in the reservoir. Requirement: Ensure that the entire filter is submerged in the solvent. 8. Fit the inner liner in the outer cap, and tighten the reservoir s cap. 9. Place the reservoir in the solvent tray. Installing the columns and tubing To install the columns and tubing 1. In the PFC analysis kit, locate the two stainless-steel tubing assemblies, labeled Mixer to PFC Isolator and Valve to isolator column. Requirement: Remove the black, protective O-rings from the stainless-steel tubing before installing them. 2. Using the 5/8-inch and 1/4-inch wrenches, attach one end of the coiled, stainless-steel tubing assembly, labeled mixer to PFC Isolator, to the inlet of the PFC Isolator column provided with the PFC analysis kit. Installing the columns and tubing 2-7

26 3. Attach the remaining stainless-steel tubing assembly, labeled Valve to isolator column, to the PFC Isolator column outlet. Tubing, Valve to isolator column Coiled tubing, Mixer to PFC Isolator PFC Isolator column Mixer 4. Attach the free end of the coiled tubing assembly to the mixer outlet port, and the free end of the valve to isolator column tubing assembly to port 5 of the sample manager injector valve. Orient the tubing, as shown below. Port 5 Drip tray Tubing, Valve to isolator column 2-8 Setting up the System Hardware

27 Flow path through PFC isolator Injection valve port 5 Sample manager PFC Isolator flow path Binary solvent manager Isolator column Mixer Requirement: Place the ends of the Tygon tubing (running from the process waste port) and the PharMed tubing (running from the needle-clean system waste port) in the pass-through of the binary solvent manager drip tray, if you removed them to install the kit. 5. Install the 10-μL sample loop (PFC analysis kit). See also: ACQUITY UPLC System Operator s Guide for detailed instructions on installing a sample loop. Installing the columns and tubing 2-9

28 6. Install the ACQUITY UPLC BEH C mm column in the column compartment. 7. Using the 1/4-inch wrench, connect the PEEK, column-to-detector tubing assembly to the outlet of the analytical column, routing it to a waste container until the flushing procedure is completed (see Flushing the system on page 3-2). Removing the PFC analysis components You must remove the PFC analysis components to resume analyzing compounds other than PFCs. To remove the components 1. Disconnect the end of the valve to isolator column tubing from the injection valve port 5 in the sample manager. 2. Disconnect the end of the coiled mixer to PFC isolator tubing from the mixer in the solvent manager. 3. Remove the PFC Isolator with the stainless steel tubing from the system. Tip: The PEEK tubing can remain. 4. Reinstall the original stainless-steel tubing, connecting the mixer and injector. 5. Check for leaks. 6. Verify system performance. See also: ACQUITY UPLC System Operator s Guide for detailed instructions on verifying system performance. 7. Store the PFC analysis components for future use Setting up the System Hardware

29 3 Preparing the System for Analysis To minimize background PFC contamination, clean the system thoroughly before performing an analysis. Carefully prepare eluents, and wash solvents in the glassware provided with the kit. Warning: To avoid chemical exposure risk, always observe Good Laboratory Practices when you use this equipment and when you work with solvents and test solutions. Know the chemical and physical properties of the solvents and test solutions you use. See the Material Safety Data Sheet for each solvent and test solution in use. Caution: To avoid damaging the kit hardware, use the PFC analysis kit only for applications involving the analysis of perfluorinated compounds. Contents Topic Page Flushing the system 3-2 Preparing eluents and wash solvents

30 Flushing the system Before performing a PFC analysis, flush the ACQUITY UPLC system thoroughly to remove background contamination that can interfere with the analysis. Flushing is especially important when the system has been used for other types of analyses and it is also required for newly installed systems. Caution: To minimize contamination, use high-purity solvents (MS-grade or better) and 18-megaohm cm resistivity water for PFC analysis. To remove background contamination, flush both existing and newly installed systems thoroughly before performing a PFC analysis. To flush the system 1. After installing the PFC analysis kit components, prepare the solvents: a. Place all solvent lines except the seal wash in a reservoir containing four liters of 100% MS-grade methanol. b. Prepare a solution of 10% methanol by mixing 100 ml of methanol with 900 ml of deionized water in a solvent reservoir, and then place the seal wash line in the 10% methanol reservoir. c. Verify that the column outlet tubing is routed to a waste container. 2. Open the MassLynx application and log on. See also: MassLynx user documentation for instructions on how to complete basic software tasks. 3. On the MassLynx toolbar, click Instrument > MS Console. 4. In the ACQUITY UPLC System tree, click Binary Solvent Manager > Control > Prime seal wash > Yes. Result: Seal-wash priming begins and continues for 15 minutes. 5. In the ACQUITY UPLC System instrument tree, click Sample Manager, and then set the column temperature to 50 C. 6. In the ACQUITY UPLC System instrument tree, click Binary Solvent Manager. 3-2 Preparing the System for Analysis

31 7. In the binary solvent manager window, prime the solvents A1 and B1: a. Click Control > Prime A/B solvents. b. Select solvent lines A1 and B1 for 3 minutes, and then click OK. c. Click Control > Set flow. d. Enter a flow rate of 0.4 ml/min for 50% A1 and 50% B1, and then click the green check mark. 8. After four hours, repeat step 6 and step 7 for the A2 and B2 solvent lines. 9. While the system is flushing, during step 6 through step 8, prime the syringes. a. Click Sample Manager > Control > Prime syringes. b. Select Sample syringe and wash syringes, enter 99 for the number of cycles, and then click OK. Allow approximately three hours. 10. After the syringe priming cycles are completed, wash the needle. a. Click Control > Wash needle. b. Enter 500 for both Strong Wash and Weak Wash, and then click OK. c. Repeat nine times. 11. When the flushing procedure is completed, connect the Column to detector tubing (attached to the analytical column outlet) to the mass spectrometer. 12. Run five blank injections before running an analytical sample. Preparing eluents and wash solvents Successful analyses with the PFC kit require specific eluents and wash solvents. Use appropriate bottles and closures to minimize contamination and bacterial growth. Carefully prepare the wash solvents and eluents, as described below, in the appropriate glassware. Caution: Use high-purity solvents (MS-grade or better) and 18-megaohm cm resistivity water for PFC analysis. Preparing eluents and wash solvents 3-3

32 Wash solvents for PFC analyses on the ACQUITY UPLC system Solvent line Strong needle wash Weak needle wash Seal wash Solvent 1 L of 100% methanol 1 L of 25% methanol in water 1 L of 10:90 methanol/water (use 5-minute washes) Eluents for PFC analyses on the ACQUITY UPLC system Solvent line A1 B1 A2 B2 Eluent 1 L of 2 mm ammonium acetate in 5:95 methanol/water (see To prepare the solvent for line A1, below) 1 L of 100% methanol Same as B1 (can place solvent line in B1 reservoir) Same as B1 (can place solvent line in B1 reservoir) Required materials Ammonium acetate MS-grade methanol 18 megaohm cm resistivity water Solvent reservoir, 1-L, with cap (including plugs for mixing) Graduated cylinders Analytical balance To prepare the solvent for line A1 1. Weigh g of ammonium acetate on an analytical balance, and then transfer it to a 1-L solvent reservoir. 2. Using a graduated cylinder, add 50 ml of methanol to the reservoir. 3. To a clean graduated cylinder, add 950 ml of 18-megaohm cm resistivity water to the same reservoir. 4. Cap the bottle, and plug any holes, to prevent leaks. 5. Shake the solvent bottle to mix the contents. 3-4 Preparing the System for Analysis

33 4 Verifying System Performance By running a standard mixture of perfluorinated compounds on a system equipped with the PFC analysis kit, you verify that the system is performing correctly. This chapter assumes you are using the Waters TQ Detector mass spectrometer. See also: The mass spectrometer s user documentation for specific information on using it with the PFC analysis kit. Warning: To avoid chemical exposure risk, always observe Good Laboratory Practices when you use this equipment and when you work with solvents and test solutions. Know the chemical and physical properties of the solvents and test solutions you use. See the Material Safety Data Sheet for each solvent and test solution in use. Contents Topic Page Preparing the standards 4-2 Performing the verification test 4-2 Interpreting the test data

34 Preparing the standards Required materials PFC calibration standards from the analysis kit Pipetter with polypropylene tips capable of delivering 10 to 1000 μl 18-megaohm cm resistivity water MS-grade methanol Vials, mm, 2-mL, polypropylene, screw-neck, with polyethylene, septumless caps To prepare the verification standard and blanks 1. Using a pipetter, transfer 1 ml of PFC calibration standard #1 to a mixing vial. 2. Deliver 3 ml of deionized water to the vial, so that the concentration of the resulting mixture is 1.25 ng/ml. Label this vial Test solution #1. 3. Prepare a 25% methanol solution by mixing 750 ml deionized water with 250 ml methanol. 4. Transfer test solution #1 to an individual UPLC sample vial for analysis as a verification standard. 5. Transfer the 25% methanol solution to UPLC sample vials to use as analysis blanks. Performing the verification test Requirement: Before you run a test to verify whether the system is operating properly for PFC analysis, ensure that the system is properly flushed (see page 3-2). The documentation CD shipped with the system includes a predefined project file for the verification test. Copy the project folder to the MassLynx directory on the hard drive of your computer before opening the project. The method in the predefined project focuses on ten, common, perfluorinated compounds. The calibration standards provided in the kit contain six additional analytes (see page 1-2) that a modified method can include. 4-2 Verifying System Performance

35 Opening the project file The PFC_Analysis.PRO project folder contains the sample list PFC_analysis.SPL. To open the project file 1. From the system CD, copy the PFC_Analysis.PRO folder to the MassLynx directory on the hard drive (usually C:\MassLynx). 2. Ensure the ACQUITY UPLC system is powered-on. 3. Open the MassLynx application and log on. See also: MassLynx user documentation for instructions on how to perform basic software tasks. 4. Click File > Open Project, and select the project folder PFC_Analysis.PRO. Result: The project opens and the sample list PFC_analysis.SPL appears. The list contains representative test samples and data for reference. 5. Click File > Open. 6. Select the PFC_analysis_run sample list to run samples. Calibrating and tuning the mass spectrometer Through the MassLynx IntelliStart interface, you can run an appropriate reference solution for ESI-negative analysis to calibrate and tune the mass spectrometer, which involves these tasks: Preparing the instrument Setting up the Tune window and preparing the mass spectrometer s solvent delivery system Generating a calibration curve using a reference solution Tuning the system on the reference material Tips: For information about using IntelliStart, consult the MassLynx online Help and the Waters TQ Detector Operator s Guide. Quattro Micro and Quattro Premier mass spectrometers require tuning and calibration. Performing the verification test 4-3

36 To calibrate the TQ detector and edit the tune file for PFC analysis 1. In the MS Console, launch the ACQUITY UPLC Console. 2. In the system tree, select TQD, and then click IntelliStart. 3. In the IntelliStart window, click Operate, API gas, and Tune. 4. In the Tune window, click File > Open. 5. In C:/MassLynx/PFC_Analysis.PRO/ACQUDB, select the tune file Instrument.ipr, and then click Open. Result: The Tune window displays the tune values. 6. Place the bottle of API calibration solution in reservoir A. 7. On the Fluidics tab, select reservoir A, and then click Purge fluidics system. 8. Select infusion as the flow state, and then specify 10 µl/min. 9. Click to start, and then minimize the Tune window. 10. In the IntelliStart window, select Instrument Setup, and then click Start. Result: The IntelliStart Setup Parameters window opens. 11. In the IntelliStart Setup Parameters window s Instrument setup tab, enter or verify the following parameters: Instrument setup conditions Parameter Value Instrument tune masses , , , and Instrument tune file Browse to C:/MassLynx/PFC_Analysis.PRO/ ACQUDB/Instrument.ipr Target resolution MS and MSMS resolution: 0.85 Da Calibration range Low mass: 2.00 Da High mass: Da Slow speed: 6.00 s Fast speed: 0.20 s 4-4 Verifying System Performance

37 Instrument setup conditions (Continued) Parameter Calibration files Calibration reporting Fluidics Value Reference: Naineg Calibration: PFC_[date].cal Print calibration report selected Enabled; reservoir A 12. Click the Sample tune tab, and enter or verify the following parameters: Sample tune conditions Parameter Value Sample tune masses , , , and Sample tune file Browse to C:/MassLynx/PFC_Analysis.PRO/ ACQUDB/PFC_analysis.ipr Fluidics Enabled; Reservoir A; Flow state: Infusion 13. To start calibration, click the green check mark. 14. When calibration is complete, maximize the Tune window. 15. Enter these values in the Tune window: Tune window conditions Parameter Value Ionization mode ESI negative Capillary voltage 3.6 kv Cone voltage As is Extractor 3 V Source temperature 150 C Desolvation temperature 400 C Desolvation gas flow 800 L/hr Cone gas flow 0 L/hr Mode MSMS Performing the verification test 4-5

38 Tune window conditions (Continued) Parameter Collision gas Acquisition mode Value Argon, at approximately kpa ( mbar, psi); 0.1 ml/min flow rate MRM 16. Save the tune file as PFC_analysis.ipr. To prepare the chromatography method and run blank and PFC standard samples 1. Place the blank sample vial in the sample tray position 2:1 and the test solution #1 (1.25 ng/ml PFC standard) sample vial in tray position 2:2. 2. In the MassLynx PFC_analysis_run.spl sample list, open the MS file named PFC_10MRM_9min. 3. Verify that the values match those in the following table: Settings for MRM of 10 mass pairs page Value Parameter Parent (m/z) Daughter (m/z) Dwell (Secs) Cone (Volts) Coll Energy (ev) Total run time 9.00 min Channels Verifying System Performance

39 Settings for MRM of 10 mass pairs page (Continued) Value Parameter Parent (m/z) Daughter (m/z) Dwell (Secs) Cone (Volts) Coll Energy (ev) Ionization mode ESI negative Repeats 1 Span 0.2 Use tune cone settings Not selected Use tune coll energy Not selected Retention window Start: 0; End: 9 4. In the MassLynx project inlet file (PFC_inlet_9min), verify that the binary solvent manager and sample manager instrument methods match the values in the following table: UPLC conditions Parameter Value Weak wash 25% methanol in water (500 μl) Strong wash Methanol (500 μl) Seal wash 90:10 water/methanol (5 minutes) Column temperature 50.0 C Sample temperature 20.0 C Sample loop and injection 10 μl (Full Loop) volume Column ACQUITY UPLC BEH C mm Mobile phase A1 2 mm ammonium acetate in 95:5 water/methanol Mobile phase B1 100% methanol Run time 9.00 min Performing the verification test 4-7

40 UPLC conditions (Continued) Parameter Low pressure limit 0 High pressure limit psi Gradient method Value Time (min) Flow rate (ml/min) %A %B Curve Confirm that you added 5 blank samples and your verification standard samples to the sample queue. Tip: To add a new test sample to the sample queue, click Samples > Add, and then specify the appropriate sample information. Use the optimized, mass-spectrometer information. Recommendation: At system startup, always run 5 blank samples before your analysis, to equilibrate the system and monitor the level of background PFC contamination. 6. Confirm the following selections: MS tune file is PFC_analysis. Injection volume in the sample list is 10 μl. Mobile phases match the solvents in the table titled Gradient method on page Prime the A and B pumps for 3 min. 4-8 Verifying System Performance

41 8. Start the flow at 0.4 ml/min with 75% solvent A for 3 min, and inspect all connections and columns for leaks. 9. In the MassLynx sample list window, click Run. Recommendations Set up a refined MS method, with multiple MRM time windows, allowing more time to scan target MRM transitions at the target PFC peak, which yields better signal-to-noise ratios and peak detection. (See the application note in the startup CD, Quantifying Trace Levels of Perfluorinated Compounds with an ACQUITY PFC Analysis Kit.) To achieve optimal sensitivity for each PFC analyte, use IntelliStart s Develop method functions, which fine-tune the MRM transitions, cone voltage, and collision energy for each perfluorinated compound. (See the MassLynx online Help and the Waters TQ Detector Operator s Guide.) At system startup, and before each run, monitor the level of background PFC contamination, and verify the performance of the isolator column by running 5 blank injections and a PFC standard. Interpreting the test data The system is performing correctly when the following statements about your processed data are true: The retention time difference between the perfluorooctanoic acid (PFOA) analyte peak and the leading PFOA contaminant peak is at least 0.4 minutes. The PFOA and perfluorononanoic acid (PFNA) peaks are baseline-resolved. To process data from the verification standard 1. In the MassLynx window, click the line number of the standard for which you are processing data. 2. In the sample list menu bar, click Chromatogram. Result: The TIC chromatogram for the standard appears. Interpreting the test data 4-9

42 TIC chromatogram of PFC standard (ESI negative mode) 3. In the Chromatogram window, click Display > Mass, and then double-click the mass transition for PFOA (413 > 369) in the function list. 4. Select Add Trace (or Replace trace) > OK. Result: The chromatogram for PFOA appears. 5. Click Process > Smooth, and then enter 3 for the window size and 2 for the number of smoothing operations. 6. Select Mean > OK. Result: The smoothed chromatogram for PFOA appears. Tip: To delete the TIC and the unsmoothed chromatogram, click within the plot area (a colored square appears to the left of the plot), press the keyboard s Delete button, and then click OK. 7. Click Display > View Verifying System Performance

43 8. In the Chromatogram Display View window, select Dot from the Grid drop-down menu, and click OK. Smoothed PFOA chromatogram 9. Expand the view of the analyte peak and the leading contaminant peaks. Tip: To draw a box around the peaks of interest, click the top-left corner of the region of interest, and then drag diagonally downward to the axis, without extending the box below the axis. 10. Measure the distance between the apex of the PFOA peak and the half-height of the first contaminant peak. Tip: Click and drag a horizontal line between the two points. The retention time difference between the two points appears in the bottom-left corner of the chromatogram window. Interpreting the test data 4-11

44 Measuring the difference in retention time 11. Verify that the difference in retention time is at least 0.4 minutes. Tip: A distance of less than 0.4 minutes can indicate a problem (see Potential analysis problems and probable solutions on page 6-2). 12. In the Chromatogram window, click Display > Mass, and then double-click the mass transition for PFNA in the Function list (463 > 419). 13. Select Add Trace > OK. Result: The chromatogram for PFNA appears. 14. Click Process > Smooth, and then specify 3 as the window size and 2 as the number of smooths. 15. Select Mean > OK. Result: The smoothed chromatogram for PFNA appears. Tip: To delete the unsmoothed PFNA chromatogram, click within the plot area (a colored square appears to the left-hand side of the plot), and then press Delete > OK. 16. Click Display > View. 17. In the Chromatogram Display View window, select Overlay Graphs > OK Verifying System Performance

45 PFOA and PFNA peaks 18. Examine the PFOA and PFNA peaks in the plots that you overlaid, and verify they are baseline-resolved. Tip: Peaks not baseline-resolved can indicate a problem (see Potential analysis problems and probable solutions on page 6-2). Interpreting the test data 4-13

46 4-14 Verifying System Performance

47 5 Preparing Samples The sample preparation procedure in this chapter serves as a guideline for preparing a surface water sample for PFC analysis. Typical sample preparation procedure Caution: To avoid sorption of PFC analytes on the walls of containers, use only polypropylene sample vials. To limit contamination, wash lab ware with nonphosphate, alkaline soap, and rinse it well with warm water. Rinse with methanol prior to use. If previous use is unknown, see Thorough cleaning of solvent reservoirs on page 6-4. Required materials Oasis HLB PLUS extraction cartridge, 225 mg, 60 μm SPE extraction manifold MS-grade methanol 18-megaohm cm resistivity water PFC Internal Standard from the analysis kit, 1-ng/mL dilution (page 1-5) Surface water sample Nitrogen gas supply Typical sample preparation procedure 5-1

48 Requirement: The system requires a supply of dry, oil-free nitrogen with a purity of at least 95%. The use of nitrogen cylinders is acceptable. Caution: If copper tubing is used for the nitrogen line, the copper must be chemically cleaned; if stainless steel tubing is used, the stainless steel must be medical grade. Ensure that there are no soldered or brazed joints in the line, as these may result in contamination of the instrument with tin or lead oxide. Any joints in the nitrogen line must be compression fittings. Centrifuge tube, 15-mL polypropylene Pipetter with polypropylene tips capable of delivering 10 to 1000 μl Nitrogen evaporator or nitrogen evaporator with a heated water-bath evaporator capable of attaining 25 to 60 C and 69 to 103 kpa (0.69 to 1.03 bar, 10 to 15 psi) nitrogen. Sample vials, mm, 2-mL, polypropylene, screw-neck, with polyethylene, septumless caps Vortex mixer Polypropylene bottle with cap To prepare a sample for PFC analysis 1. Set up the SPE extraction manifold following the manufacturer s recommendations. 2. Place Oasis HLB SPE cartridges onto the extraction manifold. 3. At a flow rate of less than 10 ml/min, pass 10 ml of methanol through the Oasis cartridge to condition it. 4. Pass 10 ml of 18-megaohm cm resistivity water through the cartridge. 5. Measure 500 ml of water sample into the polypropylene bottle. 6. Using a pipetter, add 50 μl of 1-ng/mL internal standard solution to the sample. 7. At a flow rate of 5 ml/min, pass 500 ml of the water sample (including the internal standard) through the Oasis cartridge. 8. To purge the cartridge and remove water, pass nitrogen through it until the cartridge returns to its original (dry) color, from top to bottom (approximately 20 minutes). 5-2 Preparing Samples

49 9. At a flow rate of 1 ml/min, pass 2 ml of methanol through the cartridge, and collect the solvent in a 15-mL polypropylene centrifuge tube. 10. Using a nitrogen/bath evaporator set to 35 C, evaporate the eluent in the tube until it reaches a total volume of 500 μl. 11. Using a pipetter, deliver 300 μl of the methanol eluent to a UPLC sample vial. 12. Deliver 900 μl of 18-megaohm cm resistivity water to the same vial. Result: The target sample mixture is 1:3 eluent/water. 13. Using a vortex mixer, mix the vial for 5 seconds or until the sample is uniform. Typical sample preparation procedure 5-3

50 5-4 Preparing Samples

51 6 Troubleshooting See page 6-2 for information about how to solve potential analysis problems. See also: ACQUITY UPLC System Operator s Guide for general information on troubleshooting the system. Controlling Contamination in UltraPerformance LC/MS and HPLC/MS Systems (part number ). Contents Topic Page PFC analysis troubleshooting 6-2 Thorough cleaning of solvent reservoirs

52 PFC analysis troubleshooting Consult the table below for help solving problems with your analysis. Potential analysis problems and probable solutions Symptom Possible cause Solution Contaminant peaks are larger than expected and are comparable to analyte peaks Retention time difference between PFOA (analyte) peak and leading PFOA-contaminant peak is less than 0.4 minutes; analyte peak shapes are normal Retention time difference between PFOA (analyte) peak and leading PFOA-contaminant peak is less than 0.4 minutes; noticeable broadening of peaks System has not been sufficiently flushed Incorrect flow rate Incorrect solvent composition PFC Isolator column is not performing correctly Analytical column is not performing correctly Repeat the methanol flushing procedure (page 3-2) until the background contamination is at a reasonable level. Verify the method. Adjust the flow rate to the correct value. Verify the method and the solvent supply. Adjust the solvent composition as needed. Replace the PFC Isolator column. Compare current resolution of PFOA and PFNA peaks to the resolution at installation. Verify column efficiency. Replace column if needed. 6-2 Troubleshooting

53 Potential analysis problems and probable solutions (Continued) Symptom Possible cause Solution PFOA and PFNA peaks are not baseline-resolved PFC peaks appear in blank runs along with delayed background contaminant peaks Incorrect flow rate Incorrect solvent composition Analytical column is not performing correctly PFC contamination in injector PFC contamination in sample vial PFC contamination in solvents Verify the method. Adjust the flow rate to the correct value. Verify the method and the solvent supply. Adjust the solvent composition as needed. Compare current resolution of PFOA and PFNA peaks to the resolution at installation. Verify column efficiency. Replace column if needed. Repeat the flushing procedures specific to the sample manager (page 3-3). Prime the syringes and wash the needle until the peaks no longer appear. Obtain clean sample vials. For best results, use polypropylene vials and caps. Avoid any vial with a Teflon-lined cap. Use higher purity solvents for your analysis. See the Cautions, below. Caution: To avoid contamination, use MS-grade solvents and 18-megaohm cm resistivity water for your analysis. Purify the solvents by solid-phase extraction (Waters Oasis MAX or HLB cartridges, for example), if necessary. PFC analysis troubleshooting 6-3

54 Caution: Replacing check valves can increase the background PFC contamination in the system. To avoid additional contamination, flush the system thoroughly after replacing check valves (page 3-2). Thorough cleaning of solvent reservoirs When a container s history is unknown, you must clean it thoroughly. To thoroughly clean solvent reservoirs 1. Sonicate with MS-quality 10% formic or nitric acid. 2. Sonicate with water. 3. Sonicate with methanol. 4. Sonicate with water. 5. Repeat steps 1 through 4, twice. 6. Rinse with organic solvent, MS-grade water, and, finally, with the solvent that the reservoir will contain. Recommendations: Do not wash reservoirs in detergent, alongside other glassware, or in washing facilities that can retain detergent residue. Store glassware separately from common-use glassware. To clean solvent reservoirs contaminated with microbial growth 1. Treat the reservoirs in an autoclave. 2. Remove and replace all filters and tubing between the solvent reservoir and the instrument. 3. Purge the system with methanol, and let it stand overnight. 6-4 Troubleshooting

55 A Safety Advisories Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments. Their corresponding user guides also include the hazard symbols, with accompanying text statements describing the hazards and telling you how to avoid them. This appendix presents all the safety symbols and statements that apply to the entire line of Waters products. Contents Topic Page Warning symbols A-1 Caution symbol A-5 Warnings that apply to all Waters instruments A-5 Electrical and handling symbols A-12 Warning symbols Warning symbols alert you to the risk of death, injury, or seriously adverse physiological reactions associated with an instrument s use or misuse. Heed all warnings when you install, repair, and operate Waters instruments. Waters assumes no liability for the failure of those who install, repair, or operate its instruments to comply with any safety precaution. Task-specific hazard warnings Warning symbols alert you to the risk of death, injury, or seriously adverse physiological reactions associated with an instrument s use or misuse. Heed all warnings when you install, repair, and operate Waters instruments. Waters assumes no liability for the failure of those who install, repair, or operate its instruments to comply with any safety precaution. Warning symbols A-1

56 Task-specific hazard warnings The following warning symbols alert you to risks that can arise when you operate or maintain an instrument or instrument component. Such risks include burn injuries, electric shocks, ultraviolet radiation exposures, and others. When the following symbols appear in a manual s narratives or procedures, their accompanying text identifies the specific risk and explains how to avoid it. Warning: (General risk of danger. When this symbol appears on an instrument, consult the instrument s user documentation for important safety-related information before you use the instrument.) Warning: (Risk of burn injury from contacting hot surfaces.) Warning: (Risk of electric shock.) Warning: (Risk of fire.) Warning: (Risk of needle puncture.) Warning: (Risk of injury caused by moving machinery.) Warning: (Risk of exposure to ultraviolet radiation.) Warning: (Risk of contacting corrosive substances.) Warning: (Risk of exposure to a toxic substance.) Warning: (Risk of personal exposure to laser radiation.) Warning: (Risk of exposure to biological agents that can pose a serious health threat.) A-2 Safety Advisories

57 Warnings that apply to particular instruments, instrument components, and sample types The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts. Burst warning This warning applies to Waters instruments fitted with nonmetallic tubing. Warning: Pressurized nonmetallic, or polymer, tubing can burst. Observe these precautions when working around such tubing: Wear eye protection. Extinguish all nearby flames. Do not use tubing that is, or has been, stressed or kinked. Do not expose nonmetallic tubing to incompatible compounds like tetrahydrofuran (THF) and nitric or sulfuric acids. Be aware that some compounds, like methylene chloride and dimethyl sulfoxide, can cause nonmetallic tubing to swell, which significantly reduces the pressure at which the tubing can rupture. Mass spectrometer flammable solvents warning This warning applies to instruments operated with flammable solvents. Warning: Where significant quantities of flammable solvents are involved, a continuous flow of nitrogen into the ion source is required to prevent possible ignition in that enclosed space. Ensure that the nitrogen supply pressure never falls below 690 kpa (6.9 bar, 100 psi) during an analysis in which flammable solvents are used. Also ensure a gas-fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails. Mass spectrometer shock hazard This warning applies to all Waters mass spectrometers. Warning: To avoid electric shock, do not remove the mass spectrometer s protective panels. The components they cover are not user-serviceable. Warning symbols A-3

58 This warning applies to certain instruments when they are in Operate mode. Warning: High voltages can be present at certain external surfaces of the mass spectrometer when the instrument is in Operate mode. To avoid non-lethal electric shock, make sure the instrument is in Standby mode before touching areas marked with this high voltage warning symbol. Biohazard warning This warning applies to Waters instruments that can be used to process material that might contain biohazards: substances that contain biological agents capable of producing harmful effects in humans. Warning: Waters instruments and software can be used to analyze or process potentially infectious human-sourced products, inactivated microorganisms, and other biological materials. To avoid infection with these agents, assume that all biological fluids are infectious, observe Good Laboratory Practices, and consult your organization s biohazard safety representative regarding their proper use and handling. Specific precautions appear in the latest edition of the US National Institutes of Health (NIH) publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL). Chemical hazard warning This warning applies to Waters instruments that can process corrosive, toxic, flammable, or other types of hazardous material. Warning: Waters instruments can be used to analyze or process potentially hazardous substances. To avoid injury with any of these materials, familiarize yourself with the materials and their hazards, observe Good Laboratory Practices (GLP), and consult your organization s safety representative regarding proper use and handling. Guidelines are provided in the latest edition of the National Research Council's publication, Prudent Practices in the Laboratory: Handling and Disposal of Chemicals. A-4 Safety Advisories

59 Caution symbol The caution symbol signifies that an instrument s use or misuse can damage the instrument or compromise a sample s integrity. The following symbol and its associated statement are typical of the kind that alert you to the risk of damaging the instrument or sample. Caution: To avoid damage, do not use abrasives or solvents to clean the instrument s case. Warnings that apply to all Waters instruments When operating this device, follow standard quality control procedures and the equipment guidelines in this section. Caution symbol A-5

60 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. Important: Toute modification sur cette unité n ayant pas été expressément approuvée par l autorité responsable de la conformité à la réglementation peut annuler le droit de l utilisateur à exploiter l équipement. Achtung: Jedwede Änderungen oder Modifikationen an dem Gerät ohne die ausdrückliche Genehmigung der für die ordnungsgemäße Funktionstüchtigkeit verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des Systems führen. Avvertenza: qualsiasi modifica o alterazione apportata a questa unità e non espressamente autorizzata dai responsabili per la conformità fa decadere il diritto all'utilizzo dell'apparecchiatura da parte dell'utente. Atencion: cualquier cambio o modificación efectuado en esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorización del usuario para utilizar el equipo. 注意 : 未經有關法規認證部門允許對本設備進行的改變或修改, 可能會使使用者喪失操作該設備的權利 注意 : 未经有关法规认证部门明确允许对本设备进行的改变或改装, 可能会使使用者丧失操作该设备的合法性 주의 : 규정준수를책임지는당사자의명백한승인없이이장치를개조또는변경할경우, 이장치를운용할수있는사용자권한의효력을상실할수있습니다. 注意 : 規制機関から明確な承認を受けずに本装置の変更や改造を行うと 本装置のユーザーとしての承認が無効になる可能性があります A-6 Safety Advisories

61 Warning: Use caution when working with any polymer tubing under pressure: Always wear eye protection when near pressurized polymer tubing. Extinguish all nearby flames. Do not use tubing that has been severely stressed or kinked. Do not use nonmetallic tubing with tetrahydrofuran (THF) or concentrated nitric or sulfuric acids. Be aware that methylene chloride and dimethyl sulfoxide cause nonmetallic tubing to swell, which greatly reduces the rupture pressure of the tubing. Attention: Manipulez les tubes en polymère sous pression avec precaution: Portez systématiquement des lunettes de protection lorsque vous vous trouvez à proximité de tubes en polymère pressurisés. Eteignez toute flamme se trouvant à proximité de l instrument. Evitez d'utiliser des tubes sévèrement déformés ou endommagés. Evitez d'utiliser des tubes non métalliques avec du tétrahydrofurane (THF) ou de l'acide sulfurique ou nitrique concentré. Sachez que le chlorure de méthylène et le diméthylesulfoxyde entraînent le gonflement des tuyaux non métalliques, ce qui réduit considérablement leur pression de rupture. Vorsicht: Bei der Arbeit mit Polymerschläuchen unter Druck ist besondere Vorsicht angebracht: In der Nähe von unter Druck stehenden Polymerschläuchen stets Schutzbrille tragen. Alle offenen Flammen in der Nähe löschen. Keine Schläuche verwenden, die stark geknickt oder überbeansprucht sind. Nichtmetallische Schläuche nicht für Tetrahydrofuran (THF) oder konzentrierte Salpeter- oder Schwefelsäure verwenden. Durch Methylenchlorid und Dimethylsulfoxid können nichtmetallische Schläuche quellen; dadurch wird der Berstdruck des Schlauches erheblich reduziert. Warnings that apply to all Waters instruments A-7

62 Attenzione: fare attenzione quando si utilizzano tubi in materiale polimerico sotto pressione: Indossare sempre occhiali da lavoro protettivi nei pressi di tubi di polimero pressurizzati. Spegnere tutte le fiamme vive nell'ambiente circostante. Non utilizzare tubi eccessivamente logorati o piegati. Non utilizzare tubi non metallici con tetraidrofurano (THF) o acido solforico o nitrico concentrati. Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano rigonfiamenti nei tubi non metallici, riducendo notevolmente la pressione di rottura dei tubi stessi. Advertencia: se recomienda precaución cuando se trabaje con tubos de polímero sometidos a presión: El usuario deberá protegerse siempre los ojos cuando trabaje cerca de tubos de polímero sometidos a presión. Si hubiera alguna llama las proximidades. No se debe trabajar con tubos que se hayan doblado o sometido a altas presiones. Es necesario utilizar tubos de metal cuando se trabaje con tetrahidrofurano (THF) o ácidos nítrico o sulfúrico concentrados. Hay que tener en cuenta que el cloruro de metileno y el sulfóxido de dimetilo dilatan los tubos no metálicos, lo que reduce la presión de ruptura de los tubos. 警告 : 當在有壓力的情況下使用聚合物管線時, 小心注意以下幾點 當接近有壓力的聚合物管線時一定要戴防護眼鏡 熄滅附近所有的火焰 不要使用已經被壓癟或嚴重彎曲管線 不要在非金屬管線中使用四氫呋喃或濃硝酸或濃硫酸 要了解使用二氯甲烷及二甲基亞楓會導致非金屬管線膨脹, 大大降低管線的耐壓能力 A-8 Safety Advisories

63 警告 : 当有压力的情况下使用管线时, 小心注意以下几点 : 当接近有压力的聚合物管线时一定要戴防护眼镜 熄灭附近所有的火焰 不要使用已经被压瘪或严重弯曲的管线 不要在非金属管线中使用四氢呋喃或浓硝酸或浓硫酸 要了解使用二氯甲烷及二甲基亚枫会导致非金属管线膨胀, 大大降低管线的耐压能力 경고 : 가압폴리머튜브로작업할경우에는주의하십시오. 가압폴리머튜브근처에서는항상보호안경을착용하십시오. 근처의화기를모두끄십시오. 심하게변형되거나꼬인튜브는사용하지마십시오. 비금속 (Nonmetallic) 튜브를테트라히드로푸란 (Tetrahydrofuran: THF) 또는농축질산또는황산과함께사용하지마십시오. 염화메틸렌 (Methylene chloride) 및디메틸술폭시드 (Dimethyl sulfoxide) 는비금속튜브를부풀려튜브의파열압력을크게감소시킬수있으므로유의하십시오. 警告 : 圧力のかかったポリマーチューブを扱うときは 注意してください 加圧されたポリマーチューブの付近では 必ず保護メガネを着用してください 近くにある火を消してください 著しく変形した または折れ曲がったチューブは使用しないでください 非金属チューブには テトラヒドロフラン (THF) や高濃度の硝酸または硫酸などを流さないでください 塩化メチレンやジメチルスルホキシドは 非金属チューブの膨張を引き起こす場合があり その場合 チューブは極めて低い圧力で破裂します Warnings that apply to all Waters instruments A-9

64 Warning: The user shall be made aware that if the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. Attention: L utilisateur doit être informé que si le matériel est utilisé d une façon non spécifiée par le fabricant, la protection assurée par le matériel risque d être défectueuses. Vorsicht: Der Benutzer wird darauf aufmerksam gemacht, dass bei unsachgemäßer Verwenddung des Gerätes die eingebauten Sicherheitseinrichtungen unter Umständen nicht ordnungsgemäß funktionieren. Attenzione: si rende noto all'utente che l'eventuale utilizzo dell'apparecchiatura secondo modalità non previste dal produttore può compromettere la protezione offerta dall'apparecchiatura. Advertencia: el usuario deberá saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante, las medidas de protección del equipo podrían ser insuficientes. 警告 : 使用者必須非常清楚如果設備不是按照製造廠商指定的方式使用, 那麼該設備所提供的保護將被消弱 警告 : 使用者必须非常清楚如果设备不是按照制造厂商指定的方式使用, 那么该设备所提供的保护将被削弱 경고 : 제조업체가명시하지않은방식으로장비를사용할경우장비가제공하는보호수단이제대로작동하지않을수있다는점을사용자에게반드시인식시켜야합니다. 警告 : ユーザーは 製造元により指定されていない方法で機器を使用すると 機器が提供している保証が無効になる可能性があることに注意して下さい A-10 Safety Advisories