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1 The Waters GPC Cleanup System uses two Envirogel GPC Cleanup Columns to perform routine cleanup of environmental samples as specified by EPA Method 3640A, GPC Cleanup. The columns, 19 x 150 mm and 19 x 300 mm, are connected in series. A 4.6 x 30 mm Guard Column is available for customers interested in using a guard column in front of the column set. The Envirogel columns: are packed with high-performance, fully-porous, highly cross-linked, styrene divinylbenzene copolymer particles. contain 100 Å pore size material, with a nominal particle size of 15 μm. are shipped with methylene chloride as the mobile phase. the same three columns packed in 50/50 ethyl acetate/ cyclohexane are also available. Contents I. Installing the Columns a. Connecting the Column b. Equilibrating the Columns II. Calibrating the Columns a. Preparing the Calibration Solution b. Procedure c. Injecting the Calibration Solution d. Calculating Peak Resolution e. Requirements III. Column Care and Use Guidelines IV. Column Storage a. Short-term b. Long-term V. Care and Maintenance VI. Ordering Information VII. Warrenty/Service Information

2 I. INSTALLING THE COLUMNS When installing the Waters GPC Cleanup System for the first time, refer to Chapter 2 Preparing the System for instructions on connecting the fluid lines and purging the system. This section describes: Connecting columns to the system Equilibrating the columns Do not connect the columns to the injector or detector until the GPC Cleanup System is purged with methylene chloride. Refer to the Waters GPC Cleanup System Operator s Guide for more information. a. Connecting Columns To connect the columns to the system: 1. Remove the end plugs from the columns and save them for use when storing the columns. 2. Place a ring stand or other column mounting device between the injector and detector. Mount the columns so that sample flows through the columns as shown in Figure Connect the tubing from the injector outlet to the column inlet on the small column. Using a wrench, tighten the connection 1/4 to 1/2 turn beyond hand-tight. Do not over-tighten the fittings; over-tightening damages the connection. 4. Connect the outlet of the smaller column to the inlet of the larger column, using the inch i.d., U-shaped, and column joining tube. 5. Connect the column outlet tubing (supplied separately with the detector) to the outlet of the larger column. Place a waste collection flask at the outlet tubing from the last column. 6. Set the pump flow rate to 1 ml/min, and collect column effluent for 1 minute. 7. Increase the flow rate 1 ml/min, and collect column effluent for 1 minute. Repeat until the flow rate is 5 ml/min. 8. Allow solvent to flow at 5 ml/min, until approximately 100 ml of solvent is collected. 9. Set the pump flow rate to 0.0 ml/min. Allow the system pres sureto return to 0 psi (approximately 30 seconds). 10. Connect the column outlet tubing to the sample inlet connection on the detector. b. Equilibrating the Columns To equilibrate the columns: 1. Resume flow as in steps 6 and 7 above, until the flow rate is 5 ml/min. 2. Adjust the absorbance level to 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 you should observe no large fluctuations in pump pressure (as indicated by the pump pressure transducer). Typical pump pressures are approximately 200 psi at flow rates of 5 ml/min. Figure 1. Column Installation. 2

3 4. Continue the flow until a stable absorbance reading is obtained. The columns are equilibrated when the change in the absorbance reading is AU from zero after 500 ml of solvent passes through the column. 5. Read just the absorbance level to zero, if necessary II. CALIBRATING THE COLUMNS The Envirogel GPC Cleanup columns are calibrated using a calibration solution. Calibrating the columns involves: Preparing the calibration solution Injecting the calibration solution Calculating peak resolution If you are using an injector with a 5 ml sample loop, use the calibration method listed in the EPA method. If you are using the Waters GPC Cleanup System, use the calibration method described in this section. Since the Waters GPC Cleanup System performs 2 ml sample injections, you must use a calibration solution that is 2.5 times more concentrated than the calibration solution listed in the EPA method. A 2 ml injection of the concentrated calibration solution achieves mass loading equal to a 5 ml injection of the EPA method calibration solution. a. Preparing the Calibration Solution Mix the calibration compounds into low acid methylene chloride (pesticide quality or equivalent). Refer to the Waters GPC Cleanup System Operator s Guide, Chapter 2 Preparing the System for the procedure to determine the acid level of methylene chloride. One liter of calibration solution requires the following quantities of compounds: Compound mg/l Corn oil 62,500 Bis(2-ethylhexyl)phthalate 2,500 Methoxychlor 500 Perylene 50 Sulfur 200 Reduce the quantities for smaller volumes of solution. b. Procedure To prepare the calibration solution: 1. Weigh the Corn oil into a beaker. Since the sulfur is more soluble in warm com oil than methylene chloride, add the sulfur to the corn oil. Warm the mixture until the sulfur dissolves. 2. Add the remaining compounds listed in the table above to a volumetric flask. 3. Add the warm corn oil-sulfur mixture to the volumetric flask. 4. Add methylene chloride to bring the volume of solution to one liter. 5. 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. 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 GHP Acrodisc 0.45 μm filter before injection. 3

4 c. Injecting the Calibration Solution To inject the calibration solution: 1. Filter approximately 10 ml of calibration solution through a GHP Acrodisc 0.45 μm filter. d. Calculating Peak Resolution To calculate the resolution between the peaks, measure the resolution height of the valley between the peaks, and the height of the smaller peak (Figure 3). 2. For 717 autosamplers, fill a WISP sample vial with 3 to 4 ml of calibration solution, and place the vial in the carriage at position For manual injectors with 2 ml sample loop, fill a 10 ml syringe with calibration solution. Inject 2 ml of solution into the sample loop. 4. Inject the calibration solution by either: Pressing the RUN/STOP key on the autosampler, or Switching the injector to INJECT The initial peak for corn oil appears approximately 10 to 12 minutes after injection. The final peak for sulfur appears approximately 25 minutes after injection. Figure 2 shows a sample calibration chromatogram. Figure 3. Peak Resolution. Calculate the resolution by: %R = 100 (1 - A/B) Where: R = resolution in percent A = height of the valley B = height of smaller peak e. Requirements EPA Method 3640A requires that: All peaks are observed and symmetrical. The observed resolution between the following peaks is greater than 85%: Corn oil and phthalate peaks Phthalate and methoxychlor peaks Methoxychlor and perylene peaks The observed resolution between the perylene and sulfur peaks is greater than 90%. Figure 2. Sample Chromatogram 4

5 Due to variations in detector sensitivities and cell volumes, you may need to dilute the calibration solution to achieve the specified resolution. Refer to the Waters GPC Cleanup System Operator s Guide, Chapter 3 Calibrating the System for information on setting fraction collection times. III. COLUMN CARE AND USE GUIDELINES Gel permeation chromatography columns have a finite lifetime directly related to their care and use. Column life is reduced by contamination from samples and eluents, frequent solvent changeover, and improper handling and storage. For best results, observe the following guidelines: Protect the column from vibration and mechanical shock. IV. COLUMN STORAGE Refer to the Waters GPC Cleanup System Operator s Guide for system shutdown and storage procedures. a. Short-term During short-term column storage (storage overnight or a weekend), ensure that columns do not dry out. Leave the system power ON, and reduce the flow rate to the column to 0.5 ml/min. Divert the detector output to waste. Recalibrate the system upon startup. b. Long-term Follow the long-term shutdown procedure in the Waters GPC Cleanup System Operator s Guide. Return the column to its box with the end plugs firmly in place. Store the column at room temperature. Do not exceed flow rates of 9 ml/min or pressures greater than 1000 psi. Do not use mobile phases which include water, ketones, alkanes, or alcohols (except hexafluoroisopropanol). Protect the column from rapid changes in pressure, which can result from rapidly changing the flow rate of the solvent. Change flow rates in increments no more than 11 ml/min each minute. Dedicate columns to a specific application. Frequently switching samples and solvents accelerates column deterioration and loss of resolution. Be sure that columns do not dry out. Replace the end plugs when storing the columns. 5

6 V. CARE AND MAINTENANCE General care and maintenance procedures are addressed in the Waters GPC Cleanup System Operator s Guide. Additional symptoms that you may observe are listed in Table 1. Recalibrate columns after any cleaning procedure. Table 1. Troubleshooting Problem Possible Cause Corrective Action High pressure shutdown Clogged fluid path Change all filters in fluid path. Filter all samples and solvents before use. Replace all tubing. Flush column at 2 m by reversing direction of flow through column. Replace column. Low operating pressure Leaks Tighten all fluid connections. Insufficient solvent Refill solvent reservoir. Air in-line Purge system. Retention times too long Pump flow rate set too low Check flow setting and verify output. Leaks Tighten fluid connections. Wrong columns Verify length and diameter. Retention times too short Pump flow rate set too fast Check flow rate setting and verify output. Data collection device improperly connected Ensure data collection starts at inject signal. Wrong columns Verify length and diameter. No peaks, poor recovery Solvent flow too low Verify pump pressure. Injector blocked or leaking Check for leaks or blockage. Detector malfunctioning Check wavelength, sensitivity, signal output cables. Broad, smeared peaks, or peaks appear as doublet Retention times shift more than 5% Rearrangement of packed bed or void in the column packing Blockage or leak in injector sample loop In-line or inlet filter is partially clogged Change in flow rate Change in laboratory temperature 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 not corrected after 10 injections, replace column. 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 cyclohexane:methylene chloride. Change the inlet filters. Check flow rate. Stabilize laboratory temperature or column temperature. 6

7 Sales Offices: Austria and European Export (Central South Eastern Europe, CIS and Middle East) Australia Belgium Brazil Canada China CIS/Russia Czech Republic Denmark Finland France (33) Germany Hong Kong Hungary India and India Subcontinent Ireland Italy Japan (81) Korea (82) Mexico The Netherlands +31 (0) Norway Poland (48) Puerto Rico Singapore Spain Sweden Switzerland Taiwan United Kingdom Waters Corporation. Waters, The Science of What s Possible and Envirogel are trademarks of Waters Corporation. Acrodisc is a registered trademark of Pall Corporation. Teflon is a registered trademark of the E.I. du Pont do Nemours Company. All rights reserved. October 2007 WAT Rev 4 VW-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

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