Colorado Department of Public Health and Environment ... · Colorado Department of Public Health and Environment . Laboratory Services Division . Toxicology Laboratory . Toxic Vapors,

Colorado Department of Public Health and Environment

Laboratory Services Division Toxicology Laboratory

Toxic Vapors, GC/MS, Urine/Blood/Tox-TrapsTM Revision 0 DECEMBER 2004

TITLE ........................................................................................................................................................................... 1

REFERENCES ............................................................................................................................................................ 1

METHOD ..................................................................................................................................................................... 1

PRINCIPLE ................................................................................................................................................................. 1

SAMPLE ...................................................................................................................................................................... 1

SAFETY ....................................................................................................................................................................... 2

EQUIPMENT .............................................................................................................................................................. 2

REAGENTS ................................................................................................................................................................. 3

SAMPLE PREPARATION ........................................................................................................................................ 3 URINE PREPARATION .................................................................................................................................................. 3 TOX-TRAP PREPARATION ........................................................................................................................................... 4 BLOOD PREPARATION ................................................................................................................................................ 5

PROCEDURE .............................................................................................................................................................. 5 PREPARATION ............................................................................................................................................................. 5 GC/MS INSTRUMENT SET UP ..................................................................................................................................... 6

GC/MS Parameters ................................................................................................................................................ 6 SAMPLE SEQUENCE AND GC/MS ANALYSIS. ............................................................................................................. 8

ANALYTICAL REPORTING ................................................................................................................................... 8

TROUBLESHOOTING AND MAINTENANCE ..................................................................................................... 9 LEAK CHECKING ......................................................................................................................................................... 9 TUNE FAILURE ............................................................................................................................................................ 9 NO COMMUNICATION WITH MASS SPECTROMETER .................................................................................................. 10 MASS SPECTROMETER SOURCE PROBLEMS .............................................................................................................. 10 INSTRUMENTAL BACKGROUND PROBLEMS. ............................................................................................................. 12 REGULAR MAINTENANCE ......................................................................................................................................... 12

Checking the GC inlet for leaks. .......................................................................................................................... 12 Injection Port Maintenance ................................................................................................................................. 14

POLLUTION PREVENTION .................................................................................................................................. 16

WASTE MANAGEMENT ........................................................................................................................................ 16

APPENDIX 1 ............................................................................................................................................................. 18

Division: Laboratory Services File: SOPVOCHEAD Unit: Chemistry Revision: 0 Section: Toxicology Date: December 2004

Standard Operating Procedure Page 1 of 22

TITLE Qualitative Determination of Toxic Vapors in Urine, Tox-TrapsTM and Blood, by Gas-Chromatography and Mass-Spectrometry REFERENCES Shimadzu GC-17A Ver. 3 User’s Manual. 1995. Shimadzu Corporation.

Tekmar 7000 Headspace Autosampler Instruction Manual. 1991. Tekmar Company. Compressing the Graphite Ferrule. Shimadzu document 4-19-03. 2003. Shimadzu Corporation. METHOD Gas chromatographic separation of analytes in the analytical headspace of toxicological samples followed by mass-spectrometric analysis. PRINCIPLE Volatile organic compounds are concentrated in the analytical headspace of a 3-mL urine or solubilized ToxTrapTM sample or a 0.5-mL blood sample by heating the sample to 85oC for 20 minutes. These components are purged into a heated injection valve assembly and rapidly desorbed into a capillary gas chromatography column interfaced to a mass spectrometer. SAMPLE 3 mL of urine, 0.5 mL of blood, or the solubilized silica component of a Tox-TrapTM, stored at 4oC. Holding time is fourteen days after sampling. CALIBRATED COMPOUNDS Compound MW Quant Ion CAS # acetone 58.08 43 67-64-1 n-butyl alcohol 74.12 41 71-36-3 cyclohexanone 98.14 55 108-94-1 ethanol 46.07 45 64-17-5 ethyl acetate 88.11 43 141-78-6 ethylbenzene 106.17 91 100-41-4 diethyl ether 74.12 45 60-29-7 MIBK 100.16 43 108-10-1 o-xylene and p-xylene 106.17 91 106-42-3 m-xylene 106.17 91 108-38-3 isopropyl alcohol 60.10 45 67-63-0



SAFETY Use routine precautions found in the Chemical Hygiene Plan (Appendix I – Safety Manual) when working in the laboratory. Follow the Bloodborne Pathogens Exposure Control Plan (Appendix G – Safety Manual), when working with biological fluids or tissues. Read all Material Safety Data Sheets before handling unfamiliar reagents. EQUIPMENT 1. Shimadzu GC-17A gas chromatograph using a Restek DB-VRX column or equivalent with

integrated QP 5000 Shimadzu Mass Spectrometer and:

a) Tekmar 7000 Automated Headspace Sampling System b) Shimadzu GC/MSolutions Mass Spectral Software c) AMDIS (Automated Mass-Spectral Deconvolution and Identification System) Software,

National Institute of Standards and Technology 2. Thermal Conductivity Leak Detector. 3. Glass Headspace Vials, 20-mL capacity. Note: Inspect lip and cap threads of sampling and headspace vials for gaps or cracks that may cause sample loss or septa leakage under pressure. 4. 10 and 25 µL micropipettes, for standards preparation. 5. 5 mL pipette, for sample aliquots 6. Min-Inert caps and vials for frozen standard storage. 7. Restek RTX-VMS column, 30 meter length x 0.25 µm inner diameter x 1.4 µm film

thickness, part no. 19915, or equivalent. 8. Shimadzu inlet wrench, part no. 221-41571. 9. Shimadzu Thermolite plug septa, Restek part no. 20372. 10. Shimadzu splitless glass insert, part no. 221-41544, or equivalent. 11. Shimadzu graphite ferrule for glass insert, part no. 221-15563-91, or equivalent. 12. Shimadzu split/splitless ferrule adjuster, part no. 221-41532-91.



13. Shimadzu column nut, part no. 221-32705. 14. Shimadzu 0.4 mm column ferrule, part no. 221-32126-04. REAGENTS 1. Methanol, Optima, HPLC, or Nano-grade, Keep separate from all other solvents, especially

those used in semivolatiles analysis. 2. Helium Ultra high purity, >99.9999%. 3. Reagent water, 18.3 M-ohm or better, use ultra-purified water from the ELGA purification

system in room 131. 4. Standards mix, Ultra Scientific F003 List Mixture, Catalog number FLM-003 or similar. 5. Standards mix, Ultra Scientific Alcohols Mixture, Catalog number FLM-002 or similar. 6. Internal Reference/Surrogate Standard Mix, 1,4-chlorofluorobenzene solution, 600 ppm. (25

µL neat 1,4-CFB to 50 mL methanol) Store in a refrigerator at 4o C for up to 1 year. SAMPLE PREPARATION

1. Samples will be prepared in clean, 20-mL sample vials on the day of the analysis. All

samples and standards will be brought to room temperature at the time of sample preparation. 2. The first vial prepared and analyzed will be a “clean blank” containing approximately 5 mL

reagent water with no internal standard. This “clean blank” cleans and conditions the GC column for further analysis.

3. All samples following the clean blank are prepared in accordance with the three procedures

below. If a sample type is not present in the batch, skip the preparatory section for that sample.

Urine Preparation

1. Dispense 3-mL aliquots of each sample into clean headspace vials and cap each vial with a

rubber septa (DO NOT CRIMP SEAL). 2. Dispense 3-mL aliquots of negative urine into two empty headspace vials vials and cap each

vial with a rubber septa. 3. Into one of the vials prepared in step two, micropipette 10 µL of Alcohol Mix standard and 5

uL F003 List mix standard.



4. Micropipette 10 µL of the internal standard stock solution into each of the calibration standards, blanks, and samples prepared above. Quickly cap and crimp all vials to prevent analyte losses.

5. Crimp the headspace vials securely.

a) Push the gray septa into the vial opening till it is firmly set. b) Place an aluminum-sealing ring over the septa.

c) Ensure that the sealing ring is centered.

d) Crimp the sealing ring with a headspace vial-crimping tool by placing the head of the tool

over the ring and septa while squeezing the handles tightly closed.

e) Turn the vial 90o and crimp again.

f) Check the seal by twisting the aluminum-sealing ring. If the cap is firmly in place (does not twist easily), the vial is sealed well.

Tox-Trap Preparation

1. Dispense 3-mL aliquots of deionized water for each Tox-trap, one Tox-trap blank, one DI

water blank, and one DI water mix into clean headspace vials and cap each vial with a rubber septa (DO NOT CRIMP SEAL).

2. Into the DI water mix vial prepared in step one, micropipette 10 µL of Alcohol Mix standard

and 5 uL F003 List mix standard. 3. One trap at a time, remove the cap from the sample-adsorbent end of each Tox-trap to be

analyzed, remove the plastic retainer and glass wool with forceps. Tapping gently, empty the entirety of the silica-gel adsorbent from the trap into the headspace vial and cap each vial with rubber septa.

4. Micropipette 10 µL of the internal standard stock solution into each of the calibration

standards, blanks, and samples prepared above. Quickly cap and crimp all vials to prevent analyte losses.

5. Crimp the headspace vials securely.

g) Push the gray septa into the vial opening till it is firmly set. h) Place an aluminum-sealing ring over the septa. i) Ensure that the sealing ring is centered.



j) Crimp the sealing ring with a headspace vial-crimping tool by placing the head of the tool

over the ring and septa while squeezing the handles tightly closed. k) Turn the vial 90o and crimp again.

6. Check the seal by twisting the aluminum-sealing ring. If the cap is firmly in place (does not

twist easily), the vial is sealed well. Blood Preparation

1. Dispense 0.5-mL aliquots of sheep’s blood for each one blank, and one mix into clean

headspace vials and cap each vial with a rubber septa (DO NOT CRIMP SEAL). 2. Dispense 0.5-mL of sample into headspace vial. 3. Dispense one 0.5-mL aliquot of DI water into each headspace vial. 4. Into both the blood mix vial and the DI water mix vial prepared in steps one and two,

micropipette 10 µL of Alcohol Mix standard and 5 uL F003 List mix standard. PROCEDURE Preparation

Prepare the Tekmar 7000 Headspace Autosampler and GC Column for Sample Processing 1. If it has not already been done, select Method 1 on the autosampler.

a) Press the F1 (Method) key on the Tekmar front panel. b) Press 1 on the numeric keypad. c) Press F2 (Run), and the method will start, as above. Note that normal cooling times are

approximately 1 hour, so plan accordingly (this is a great time to prepare your samples and standards).

Tekmar 7000 Headspace Autosampler Parameters for Method 1 Parameter Value Line Temperature 175o C Valve Temperature 175o C Mount Temperature 175o C GC Start Option Start of Desorb GC Cycle Time 20 minutes



Parameter Value Desorb Time 1 minute 2. Reset the column temperature to 35o C On the Shimadzu GC keypad select the Col button,

followed by 3, 5, and Enter. 3. Select F2 (A/S) to change to the Auto-sample Set-up menu in the Tekmar 7000 LCD panel. 4. Select F2 (Sched.) to select the Schedule Editor. 5. Enable the schedule function (if not already enabled) by pushing F3 () twice and then the Y

key on the numeric keypad (this is also the 7 key). 6. Enter the number of the last sample vial by pushing F3 () until under the column headed

Stop, and pressing the Enter key. 7. Press F3 () on the keypad. 8. Press the Enter key, followed by the sample sequence start number (generally # 1) and again

press Enter. 9. Press F3 () on the keypad. 10. Press the Enter key, followed by the sample sequence end number (the number of the last

vial in the autosampler) and again press Enter. 11. Press F4 twice to return to the Main Menu. GC/MS Instrument Set Up GC/MS Parameters

Solvent delay 1.50 min Mass scan parameters 35-450 Sampling # 2 (less than 1 per second) Inlet Temperature 175°C Injector Temperature 175°C Gas Pressure 25 cm/sec 11.6 psi constant flow mode Purge injector splitless mode Carrier gas He (UHP Grade) Column temp program initial 35°C for five minutes

20 degrees/min to 220°C MS Source Temperature 230o C



1. Ensure that the valve on the Shimadzu helium tank attached to the VOC apparatus is open and that the regulator registers a pressure of 500 psi or greater and the second regulator is at least 80 psi. If the gas is less than 500 psi, replace the helium tank.

2. Turn on the computer workstation and cancel the password entry routine for LAN logon. 3. Select the GCMS Real Time Analysis icon on the windows desktop to start the GC/MS

software. 4. Select Wizard… from the Edit Menu. 5. From the next three pop-up windows, make these selections, in order:

a) New Batch Table b) Line 1 for the Batch Type

c) Unknown Only

d) C:\GCMSsolution\Data\Methods\HSTV.qgm for the method file. This selection can

be made from a file explorer type browser by pressing the green folder icon at the right of the text box.

e) Check both Quantitative and Qualitative analysis.

f) Select the Next > button at the bottom of the window.

g) 1 Vial

h) The number of vials in your prepared batch for the Sample Count.

i) 0 for the Injection Volume.

j) Delete the contents of the text box Sample Name.

k) Use MMDDYY01 format for Sample ID and check the Auto-Increment box. Example:

for samples analyzed on March 2, 2006 enter 030206001.

l) Select the Next > button at the bottom of the window.

m) Use MMDDYY01 format for Data File Name and check the Auto-Increment box. Example: for samples analyzed on March 2, 2006 enter 030206001.



n) Check the Report Out box and select C:\GCMSsolution\Data\Reports\TVreport.qgr as the report file by clicking on the data explorer button to the right of the text box and using the browse feature.

6. Review the batch table generated by the wizard. If no Tuning files are listed in the column

labeled Tuning File select VO.qgt by clicking the mouse button at the right side of the first cell and choosing from the list offered. Copy the tuning file to all rows of the column by right-clicking the mouse and selecting Copy in the first cell and Paste in each successive cell.

7. From the Assistant Bar, select the Tuning icon from the Top menu (Real Time appears at the

top of the Assistant Bar). 8. Save the Batch File, if requested to do so. 9. Select the green right arrow button from the Assistant Bar or the Menu at the top of the

Tuning Window to start the Auto-tuning process. 10. Wait until all tuning adjustments and calibrations are completed (approximately 3 minutes). 11. Select Save Tuning File from the File menu.

Sample Sequence and GC/MS Analysis.

1. Return to the Batch Processing window by selecting the Batch Table tab, or selecting Batch

Processing from the Assistant Bar under Top. 2. Press the Start button (green right arrow). 3. At this point, the software will begin preparation of the run. After a few seconds, the system

will pause and a message box will appear stating that the run will begin as soon as the sampler is ready.

4. On the Tekmar 7000 autosampler check to see if “Press Start to Begin” is visible in the LCD

Panel. When this message appears, press the Start button on the keypad. 5. The question “Are applicable GC Times Correct?” will appear, select F1 (Yes). 6. Wait for the run to begin automatically, allowing the full amount of time for the solvent

delay to elapse (a prompt will indicate if you want to bypass, click the No button or ignore the prompt – it will disappear after the time indicated has passed).

ANALYTICAL REPORTING 1. All analytical reports should print on LARS_LAS#18 in the chemistry area.



2. Verify that the analytical reports are complete, and create results lines on the Toxic Vapor Analysis sample log sheet for each of the control samples (blanks and mixes).

3. Indicate the number of positive hits for calibrated compounds in each of the analytical mixes. 4. Transfer compound results for each analytical sample not present in matrix-representative

blank samples to the Toxic Vapor Analysis sample log sheet. NOTE: Verify that compounds reported in patient samples are not of biogenic origin. In general, ignore ketones and aldehydes in urine, and all compounds that have a lower analytical response (area counts) than the internal standard. Report ethanol or acetone only if concentrations are at least 1000x that of the internal standard (area counts) and confirm all alcohol results reports with a quantitative analysis by GC/FID. TROUBLESHOOTING AND MAINTENANCE

Leak checking Several connections on the GC/MS system should be checked for leaks if elevated levels of air, nitrogen, and/or water are found in the system by an air and water check. Check the following areas for leaks with a calibrated thermal-conductive leak check meter, NEVER use Snoop™ or a film liquid product. 1. The sample injection port. Check for tightness of the septum nut; it should be neither loose

nor more than a quarter turn past finger-tight. Use caution when adjusting because the injection port is heated and may be extremely hot to the touch.

2. The inlet/capillary column connection. Check that the capillary column nut is tightened

securely counter-clockwise and that the graphite/vespel ferrule is seated well within the inlet/column connection. Tighten only until resistance is evident and then tighten ¼ turn more.

3. The capillary column/mass spectrometer connection. Check to make sure the capillary

column nut is tightened securely clockwise and that the pure graphite ferrule is seated well within the connection. This column nut tends to be the biggest cause of air and water leaks into the system and has a tendency to become loose if the heat on the transfer line connection is changed.

4. Check all helium line connections between the pressure regulator on the helium tank and the

GC inlet. Tighten or replace fittings/ferrules as necessary. Tune failure A proper tune can be difficult to achieve for many reasons. Check through this list before calling maintenance personnel.



1. Tune solution. Be certain the tune solution has not expired and that it has not been exposed

to room temperature for long periods. Prepare a new solution first to replace a failed tune mix.

2. High background. Set the temperature higher during the tune operation, this can reduce the

always-present GC background by restricting column flow into the MS. 3. Software autotune problems. Attempt to tune the instrument with a variety of different tune

features. Remember that the software is basing the instrumental tune on a combination of starting ion ratios followed by a series of statistical calculations based upon those initial factors. As the initial factors change during each successive tune operation, so do the final values.

No communication with Mass Spectrometer 1. Instrument will not tune, corrupt data file created with starting time value repeated

continuously.

2. Connecting cable loose or disconnected. Check all instrumental connections between the GC/MS system and the analysis computer. Reconnect any loose or disconnected interface cables or connection retaining screws.

Mass Spectrometer Source Problems 1. Double-click the Vacuum Control icon in the Class-5000 main menu. 2. Select the Auto-Shutdown command from the Shutdown menu. 3. Allow the mass spectrometer to vent (release its vacuum) fully; this process will take

approximately 30 minutes. An estimated time to completion will appear in a small window that opens up on the computer monitor.

4. Remove the dust cover from the mass spectrometer. 5. Pull the mass spectrometer front plate open carefully. There may be a slight vacuum left in

the mass spectrometer so the sound of air entering is common at this point. 6. Note: Use nylon cloth gloves to handle all hardware inside the mass spectrometer.

Fingerprint and skin oils can contaminate the interior and be almost impossible to remove.

7. Remove the filaments from the ion source by loosening the hex screws at the top and bottom

of the vacuum chamber (2 per filament), just inside the door seal. Slide each filament toward you and away from the ion source carefully, and remove them from the vacuum chamber.



Examine the filaments for severe discoloration or filament burnout, replace with new filaments if necessary.

8. Remove the “S” and “N” magnets from above and below the ion source, respectively. 9. The ion source is attached to the inside of the side plate. Disconnect the brown colored wire

from the ion source. 10. Loosen the thumbscrew holding the ion source in place by turning clockwise until the ion

source box push rod has retracted fully to the right side of the vacuum chamber. Pull the ion source out of the mass spectrometer by sliding it to the right, using clean cloth towels and nylon gloves to protect from surface contamination. The source body may need to be loosened by carefully wiggling it up and down. Remove the source body by pulling it to the right, then toward you and out of the vacuum chamber.

11. On a clean cloth or paper wipe, remove the four screws attaching the ion lens plate to the

source body. Be careful not to lose or damage any of the stainless steel or ceramic washers that attach the ion lens to the source body.

12. Unscrew and remove the filament clips from the source body. 13. Clean all metallic parts to a high metallic luster by polishing with 3600 grit followed by

12000 grit sanding cloths. No blackening or discoloration should be present upon the source body or ion lens when polishing is complete.

14. Immerse the source body (NEVER the repeller or any ceramic parts) in acetone and clean in

an ultrasonic bath. 15. Rinse the source body with clean acetone and repeat the above step. 16. Allow the source to dry at room temperature in a clean environment for 6 to 8 hours or in a

200o C oven for ten to fifteen minutes. 17. Reassemble the source body, taking extreme care not to expose any clean parts to sources of

contamination on work surfaces. 18. Carefully replace the aluminum foil that separates the source body from the heated transfer

line assembly. Ensure that the shaped-foil piece is in place around the pins on the transfer line assembly.

19. Place the reassembled source back into the mass spectrometer. 20. Reconnect all wires to their appropriate source connections.



21. Re-tighten the ion source box push-rod by turning the set-screw counter-clockwise until the source body and push-rod are firmly in place.

22. Replace the “S” and “N” magnets to positions above and below the ion source, respectively. 23. Close the front plate and tighten the set-screw firmly. Pump down the MS by selecting Auto-

Startup from the Startup menu in the Vacuum Control window. 24. Allow the mass spectrometer to pump down (regenerate its vacuum) fully, this process will

take approximately 15 minutes. An estimated time to completion will again appear in a small window that opens up on the computer monitor.

25. Allow approximately 4 hours for equilibration before using the instrument. 26. Perform an air and water check before operating the GC oven. If air and or water values are

above 5% for any parameter, check for vacuum and seal leaks. Instrumental Background Problems. 1. Make certain the water supply for reagent water is uncontaminated. Deionized water

processed through resins and purifying apparatus commonly contains small quantities of acetone and other contaminants.

2. Teflon linings and valve coatings can leak organic volatile contaminants into apparatus.

Make note of all instrumental changes in the daily maintenance logs and use these to track the source of changes that might have introduced contaminants.

3. The instrumentation commonly produces contaminants while sitting that build up in the

concentrator trap (tetrahydrofuran being the most common). Run a “clean” blank always as a first sample to rid the concentrator of residual contaminants.

Regular Maintenance 1. Several chromatographic problems (see troubleshooting, above) may appear when the

column is in need of routine maintenance. Even without these problems, routine maintenance should be performed on all instrumental systems every six months, while specialized parts (septa, column ends) may need more maintenance.

Checking the GC inlet for leaks. 1. Supply carrier gas with a delivery pressure of greater than 600kPa to the carrier inlet at the

back of the GC17A. 2. Cool the GC oven to 45o C by selecting the COL key on the GC keypad, followed by the 4, 5,

and ENTER keys.



3. Cool the GC injector to 45o C by selecting the INJ key on the GC keypad, followed by the 4,

5, and ENTER keys. 4. Equilibrate the injector and GC oven for five minutes. 5. One recommended method is to equilibrate the injector and also turn off the GC Oven

circulation fan after the GC Oven has been cooled to ambient temperature. The circulation fan is turned off by pressing COL, OFF, and ENTER.

6. Turn off the Automatic Flow Controller (AFC) by pressing the GC keypad buttons FLOW1,

OFF, ENTER. 7. Remove the capillary column from the injection port outlet. 8. Plug the injection port column connection with a new column-to-injector graphite ferrule, not

removing the stainless steel center pin in the ferrule. 9. Turn on the AFC by pressing the FLOW1, ON, ENTER buttons. Press the FLOW1 button

and set the Pressure to 50 kPa and the Flow to 50 ml/min. 10. Allow the carrier gas to purge through the injector and out of the split and septum purge

vents for at least one minute. 11. Press the FLOW1 button, leave the Pressure at 50 kPa and set the Flow to 0 ml/min by

pressing 0 and ENTER. 12. Plug the split vent and septum purge vent both with female G plugs using 3 aluminum

gaskets (Shimadzu part no. 201-35183-00). 13. Press the FLOW1 button and set the Pressure to 0 kPa and the Flow to 50 ml/min. 14. When the pressure reaches approximately 325 kPa press FLOW1, OFF, ENTER. 15. Turn off the carrier gas supply valve located on the back of the GC. If your GC17A does not

have a carrier gas shutoff valve then shut off the carrier gas somewhere after the tank pressure regulator and before the Carrier In fitting on the back of the GC.

16. Press the FLOW1 button and set the Pressure to 0 kPa and the Flow to 50 ml/min. 17. When the pressure reaches approximately 325 kPa press FLOW1, OFF, ENTER. Also, turn

off the carrier gas supply valve located on the back of the GC.



18. Press the MONIT button. The GC keypad display should read approximately 300 to 400 kPa. If this pressure reads higher than 400 kPa open the septum purge vent plug slightly to adjust the pressure below 400 kPa.

19. Give the GC at least 5 minutes for pressure equilibration before the next step. 20. After pressure equilibration the MONIT pressure reading should drop no more than 3 kPa in

one hour. 21. When finished, slowly release pressure first through the septum purge line vent plug to

prevent the glass liner packing from being displaced inside the injector liner. Injection Port Maintenance

1. Turn down the inlet temperature on the GC by pressing the INJ key on the keypad at the

upper right hand corner of the GC oven (second instrument control row, third button) followed by the 3, 0, and ENTER keys.

2. Turn the GC oven off by pressing the COL key on the keypad, followed by the OFF and

ENTER keys. 3. Wait until the temperatures drop below 45o C in both the inlet and the oven before continuing

with maintenance. 4. Turn off the gas flow on the GC by pressing the FLOW1 button. Press the OFF key and

ENTER. The LCD display at the top of the keypad should indicate the flow status is off. 5. After the pressure has been entirely released from the inlet and the temperatures have

decreased, remove the septum nut by turning it counterclockwise. The septum rests in a cup underneath this nut at the top of the GC inlet port.

6. If the septum is silicone rubber, analyze the rubber for degradation, silicone oil bleed, or

heavy coring (evidenced by single holes in the septa larger than 0.5 mm in size). Generally septa need replacing on VOC machines every six months.

7. Remove the GC column end from the bottom of the inlet with a ¼ inch wrench. Turn the nut

counter-clockwise to loosen it. Be careful not to bend the column too far or tighten the nut instead of loosening, as this may damage the column or column nut threading.

8. Using a silicone septum, cap the open end of the column quickly by inserting the column end

into the soft silicone material to prevent air leakage into the mass spectrometer. Some leakage will occur, but this action will keep it to a minimum.

9. Remove the inlet liner from the top of the inlet by using the GC inlet wrench (large end) to

remove the inlet weldment (attached to the incoming gas lines).



10. Examine the inlet liner for damage, abrasions, discoloration, or contamination. If it is

unserviceable or more than 6 months old, replace the liner. 11. The Shimadzu inlet port liner requires the use of a compression tool to attach a 5 mm

graphite/vespel ferrule assembly. Follow the directions in appendix 1 of this SOP for the attachment of this ferrule.

12. Reinsert the inlet liner, tightening the inlet weldment with a ½ inch wrench until the nut is

secure and unable to be loosened by hand. 13. Examine the first 6 inches of the column (the end that was connected to the inlet) for damage,

abrasions, discoloration, or contamination. If the column is noticeably degraded, chromatograms are indicating high degrees of tailing in many compounds, or the column has not been serviced for a year, clip six inches from the end of the column. a) Locate a column-clipping tool in the GC toolkit. b) Selecting a point about six inches from the end of the column, score the column lightly

with the clipping tool. c) Stressing the column at both sides of the score, break the column and discard the

degraded end. d) Check the edge of the column with a magnifying lens, if possible, to ensure the break was

clean and that the edges of the break are perpendicular to the edge of the column. 14. Even if the column was not in need of the above clipping procedure, clip about ½ to 1 inch

off of the end of the column AFTER sliding the GC column nut and a new 0.4 mm (hole diameter) graphite/vespel ferrule over the open end, in that order. Make certain the GC column nut’s threads and the ferrule’s tapered end are facing the end of the column.

15. Locate the “S” labeled ferrule adjuster in the GC toolkit and push the capillary column

through the ferrule adjuster until approximately 10 mm of column sticks out from the open end.

16. Slide the slit of the column nut over the column behind the ferrule and hand-tighten the entire

assembly. 17. When completely hand-tight, tighten the assembly another ¾ turn with a wrench. 18. Clip the end of the column projecting from the tip of the ferrule adjuster.



19. Remove the column end from the inlet again and check the ferrule and column end for snug fit (column end should not be able to slide back and forth within the 0.4 mm hole in the ferrule).

20. Reinsert the column end and ferrule into the GC inlet/column connection and tighten the GC

column nut fully ½ turn past initial resistance.

POLLUTION PREVENTION

1. Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Numerous opportunities for pollution prevention exist in a laboratory operation. The United States Environmental Protection Agency (US EPA) has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, laboratory personnel should use pollution prevention techniques to address waste generation. When wastes cannot feasibly be reduced at the source, the US EPA recommends recycling as the next best option.

2. The quantity of chemicals purchased should be based on expected usage during the shelf life

and disposal cost of unused material. Actual reagent preparation volume should reflect anticipated usage and reagent stability.

3. For information about pollution prevention that may be applicable to laboratories, consult

“Less is Better: Laboratory Chemical Management for Waste Reduction”, available from the American Chemical Society’s (ACS) Department of Governmental Regulations and Science Policy, 1155 16th Street NW, Washington D.C. 20036, (202) 872-4477.

WASTE MANAGEMENT

1. The US EPA requires that laboratory waste management practice be consistent with all

applicable rules and regulations. Excess reagents, samples and method process wastes should be characterized and disposed of in an acceptable manner. The agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the letter and spirit of any waste discharge permits and regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste identification rules and land disposal restrictions. For further information on waste management consult the “Waste Management Manual for Laboratory Personnel”, available from The American Chemical Society at the address listed above.

2. Dispose of all samples in the laboratory sink with a large volume of excess water after

neutralization to pH 5-9 with sodium bicarbonate (NaHCO3).



3. Dispose of methanol reagent waste in the VOC waste container in the hood in room 129. When this container fills, send it with a statement of contents to the RCRA waste room in shipping/receiving. Give a copy of the statement of contents to the shipping manager.



APPENDIX 1 Ferrule Compression (Shimadzu Document 4-19-03 brd) This document describes how to compress a 5mm graphite ferrule on an injection port glass insert. Correctly compressing the graphite ferrule on the glass insert is an essential requirement for an injection port that will not leak. This document is specifically written for the GC-17A split/splitless injector but the same compressing is also necessary when a graphite ferrule is used on other injection ports. Below is a picture of the parts required to compress the 5mm graphite ferrule on a glass insert. In addition to parts A, B and C, two 12mm wrenches (086-03011-00) are also used in this procedure.

A. MM5 male Fitting, P/N 221-32510-00 (approximately $8 each)

B. Back Ring or Back Ferrule, sold only as an included part in the Glass Column Joint Kit P/N 221-15561-91 (approximately $23)

C. MF female Nut, P/N 221-32790-00 (approximately $6 each)

D. Graphite Ferrule, 5.1mm ID, P/N 221-46403-92 (approximately $50 for 4 ferrules)

E. Glass Insert or Inlet Liner, P/N 220-90784-00 (approximately $160 for 5 inserts) for split or splitless injection, deactivated insert with deactivated fused silica wool

PROCEDURE 1. On the topside of the glass insert, assemble all the parts exactly as the sequence shown

above. Firmly finger tighten together parts A, B, C and D. The top end of the glass insert should be lined up flush with the surface of the nut, as shown below, before tightening with the wrenches in the next step.



2. Using the 12mm ends of two 10mm/12mm open end wrenches tighten the male fitting and

nut together by squeezing the wrenches together. This action is shown starting with the left picture and ending with the middle picture below. Then slide the right side wrench off the nut and rotate the 12mm end of the wrench around 180° and slide it back onto the nut. (The dark wrench in the pictures is the right side wrench.) The result of this action is shown in the right picture below. This makes an angle between the wrenches for successive compressions after the first. A total of 5 compressions, the initial and 4 successive compressions, should be enough to compress the graphite ferrule correctly. The angle between the wrenches for the initial compression is shown in the left picture and the angle between the wrenches for successive compressions is shown in the right picture. Always keep the 12mm ends of the two wrenches close together or touching against each other to prevent stress that could break the glass insert.

3. Remove the male fitting, nut and back ring from the glass insert. This involves using the

wrenches to loosen the nut from the male fitting and also carefully grasping the edge of the back ring to pull it away from the graphite ferrule



From the side perspective, the metal band of the graphite ferrule should be positioned just a couple of millimeters below the top of the glass insert. When installed in the injector, the glass insert will be pushed down until the top of it is about 1mm above the metal band. After compressing then, the top of the glass insert should be slightly more than 1mm above the metal band as shown in the left picture below. The right picture below shows the compressed graphite ferrule on a glass insert that is halfway inserted into the injector.

The compressed graphite ferrule should have a shiny graphite surface on top, as shown below.

Compressing the graphite ferrule according to this document’s procedure is imperative for 2 reasons. Think of the Back Ring as a piston that presses down the whole of the graphite surface as it compresses. Compressing the whole of the graphite surface results in solid graphite with



the highest level of graphite in the metal band. The top of the injector has a circular edge underneath it that must make a gas tight seal into the top of the graphite ferrule as the injector nut is tightened. The circular edge pressed into the top surface of the graphite ferrule seals the top of the injector to the body of the injector and holds the pressurized carrier gas in the injector.

Reason #1 is that sample and carrier gas will leak out and destructive oxidizing air will leak in to the injector if the graphite ferrule is not compressed correctly by this document’s procedure. And further, any time that the injector top is loosened or removed, the circular edge is pulled out of the graphite ferrule and a new one should be compressed on the glass insert. Reason #2 is that the graphite must compress and seal around the glass insert because the ferrule must block split flow from making a shortcut on the outside of the glass insert to the split line. The split flow must flow down through the inside of the glass insert from top to bottom with the column flow for split or splitless analysis to work correctly and reproducibly. If the graphite ferrule is not compressed tight around the glass insert then gas can flow on the outside of the glass insert between it and the graphite ferrule directly to the split line and out to the split vent. This will change the split ratio and also disrupt flows of carrier gas and sample throughout the injector.



Written by: Patrick Ayres Date: 12/07/2004 Procedure Author Approved by:

Cynthia Burbach

Date:

12/13/2004

Section Supervisor

Laurie Peterson-Wright

Date:

12/14/2004

Program Manager

James Beebe

Date:

12/15/2004

QA Officer

David A. Butcher

Date:

12/15/2004

Division Director Effective Date: 12/15/2004 Deleted From Service Date:

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