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1. 1 2 3 Sample Preparation Suggestions 1 2 3 1 Calibration Standards The signal produced by the detector is unique for each compound and must be compared to a known sample for identification and quantification Calibration standards must therefore be used and are typically run as the first injections each day or with each set of samples The lowest concentration standard should be run first and should be at or slightly below the lowest expected sample concentration The highest standard should be at or slightly above the highest expected sample concentration and should be run as the last standard The remaining standards should be evenly distributed between the highest and lowest standards and there should be a minimum of 3 standards in total 1 2 3 2 Internal Standards If samples are diluted or extracted before analysis a set of control standards should be processed in the same way to determine the effects of sample preparation Alternatively each sample can be spiked with a known amount of a standard and the final calculated concentrations following chromatography can be normalized based on the concentration of this internal standard Use of an internal standard overcomes problems such as inconsistent injection volumes and is highly recommended for GC analyses 1 3 Varian 3800 GC Software Overview The Saturn GC MS is controlled using the Workstation v 5 51 software This software is used to develop and control methods run si2. add timed events and reintegrate if required 1 2 Open up Interactive graphics using the button the Star toolbar it looks like a blue chromatogram Select the file s that you would like to view and press lt lt Add to List Then click on Open Files If you place the mouse over each of the tool buttons in this window but do not click on the button you will be able to view a brief description of what each button does To zoom in on a given area of the chromatogram click on the left mouse button and then drag over the area to zoom Varian GC Standard Operating Procedure 18 4 To view the peak area data for a given chromatogram left click on the desired trace and select View GC Results Only Or View Standard GC Report 5 If more than one file is open the data can be overlaid or viewed separate chromatograms by using the Tile Chromatograms and Overlay Chromatograms buttons If the data is overlaid click on the coloured squares at the top of the window to select which trace is on top and to view the baseline and peak start and end indicators 6 If you are not satisfied with the automatically identified peak start and end points left click on the triangle that indicates the start end and drag it to the desired position Then press the Reintegrate Now button and view the GC results to see the recalculated peak area 7 Right click on the area below the chromatogram to add Time Ev
3. are not suitable for GC analysis Most organic compounds can be analyzed by GC as long as they are not thermally labile while most inorganic compounds typically are not volatile enough Summarized from Rood 2007 1 2 2 Instrumentation The primary components of a GC include the carrier and detector gases flow controllers or valve an injection port the column and oven a detector and a recorder Figure 1 1 Flow Control V Molecular Detector Computer or Integrator Detector Gas Carrier Gas Figure 1 1 Basic Gas Chromatograph Varian GC Standard Operating Procedure 3 1 2 2 1 Carrier and Detector Gases The mobile phase is composed of an inert carrier gas that sweeps past the injector and transports the sample components through the column The most common carrier gas is helium although nitrogen argon and even hydrogen can be used Carrier gas selection depends on the detector in use column characteristics the sample matrix safety considerations and cost If capillary columns are used the most popular carrier gases are helium and hydrogen which provide high efficiency high theoretical plate count over a wide range of flow rates Nitrogen which can provide very high efficiency but only for very low flow rates is commonly used with packed columns Grob and Barry 2004 1 2 2 2 Injectors Injectors fit into two main classes vapourization and on
4. be designed to accommodate two types of injectors two columns and two different detectors Table 1 1 lists some of the common detectors used in gas chromatography The Varian GC is equipped with an FID and a mass spectrometer In the FID organic compounds burn in a hydrogen and air flame and produce ions and electrons which conduct electricity through the flame The resulting current is proportional to the amount of the compound The mass spectrometer converts gaseous molecules to ions which are then separated by a quadrupole mass filter and detected The mass spectrometer is discussed in greater detail in the Varian GC MS Standard Operating Procedure Varian GC Standard Operating Procedure 6 Table 1 1 Common detectors for a GC Sheffield Hallam University m Limit of Dynamic Detector Selectivity Detection range EE zao Most organic compounds 100 pg 107 Universal detector for non in 107 conductivity TCD organic compounds 9 Halides nitrates nitriles p capture peroxides anhydrides 50 fg 10 organometallics Nitrogen I 6 phosphorus Nitrogen phosphorus 10 pg 10 Sulphur phosphorus tin boron arsenic germanium 100 pg 10 selenium chromium Aliphatics aromatics RN ketones esters aldehydes oo amines heterocyclics 2 pg 107 organosulphurs some organometallics Mass spectrometer Universal detector for 7 MS ionizable compounds ss 19 Varian GC Standard Operating Procedure
5. Residence Time 0 vi SPME refers to Solid Phase Microextraction please contact the Instrumentation Technician if you would like to use this technique d Under Liquid Sampling Options i Select Solvent A Wash Solvent B Wash A then B Wash or No Wash No Wash may result in significant sample carry over and cross contamination Note Make sure the solvents used for washing are miscible with each other and with the sample solvent ii The syringe should be set at 10 uL this is the syringe that is installed on the 8200 7 Under 3800 GC Control Click on Column Oven to set up the temperature programming for the run a Make sure the Column Oven Coolant is set to OFF b Set the stabilization time between 0 5 and 2 minutes the oven will stabilize at the set temperature for this amount of time before each injection C Usethe table to program the column temperature for the run DO NOT set the temperature above 300 C Please contact the Instrumentation Technician if you think you need to use a higher temperature i Enter the initial column temperature and hold time in the first row a rate cannot be set for the initial temperature ii Enter the second temperature ramp rate and hold time in the second row After the hold time on the first temperature has elapsed the oven will ramp to the second temperature at the rate specified in the second row and then remain at that temperature for the hold time listed in the second r
6. column Vapourization injectors can be split splitless or direct and on column can be direct or cooled The overall purpose of the injector is to deposit the sample onto the column in the narrowest band possible with minimal discrimination between sample components Vapourization Split splitless injectors are heated and cause the sample to vapourize before it enters the column Injector temperatures are usually set to ca 50 C above the boiling point of the least volatile component of the mixture The split splitless design determines the amount of sample that enters the column A splitless injector is typically used for trace level analysis and transfers the entire sample into the column A split injector sweeps a predetermined potion of the sample out of the injector through the split vent and the remaining small portion of the sample enters the column A split injection allows a reasonable amount of concentrated sample 0 1 to 10 ug to be injected without overloading the Figure 1 2 Varian 1079 column Injection speed should be as high as possible split splitless injector for a split injection and can be slower for a splitless Varian GC MS Manual injection On column injectors deposit the liquid sample directly onto the front of the column or a sort piece of deactivated silica called the retention gap The sample then vapourizes as the temperature of the injector is increased Cool on column injections are ideal fo
7. the Instrumentation Technician for assistance 14 Save the method 15 Proceed to Section 6 2 to active the method and begin analyses Varian GC Standard Operating Procedure 16 6 2 Sample Analysis 1 2 3 6 T Activate the desired method by clicking on third folder icon from the left on the system control tool button or using File Activate Method Open the valves on the hydrogen and air cylinders Check the gas cylinder levels and fill in the log book each cylinder must have more than 200 psi total pressure and have enough gas to complete the desired analyses The cylinder delivery pressures should already be set as follows and should not require adj ustment a Hydrogen 40 psi b Air 60 psi c Helium 80 psi If the actual delivery pressures are not the same as those listed above contact the Instrument Technician for assistance DO NOT adjust them yourself Check the rinse solvent on the far left side of the instrument and refill the bottle if needed Cover the bottle with a gloved hand and refill in the fumehood While the instrument is equilibrating create a sample list by selecting File gt New SampleList a Name the SampleList and save it in you lab folder b Under Sample Name give a descriptive name for the sample or standard c Under Sample Type select Analysis d Under Inj indicate the number of injections to be made from each sample e Under Injection Notes include any notes that would be useful fo
8. 2004 The stationary phase can be composed of extremely small solid particles or a thin liquid film coated on a solid support the mobile phase can be a liquid or gas but in the case of gas chromatography it is always a gas Samples can introduced to the gas chromatograph GC as a liquid which is then vapourized in the injector or as a gas The vapourized or gaseous sample is carried into the column with an inert carrier gas such as helium Separation is based primarily on boiling point and there temperature of the column oven must be carefully controlled The chemistry of the adsorbed stationary phase also influences separation but no to the same extent as in liquid chromatography The separated sample components exit the column at different times and are detected by the appropriate detector The final signal is sent to a computer where it can be compared to the signal from a known standard and quantified using chromatography software Varian GC Standard Operating Procedure 2 In general gas chromatography can be used to analyze any compounds that will volatilize at temperatures below approximately 350 C and not be degraded or react with other compounds at these temperatures If the volatility of a compound is not known it may be estimated based on molecular weight polarity and structure Hydrocarbons with molecular weights over 500 have be analyzed by GC while small sugars and amino acids with many polar amine and hydroxyl groups
9. CEDURES 4 1 Accidents If you notice anything unusual concerning GC operation smells noises etc contact the Instrumentation Technician immediately All incidents must be reported to the Instrumentation Technician and if applicable a student s supervisor The Instrumentation Technician will insure that all accidents incidents and near misses involving instruments are reported as promptly as possible to the Environmental Occupational Health and Safety EOHS Office via the WLU Employee Accident Incident Occupational Disease Report form www wlu ca eohs forms All incidents that result in critical injuries must be reported immediately to the EOHS Office by telephone Additional details regarding incident reporting can be found in the WLU Accident Incident Procedure www wlu ca eohs 4 2 Spills The WLU Laboratory Health and Safety Manual provides detailed instructions for dealing with major and minor spills Do not attempt to clean up a spill if you have not been properly trained or if you are unsure of the proper procedures Before using ANY hazardous materials make sure you understand the proper clean up procedure The Environmental Occupational Health and Safety Office is also available to provide guidance at ext 2874 See the WLU Laboratory Health and Safety Manual for further details If a spill occurs ON or IN the GC contact the Instrumentation Technician IMMEDIATELY 5 WASTE DISPOSAL PROCEDURES All WHIMIS Departm
10. L UR ER Research Instrumentation User Guidelines amp Standard Operating Procedure r the Varian 3800 GC GC MS Standard Operating Procedure ii TABLE OF CONTENTS DISCLAIMER cts met ili ACKNOWLEDGEMENTS Rr dT iv LI INTRODUCTION e tac edt aet s 1 1 1 Purpose of the Standard Operating Procedure 1 1 2 BISOL V asso esee seta er 1 1 2 1 Simple Gas Chromatography e ede eue saa 1 1 2 2 Instrumentation cse T D Do YE Oe Ps eco dread o a 2 1 2 3 Sample Preparation Suggestions 7 1 3 Varian 3800 GC Software Overview 7 2 POTENTIAL HAZARDS E aote eu a ex lt orto aal iod 10 3 PERSONAL PROTECTIVE EQUIPMENT satt rete a td rh 10 4 SPILL AND ACCIDENT PROCEDURES vun a Ec De Xen d C es 11 A Accidents ag tetas 11 2 2 BITS ot sex sacs 11 5 WASTE DISPOSAL PROCEDURES RC ER E PROS 11 6 PROTOCOL sm CE ie LS bei ie Ce REA 12 6 1 Create a Method erecta pia Us tea be sd nitas 12 62 Sample Analysis tatu eal toon ud eU IMS 16 6 3 bd ste
11. and purge for an additional 60 minutes Reconnect the carrier gas line to the back of the GC Check the cylinder and fittings for leaks Replace gas purifiers as required The High Capacity Gas Purifier tube removes oxygen and water from at least 60 tanks of heavily contaminated gas containing 100 ppm of oxygen and or water one cylinder of UH 5 0 helium contains less than 2 ppm water and less than 1 ppm O2 Varian GC Standard Operating Procedure 21 9 QUICK REFERENCE GUIDE WEAR APPROPRIATE PERSONAL PROTECTIVE EQUIPMENT 1 QJ NJ 9 Create a method if required a Click on the Method Editor button b Open the GC METHOD TEMPLATE mth file C SAVE this file with a new name in the folder for YOUR LAB before making any changes d Click on Method Notes and enter a description of the method e Click on Configuration under 8200 Autosampler Control Address 26 and set up the autosampler d Under 3800 GC Control Click on Column Oven to set up the temperature programming for the run DO NOT set the temperature above 300 C e Click on Injector to set the injector temperature and split ratio Select the tab for Middle Injector f Click on Flow Pressure to set the column carrier gas flow rate Select the Middle EFC tab g Click on Detector to set the detector temperature and gas flow rates Select the Middle Detector tab Save the method Activate the desired method using File A
12. ation under 8200 Autosampler Control Address 26 a The Carrousel Type should be 48 vials b For faster sample analysis select Prep Ahead and in the EndTime box enter a time that is 2 or 3 minutes shorter than the total run time If sample analysis speed is not a concern leave the Prep Ahead box unchecked and do not enter a number in the EndTime box C Select the desired sampling mode i Standard mode the sample is injected with a solvent flush do not select if the solvent peak is large and close to the peaks of interest ii Volatile Sample Use with samples that are in a high vapour pressure solvent The sample is injected with a solvent flush slow plunger speed and no upper air gap to minimize flashing during sampling iii Neat Sample This mode minimizes the amount of solvent that is injected with the sample and the syringe is washed several times with the sample before injection iv Viscous Sample For samples that have a higher viscosity than water This mode uses slow plunger speeds and longer pauses to improve reproducibility v User Defined Allows each of the sampling options to be selected manually the recommended settings for this option are Solvent Flush Yes Syringe Wash time 20 Solvent Plug size 0 Vial Needle Depth 90 Uptake Speed 5 Upper Air Gap Yes Lower Air Gap Yes Varian GC Standard Operating Procedure 14 4 Pause Time 2 Hot Needle Time 0 Injection Rate 5 Needle
13. cedure 27 DATE NAME ExT TYPE OF MAINTENANCE FREQUENCY OF MAINTENANCE WEEKLY PROBLEMS COMMENTS
14. ctivate Method Open the valves on the hydrogen and air cylinders Check the gas cylinder levels and fill in the log book each cylinder must have more than 200 psi total pressure and have enough gas to complete the desired analyses The cylinder delivery pressures should already be set as follows and should not require adj ustment h Hydrogen 40 psi i Air 60 psi j Helium 80 psi Check the rinse solvent on the far left side of the instrument and refill the bottle if needed While the instrument is equilibrating create a sample list by selecting File gt New SampleList a In the row following the last sample under Sample Type select Activate Method then click on the Autolink button select Browse look in the Templates folder and choose the method GC Standby mth Load the samples into the autosampler tray according to the order in the SampleList You MUST use vials that are 12 x 32 mm Click on Begin in the SampleList window Observe the first injection to check for any problems and then occasionally check on the instrument as the analyses proceed 10 When the run is complete the raw data can be exported by right clicking on the chromatogram as displayed in Interactive Graphics then selecting AIA Import Export then selecting Varian to AIA then Varian GC Standard Operating Procedure 22 1 12 13 selecting the desired file and folder to save it in Then select AIA to Text
15. ent and Health and Safety guidelines must be followed for disposal of the substance to be analyzed See the WLU Laboratory Health and Safety Manual for details Varian GC Standard Operating Procedure 12 6 PROTOCOL The instructions provided in this protocol are only intended to serve as reminder all users must be trained by the Instrumentation Technician before using the GC for the first time If the GC is completely shutdown all components are turned off and the gas cylinders are closed the instrument will have to be flushed with helium and the column baked out before use Please contact the Instrumentation Technician for assistance with these procedures 1 Check the log book first to ensure that the GC MS is functioning properly 2 If the GC is in standby proceed to section 6 1 to develop a method or section 6 2 to activate a previously created method 6 1 Create a Method If you do not already have a method for analysis you will need to create one If you have already created a method proceed to Section 6 2 to activate the method and begin analyses Refer to Figure 6 1 for the following steps Method Builder Method1 Iof J Eile Edt View Window 1 x meo Sle S mj 14 4 p Location Method File Not Saved Created Modified Size 9401 bytes Method File Attributes Read only Hidden O Archive Requires Password on Save EETMethodl M
16. ents such as Solvent Reject this will allow the area under the solvent peak to be integrated but it will not appear in the GC report Varian GC Standard Operating Procedure 19 7 TROUBLESHOOTING Thermo Fisher provides very useful troubleshooting on their website www separatedbyexperience com The Instrumentation Technician is also available to assist with unusual GC results or other analysis problems 8 PREVENTATIVE MAINTENANCE Users are not to perform maintenance These procedures are carried out by the Instrumentation Technician 8 1 Daily Check the log book for any problems or concerns Check column nuts are tight Condition column daily or as required Check gas supplies 8 2 Weekly Change injector insert if necessary 8 3 Monthly Perform a leak check as follows 1 DO NOT use a soap based leak detection fluid Use an alcohol such as isopropanol or a 50 50 mixture of isopropanol and water and use a syringe to place a few drops on the desired fitting Monitor the area for bubble formation 2 Set the column oven and the injector zone temperatures to 50 C and allow them reach this temperature Remove injector septum nut and install a new septum Remove the column from the injector and seal the injector with capillary nut 03 949551 00 with a no hole ferrule 28 694590 01 5 The septum purge outlets are located on the top frame surrounding the column oven behind the column oven door Seal the s
17. eptum purge by removing the outlet fitting from the septum purge valve and replacing it with a blank off plug 16 000154 00 6 The split vent outlets are located on the left side panel of the 3800 with manual pneumatics or inside the pneumatics compartment with EFC Seal the split vent outlet by installing Swagelok union on the outlet tube and sealing it with a blank off plug 7 With all outlet ports plugged pressurize the system to 20 30 psi This can be accomplished by adjustment of the Electronic Flow Control and setting carrier gas pressure gt WwW Varian GC Standard Operating Procedure 20 8 Shut off the carrier supply at the source and monitor the displayed pressure at the GC for 15 minutes The pressure should not drop more than 0 5 psi in 15 minutes 8 4 Six Months Condition the system 8 5 Annually and As Required Change septa typically every 50 100 injections Replace the gas cylinders With typical usage A size cylinder of carrier gas should last for three to six months Before changing the carrier gas cylinder cool the system to room temperature and unplug the converter tube furnace Allow the system to cool for at least one hour with carrier gas flowing Turn off the carrier gas cylinder and disconnect the carrier gas line from the back of the GC Attach a new cylinder and purge the lines and converter tube for 30 minutes at 100 ml min Turn on the converter tube furnace
18. ethod Notes f 0200 AutoS ampler Control Address 26 T Configuration E 3800 GC Address 44 E E 3800 GC Control Sample Delivery Injector Flow Pressure Column Oven z Detector Dutput Data Acquisition El m Channel Front E E Data Handling 2 Integration Parameters Peak Table 5 Calibration Setup L Verification Setup Time Events T able E Standard Reports Print Options Results Format B Chromatogram Format Calibration Block Report Format Figure 6 1 Method Builder Window Varian GC Standard Operating Procedure 13 1 Click on the Method Editor button on the Star toolbar second button in then toolbar at the top of the screen see Table 1 1 for a illustration of this button 2 Select Open an Existing Method File Look in the Templates folder and select the GC METHOD TEMPLATE mth file This file is configured for liquid sample analysis on the GC using the autosampler and FID detector 3 SAVE this file with a new name in the folder for YOUR LAB before making any changes 4 The top most item in the tree is the name of the Method file An asterisk follows the name if the Method file has been modified since it was opened or last saved 5 Click on Method Notes and enter information related to the types of samples to be analyzed a literature reference for the method if applicable your name and supervisor and the date the method was created 6 Click on Configur
19. ewer GC Batch Batch reporting is used to create standard Reporting reports for a group of data files x Quick Start is used to activate a method and Quick Start run a single sample without generating a SampleList Varian GC Standard Operating Procedure 9 Table 1 3 System Control Toolbar Description System Control Tool Button Purpose Toolbar illustration 35 1 is y JAO Create a new automation file select a I previously created automation file edit notes for automation file Automation files are used to s View edit or reactivate an instrument startup1 mth k method Activate instrument method Edit module information for an on line er module A module is an instrument component such as the 3800 GC or the 8200 autosampler Inject a single sample Start a recalculation list sample list or SHP SFO sequence list respectively the list must already be open Pause automation The sample that is currently running will be completed and then the automation will be suspended When the Begin button is pressed the list will resume from the point when it was suspended Immediately stops automation and cancels the current run If the Begin button is pressed automation will start from the first row of the active list Varian GC Standard Operating Proced
20. hanism and are present with all stationary phases smaller compounds and compounds with low solubility will experience lower retention due to dispersion Varian GC Standard Operating Procedure 5 Stationary phases are typically composed of polysiloxanes with methyl phenyl cyanopropyl or trifluoropropyl substitutions Substituted polysiloxanes are very robust columns and usually have a very long lifetime Arylene modified polysiloxanes are not as robust but have a very low bleed and are ideal for mass spectrometry detection Polyethylene glycol PEG columns can provide unique separation characteristics for difficult mixtures but are easily damaged Porous layer open tubular PLOT columns are uniquely designed for the analysis of gases at room temperature Stationary phases can also be bonded or non bonded bonded phases are more stable and generally preferred Columns can be up to 100 meters in length with inner diameters between 0 1 to 5 mm Longer columns with smaller diameters provide better resolution but cost increases with length and capacity generally decreases as diameter decreases All stationary phases are broken down by oxygen at higher temperatures although some are more sensitive than others and great care must be taken to ensure minimal exposure by maintaining a sufficient flow of high purity dry helium and regular leak checking 1 2 2 4 Detectors A GC can be equipped with several different detectors and many models will
21. he desired column pressures for the run OR select Constant Column Flow Mode and enter the desired flow rate for the column the GC will then automatically determine the required pressure adjustments to maintain the flow rate as the temperature changes in the oven The pressure is typically set around 10 psi Note In constant pressure mode the column flow rate will decrease as column temperature increases 11 Click on Detector to set the detector temperature and gas flow rates a b C d e Select the Middle Detector tab The Detector Type must be set to FID and should not require changing Make sure the Detector Oven and Electronics are set to ON Enter the desired Detector Temperature Do not change any of the values in the Adjustments box at the bottom of the window 12 Do not change any of the settings in the Output or Data Acquisition windows 13 Under Data Handling a b C Do not change the settings under Integration Parameters Verification Set up or Time Events Table The Peak Table can be used to automatically label the peaks of interest If you know the retention times of your analytes they can be entered manually or they can be entered at a later time following the injection of test standards The Calibration Set up is used to run calibration standards and automatically quantify unknowns based on the standard curve If you would like to set up a standard curve automatically please contact
22. his manual Gena Braun Instrumentation Technician This manual was prepared for Laurier Any corrections additions or comments should be brought to the attention of the Instrumentation Technician at 519 884 0710 ext 2361 iv Issued August 2009 Revision 0 Varian GC Standard Operating Procedure 1 INTRODUCTION 1 1 Purpose of the Standard Operating Procedure This standard operating procedure SOP is NOT a substitute for training and or reading the appropriate manuals before use All principle investigators and supervisors should document that training has been received by students and staff who will be using the Varian 3800 GC A list of authorized users will be kept by the Instrumentation Technician This SOP is intended to promote consistent and safe use of the Varian 3800 GC within the Faculty of Science This SOP covers the potential hazards personal protection requirements spill and accident procedures waste disposal considerations and instrument operation for the Varian 3800 GC henceforth referred to asthe GC 1 2 Theory 1 2 1 Simple Gas Chromatography Chromatography 15 the science of separation a sample that passes though chromatographic bed or column will be distributed between a stationary phase and a mobile phase based on physical properties or chemical interactions As a result of these interactions the components in the sample travel through the column at different rates and can be separated Meyer
23. ity Environmental Occupational Health and Safety Office 2007 Laboratory Health and Safety Manual Varian GC Standard Operating Procedure APPENDIX 1 GC FID USER LOG 24 Varian GC Standard Operating Procedure 25 MIDDLE CYLINDER METHOD DATE NAME ExT SUPERVISOR PRESSURES AT NUMBER COLUMN HEAD TEMPERATURES PROBLEMS START INJ ECTIONS PRESSURE IN STANDBY Fig to INJECTOR AIR VAPOUR _ OVEN HE So TOTAL DETECTOR e INJECTOR _ _ _ ___ AIR VAPOUR _____ OVEN _ HE O O O ele TOTAL DETECTOR _____ INJECTOR _ _ _ _ ___ AIR VAPOUR _____ OVEN HEC TOTA ______ ________ Ho INJECTOR AIR VAPOUR _____ OVEN TOTAL gt DETECTOR 5 2222 ok te LIQUID INJECTOR _ AIR VAPOUR _____ OVEN _ HER LIT TOTAL _ DETECTOR 2 2 Hi 2t INJECTOR _ AIR VAPOUR _____ OVEN _ HES TOTAL 3 DETECTOR LIQUID INJECTOR AIR _ VAPOUR _____ OVEN HEren tm TOTAL 3 DETECTOR _ Hat LIQUID INJECTOR AIR 0 VAPOUR _____ OVEN _ TOTAL 3 DETECTOR ______ LIQUID __ INJECTOR VAPOUR _____ OVEN _ HE TOTAL DETECTOR Varian GC Standard Operating Procedure APPENDIX 2 PREVENTATIVE MAINTENANCE LOG 26 Varian GC Standard Operating Pro
24. ngle or multiple samples and view and modify run results The main applications are accessed via the Star toolbar those related to GC operation are listed in Table 1 2 This toolbar also has buttons for the most recently used method and the most recently viewed data file All sample analyses and methods are run through the System Control Application and this window should be left open at all times when the GC is running or in standby The System Control toolbar is located below the Star toolbar Table 1 3 illustrates the System Control toolbar and describes the function of each button Varian GC Standard Operating Procedure 8 Table 1 2 Star Toolbar Description Star Toolbar Application Button Purpose System control is used to monitor instrument status and control sample analyses This window is always left open if System Control the GC is running or in standby The Method Editor is used to view and edit instrument operation data acquisition and data handling methods Method Editor Automation File Editor allows editing of Sample Lists Recalculates and Sequences while the GC is running other methods Automation File Editor Interactive Graphics is used to view sample chromatograms adjust integration parameters and recalculates results Interactive Graphics The Report Viewer can be used to view GC results and modify the report to include a variety of method and run information GC Report Vi
25. njector currently used in the GC MS system 1 2 2 3 Capillary Columns GC columns come in a variety of styles but the most popular are capillary columns which consist of a fused silica tube that has a thin coating of chemically bonded stationary phase on the inside and a polyimide coating on the outside to provide strength Column stationary phase selection depends on sample components desired column lifetime and cost The components of the sample are primarily separated based on volatility and therefore temperature The stationary phase must be stable above the temperatures required for separation Most columns can be operated at temperatures up to 350 for a brief period or 325 for an extended period Lower temperatures can provide better separation but lead to a longer run time and peak broadening Oven temperature is often increased as a run progresses to liberate the least volatile compounds Column stationary phase does play a small roll in sample separation The polarity of the column does not significantly influence separation but does determine the capacity of the column a more polar stationary phase will have a higher capacity for polar components A less polar column such as 10096 methyl polysiloxane is typically more stable and will have a longer lifetime Column selectivity based on intermolecular forces such as dispersion dipole and hydrogen bonding does influence separation Dispersion forces are the primary selection mec
26. ow iii Enter any additional temperatures in the following rows iV Note It is recommended to ramp the temperature up at the end of each run to remove any less volatile components from the column before the next injection 8 Do not change any of the parameters under Autosampler or Sample Delivery 9 Click on Injector to set the injector temperature and split ratio a Select the tab for Middle Injector b The Injector Type should be set at 1079 and should not require changing c Make sure the Injector Oven is ON and the Injector Coolant is OFF d Set the desired injection temperature in first row of the table and enter a hold time that is equal to the Column Oven End Time shown at the bottom of the window Note Avoid ramping the Varian GC Standard Operating Procedure 15 injector temperature if possible as repeat temperature changes cause stress on the components and can cause eventual leaks Click on the Split Ratio button and enter the desired split ratio for the injection in the first row set it to off for a splitless injection Do not change the values in the second and third row Click OK For example If a 1 wl sample is injected and the split ratio is set at 4 0 25 ul of the sample will enter the column 10 Click on Flow Pressure to set the column carrier gas flow rate a b C Select the Middle EFC tab The type must be set to Type 1 and should not require changing Use the table to set t
27. r sample identification or data analysis f Indicate the injection volume for each sample 0 5 to 1 good starting point Leave the remaining columns at the default values In the row following the last sample under Sample Type select Activate Method then click on the Autolink button select Browse look in the Templates folder and choose the method GC Standby mth When the GC has run all of the samples in the SampleList it will automatically switch to the stand by method i Click on the Data Files button and select the desired folder to save the data in Leave the remaining boxes as the default values and each sample will be saved in a file according to the sample name j Click on the RecalcList button and select the option Files will not be appended to any RecalcList Load the samples into the autosampler tray according to the order in the SampleList You MUST use vials that are 12 x 32 mm Contact the Instrumentation Technician if you are unsure if your vials are the correct size Click on Begin in the SampleList window 2o Varian GC Standard Operating Procedure 17 8 9 Observe the first injection to check for any problems and then occasionally check on the instrument as the analyses proceed a The results can be viewed using the GC Report Viewer button on the Star toolbar If the integration is unsatisfactory it can be viewed and adjusted using the Interactive Graphics button See Section 6 4 fo
28. r further information on viewing data in Interactive Graphics When the run is complete the raw data can be exported by right clicking on the chromatogram as displayed in Interactive Graphics then selecting AIA Import Export then selecting Varian to then selecting the desired file and folder to save it in Then select AIA to Text select the cdf file that wasjust created and press Open select the Raw Data checkbox only click OK and save the results in the desired folder with the desired name 6 3 Standby If the GC MS will be used within the next two weeks put the GC in stand by as follows otherwise contact the Instrumentation Technician to shut the instrument down completely 1 2 3 When the run is complete if the GC is not in standby activate the standby method by selecting File gt Activate Method and choosing GC Standby mth from the Template folder Turn off the hydrogen and air cylinders using the cylinder valves do not adjust the regulators Check the programmed and actual flow rate in the Middle Detector EFC Status box and confirm that the make up gas CH 1 flow rate in the instrument is 3 and the hydrogen Ch 2 and air Ch 3 flow rates are zero Note any problems or concerns in the log book and list the number and types of injections run 6 4 Data Viewing and Analysis Interactive Graphics can be used to view previously run chromatograms and peak area data adjust peak start and end times
29. r heat sensitive samples and high boiling point compounds or other samples that experience significant discrimination in vapourization injectors Direct injections are usually done for large ID columns 0 45 to 0 53 mm Varian GC Standard Operating Procedure 4 Most injectors use liner The liner resides inside the injector and protects the injector body from the sample mixes the sample and affects the volume of sample that can be injected When the sample is injected into a vapourization injector it vapourizes rapidly and if the vapour volume is larger that the injector liner the sample may flash back into the gas lines and injector body and cause cross contamination between samples Samples volumes are typically 1 ul but should be adjusted depending on the expected vapour volume of the mixture and the loading capacity of the column Liners with frits or complex inner surfaces are usually used with split injections and create a turbulent flow path that mixes the sample as it is vapourized to minimize injector discrimination Splitless injections are typically done with a straight liner with a restriction at one or both ends Specially designed liners are required for on column injections Deactivated liners should be used in all cases Septum purge is used to prevent septum bleed components from entering the column and is usually set at a fixed rate 1 3 ml min and does not need to be changed Figure 1 3 illustrates the Varian 1079 i
30. select the cdf file that was just created and press Open select the Raw Data checkbox only click OK and save the results in the desired folder with the desired name If the GC MS will be used within the next two weeks put the GC in stand by as follows otherwise contact the Instrumentation Technician to shut the instrument down completely a When the run is complete activate the standby method by selecting File Activate Method and choosing GC Standby mth from the Template folder b Turn off the hydrogen and air cylinders using the cylinder valves do not adjust the regulators Note any problems or concerns in the log book and list the number and types of injections run Interactive Graphics can be used to view previously run chromatograms and peak area data adjust peak start and end times add timed events and reintegrate if required Varian GC Standard Operating Procedure 23 REFERENCES Grob RL Barry EF 2004 Modern Practice of Gas Chromatography Fourth Edition Wiley and Sons Inc New J ersey Rood D 2007 The Troubleshooting and Maintenance Guide for Gas Chromatographers Fourth Revised and Updated Edition Wiley VCH Weinheim Sheffield Hallam University Gas Chromatography http teaching shu ac uk hwb chemistry tutorials chrom gaschrm htm Accessed Aug 24 2009 Varian Associates Inc 1998 Saturn GC MS Workstation Version 5 2 Hardware Maintenance Operator s Manual Wilfrid Laurier Univers
31. ssi peso ook ac kins tinge tiec Fa Me yaka a ki TUE ue 17 6 4 Data Viewing and 17 Hic TROUBLESHOOTING ua u e a ty Giant reroute 19 8 PREVENTATIVE MAINTENANCE er ERE Lr 19 BD ee ie ener tees te ag ala as 19 8 2 Wak 19 Bx AMOTIBD ect 19 B 5X MOMEAS teta te baad dd 20 8 5 Annually and As at rait 20 9 QUICK REFERENCE GUIDE dota s Zug qaad aus 21 APPENDIX 1 GC FID USER EP va ree ma aan Eius 24 APPENDIX 2 PREVENTATIVE MAINTENANCE LOG 26 GC MS Standard Operating Procedure iii DISCLAIMER The materials contained in this document have been compiled from sources believed to be reliable and to represent the best opinions on the subject This document is intended to serve only as a starting point for good practices and does not purport to specify minimal legal standards No warranty guarantee or representation is made by Laurier as to the accuracy or sufficiency of information contained herein and Laurier assumes no responsibility in connection therewith Varian GC Standard Operating Procedure ACKNOWLEDGEMENTS The following individuals of Laurier contributed to the writing editing and production of t
32. ure 10 2 POTENTIAL HAZARDS The GC is connected to several gas cylinders These cylinders must remain upright and secured during use When cylinders are empty around 200 psi they must be turned off and capped before transport Cylinder replacement on the GC is done by the Instrumentation Technician only The FID is connected to a hydrogen cylinder Hydrogen is highly flammable gas and care must be taken to ensure that there are no leaks from this cylinder and that it is turned off after use The helium gas that flows into the GC column is purified and dried by a converter tube that contains zirconium This tube may explode if it is heated in the presence of air NEVER disconnect the converter tube if the converter tube oven it turned on The GC oven injector and detector operate at high temperatures Do not open the oven door or try to access the detector or injector while the instrument is set at operating temperatures 3 PERSONAL PROTECTIVE EQUIPMENT Standard laboratory protective equipment must be worn latex or nitrile gloves lab coat Closed toe and heel footwear constructed of resistant material is also required for all laboratory activities See the WLU Laboratory Health and Safety Manual for additional information on personal protective equipment http www wlu ca documents 23120 Laboratory Health 926 Safety Manual Feb 2007 Final pdf Varian GC Standard Operating Procedure 11 4 SPILL AND ACCIDENT PRO
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