Analysis of Liquid Samples on the Agilent GC-MS

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1 Analysis of Liquid Samples on the Agilent GC-MS I. Sample Preparation A. Solvent selection. 1. Boiling point. Low boiling solvents (i.e. b.p. < 30 o C) may be problematic. High boiling solvents (b.p. > 110 o C) may not fully evaporate and should be avoided. 2. Water is detrimental to the GC column so make sure that the solvent is very dry. 3. The most commonly used solvents are ethyl acetate, dichloromethane, chloroform, hexanes and diethyl ether. B. Sample concentration - The sample concentration should be <1 mg/ml to avoid overloading the mass spectrometer. The lower limit of detection is around 1 ng/ ml. Some variation in the concentration can be dealt with by adjusting the split ratio of the injector. C. Filtration - The sample should appear clear with no debris. Always filter the solution because debris can clog the injection needle. The clog may or may not be noticeable. D. Vials - Put the sample in a 2 ml vial with a septum cap. For occasional users, vials are available in the mass spec lab. If you expect to be a heavy user, ordering information is given on the last page of this write-up. 1. Only the provided/specified vials will work with the autosampler. 2. Place your sample(s) in the autosampler rack. There are 100 positions, 20 rows of 5 radiating from the center. The smallest number in the row is nearest the center and the largest number is on the outer edge. 3. Check that there is an adequate quantity of solvent in the bottle in position A of the autosampler. Generally acetone is left there. If that presents a problem with reactivity for your sample(s), place a different vial of appropriate solvent in position A during your run and replace the acetone vial in position A after your run. E. GC column - The column that is generally left in the GC is the Agilent HP5-MS, which is designed to be used with non-polar compounds. 1

2 II. Data Acquisition A. Getting started 1. If necessary, log on to the PC with the user name gcms_user. 2. DO NOT OPEN two versions of the Data Acquisition software. Check the task bar to make sure that the data acquisition window is not minimized. If it is not open, select the Data Acquisition icon at the lower right of the desktop. 3. If the Data Acquisition program was already open, the instrument should be left with parameters from a Method called IDLE.M. 4. There are two ways to collect data on this instrument. Both of them require you to set up a method which has all the parameters for the GC and the MS modules of the instrument. (1) You can load and run a method to collect data for one sample. (2) You can set up a Sequence to run multiple samples and use multiple methods. B. Setting up a Method 1. Start by loading the default starting method. Click on the Open icon in the Method section. Select Auto_Generic.M., then OK. This method will do the following: a. Move the specified vial from the autosampler rack to the injection position. b. Pump the sample up into the syringe 3 times and inject 1.0 μl of sample. c. Start the oven program and turn on the mass spec detector 3 minutes after the injection. d. Acquire mass spectra at the rate of 1.5 scans per second, scanning from 40 to 1,050 amu. Quant. Alternatively, it will follow one or more selected ions for quantitation. e. Right after the injection, the syringe is washed 3 times with solvent from the solvent A bottle. Then the vial is returned to its original position in the autosampler rack. 2

3 2. Modify the method for your sample. Click on the Edit icon in the Method section. You will be guided through a number of menus. Most of the menus require no changes and can be dismissed by pressing OK. a. Method sections to edit. Check only Instrument/Acquisition. b. Inlet and Injection Parameters. Make sure the ALS injector is selected. c. GC Parameters. There are two sub-menus in which you may want to change some parameters: (Don t press Enter or OK until you are done with all sub-menus.) 1) Inlets (Select the back inlet to use the HP-5 column): a) A reasonable setting for the inlet temperature is 280 o C, but you can change this to accommodate thermal instability of your sample. Remember that the temperature of the inlet must be hot enough to volatilize everything in your sample. b) Depending on the concentration of your sample, you can choose a split or splitless injection. If you are unsure if your sample concentration is low enough to avoid overload of the mass spectrometer, use the maximum split ratio (100:1). If your sample concentration is well characterized, you can set it to any split ratio or even splitless for low concentration. 2) Oven: a) A reasonable temperature program for an unknown is: 50 C for 1 minute; ramp from 50 C to 300 C at 20 C/minute; hold at 300 C for 3 minutes. The total run time with this program is 16.5 minutes. b) You can use a different temperature program. But, it is a good idea to end with 300 C for at least 3 minutes to assure that all materials from your sample are desorbed from the column. Do not exceed the set maximum for the column. 3) Select OK when done with all sub-menus. d. GC Real Time Plot. This pertains to non-mass spec detectors and there are none. e. MS Tune File. The file atune.u should already be selected. If not, select it. 3

4 f. MS SIM/Scan Parameters. 1) Under Real Time Plot, in the MS Window 2 section, select Spectrum or Extracted Ion. 2) Under MS Instrument Parameters. (A) Solvent delay. If your solvent boiling point is > 100 C, change the Solvent Delay from 3 minutes to 4 minutes. This means that the MS detector will turn on 3 minutes after the injection. The delay is required to avoid overloading the mass spectrometer with the solvent as it moves off the column. If you change the solvent delay, do not overwrite Auto_Generic.M. (B) Acquisition mode. Select Scan or SIM. 3) Under Edit Scan Parameters. Set up the mass range under the Plotting tab. In the Plot window #2 section, input the low mass and the high mass of a range that covers at least 1 amu. 4) Under Edit SIM parameters. Define Group. Choose resolution. Define the ions that make up each group (m/z, dwell time). g. Save Method As. 1) If you have only changed the inlet temperature and oven parameters that have been discussed above, it is OK to overwrite the method named Auto_Generic.M. 2) Otherwise, specify a different name and include your name in the method name. 3) You may also print a copy of the method for your records. Method Print method check boxes next to GC/ALS and MS Parameters OK. 4

5 C. Collecting data for a single sample (Running a method). 1. Select the Run icon in the Method section. 2. Specify the following parameters in the run window. a. Data Path. Save your data in the D:\DATA\<username> directory. Use the browse function to select and/or create sub-directories under your username. The data path must exist before you start the run or you will get an error message that says Path must exist and the run will not start. b. Operator name. c. Data File Name. A.D will be appended to the name. Do not use / in the name. d. A sample name is optional. d. Vial # where you have placed your sample. 3. Click on RUN METHOD. 4. Once the run is started, a window appears that says Override solvent delay. Always either ignore this message (it will go away at the end of the solvent delay) or select NO!!! 5. After the run is finished, load the IDLE.M method. This will leave the instrument in an idle state, which conserves high purity helium gas and leaves the column at an intermediate temperature to keep it clean. 5

6 D. Collecting data for multiple samples. (Setting up a Sequence ). 1. Open the default sequence. Click on the Open icon in the Sequence section. Select default.s., then click on Select. 2. Edit the sequence. Click on the Edit icon in the Sequence section. In the spreadsheet that opens, specify the following for each sample. a. Data Path. Save your data in the D:\DATA\<username> directory. Use the browse function to select and/or create sub-directories under your username. The path must exist before you start the run or you will get an error message and the sequence will not run. b. Method Path. Use the browse function. Do not include the method name. c. Sample type. Sample name. Vial number. Method name. Data file name. d. Click OK to close the spreadsheet window. Note that the changes are NOT saved yet. 3. Check the sequence. Click on the Check icon in the Sequence section. a. Select Full Method and make sure Overwrite Existing Data Files is NOT checked. b. Select Run Sequence, select Yes under View It?. If that shows no errors, close the log window. If there are errors, edit the sequence and check again until there are no errors. 4. Save the sequence. Click on the Save icon in the Sequence section. Specify a sequence name that contains your name, then select Save Sequence. 5. Before starting the run, open the Method named idle.m. When a sequence is finished, the instrument will re-load the Method that was open before the Sequence was started. The idle method will leave the instrument in an idle state, which conserves high purity helium gas and leaves the column at an intermediate temperature to keep it clean. 6. Start the run. Click on the Run icon in the Sequence section. In the run window: a. Make sure Full Method is checked and Overwrite Existing Data Files is NOT checked unless you want to deliberately overwrite bad data. b. Select Run Sequence. This should run all the samples in your sequence. You can use the data analysis program to view the data from completed runs while the sequence is running the rest of the samples. 6

7 III. Data Analysis A. Open the data analysis software. Data Analysis icon at the upper left of the desktop. B. Load the desired data file. In the directory area to the left, find the filename for your run (filename.d). Right click on the subdirectory and select Load. This opens the gas chromatogram (TIC) window. C. Working with the chromatogram (TIC). 1. Zoom in and out. Click and drag with the left mouse button to expand an area of the TIC. Double-click the left mouse button to zoom out to the original axis values. 2. Generate extracted ion chromatograms (XICs) for a single m/z value. a. Select the Ion Chromatograms button on the lower toolbar. b. Enter the time range and m/z values for the plot. c. To restore the display of the TIC, select the Draw Chromatogram icon at the left end of the lower toolbar. 3. Annotate the TIC/XIC window. Position cursor in desired location and push both mouse buttons at the same time. D. Working with the mass spectrum: 1. For the spectrum of a particular scan, place the mouse pointer over the appropriate retention time and double-click the right mouse button. 2. For the average of several scans within a chromatographic peak, right click and hold while dragging the mass over the scans to be averaged. 3. To subtract scans from either side of the chromatographic peak. a. Select the signal area of the chromatogram (i.e. GC peak). Right click and drag. b. Select the noise area of the chromatogram (baseline). Right click and drag. c. Push the Subtract icon on the lower toolbar. The last spectrum displayed will be subtracted from the second to last spectrum displayed and the difference spectrum will then be displayed. 7

8 E. To send the output to the printer: 1. In Windows Print Manager, select the printer you want to use as the default printer. Choose Adobe PDF to create.pdf files of your data. 2. In Data Analysis Specify the printer setup Select the printer icon on the upper toolbar Select the items (TIC, XIC, MS) you want to print. G. Analyzing multiple data files in the same window. a. View Analyze multiple data files. b. File Select multiple data files Multiple file dialog box. i. Set the right data path. ii. Highlight file name(s) and press the arrow button. iii. Press the Process button. This opens the Enter m/z values window. c. Press OK in the Enter m/z values window to load the chromatograms. d. File Print screen to print the multiple chromatograms. H. Overlay chromatograms for direct comparison. Tools Overlay Chromatograms. I. GC peak integration. a. Transfer data files from the Instrument Control PC to the Data Analysis PC. b. Display your gas chromatogram and look at the peak(s) of interest. i. Peaks should have a symmetric Gaussian shape. Excessive fronting (shark fin shape) indicates column overloading. Excessive tailing indicates excessive analyte interaction with inlet liner or column. ii. The peak to be integrated should not overlap with other peaks. If overlapping occurs consider one of the following options. (1) Optimize the GC oven program to isolate the species of interest. (2) Use the extracted ion command to generate a gas chromatogram with a single peak. (3) Collect data in the SIM mode. However, this only works if you know exactly what species you re looking for. 8

9 c. Decide which integration algorithm to use. i. The default Chemstation integrator can handle the largest variety of GC functions. This algorithm has a timed events function which allows you to change integration (turn on or off) with retention time. This is useful when you have a varying baseline from column bleed in GC. ii. The RTE integrator was designed to quickly integrate simpler (or simplified) chromatograms such as XICs or SIM data. This algorithm has special filters like "maximum number of peaks" and "area percent". This is useful if you want to integrate the top "X" peaks based on abundance or area. d. Automatic integration. All commands are located under the Chromatogram Menu. The commands are executed in the following order. i. Select integrator. The Chemstation integrator is the default. ii. Set MS Signal Integration parameters (Chemstation Integrator). Adjust initial peak width or initial threshold to increase/decrease the sensitivity of the integrator. 1. Highlight integrator event Change setting Enter. 2. Apply to integrate with new values. 3. Load / Save integration parameters. 4. OK / Cancel to exit the window. iii. Integrate. Do not use Auto-Integrate it will ignore your integration parameter settings. The RT value will appear on the TIC display. iv. View Integration Results. This displays a table of integrated values. You can copy/paste the value(s) into a word processor or spreadsheet. Integrating XICs will generate multiple tables. e. Manual integration. i. Tools Options Manual Integration. Cursor changes to crosshairs. ii. Use click and drag to draw a line across the RT range you want to integrate. iii. View integration results as above. 9

10 Fisher Scientific Part Numbers for Vials and Caps Screw cap vials: Part no: Price October 2011 Clear glass vials Pack of 100 for $ Amber glass vials with Write-on Patch Pack of 100 for $ Caps with PTFE/Silicone septa Pack of 100 for $ Crimp cap vials: Clear glass vial Pack of 100 for $ Amber glass vial Pack of 100 for $ Crimp seals with PTFE/Red rubber septa L Pack of 100 for $ Inserts for small sample sizes: Glass conical insert, 350μl - precision formed tip Pack of 100 for $

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