Gas Chromatography (GC) with Flame-Ionization Detection

1. Initialization of the GC

1. Turn on the helium carrier gas and air and adjust the pressure gauges on the instrument.
2. Turn on the column oven to a high temperature (typically 250 °C or above) to bake in the column. Do not exceed the maximum temperature of the column. This will remove any contaminants. Let it bake for at least 30 min before running a sample.

2. Making a Methods File

1. In the software controlling the instrument, input all the desired values for a methods file. First, set the autosampler settings. Set the number of pre-run rinses, post-run rinses, and rinses with sample. These rinses clean the column between different samples.
2. The amount injected is typically 1 µL. A split ratio is usually set because injecting all of a sample might overload the column. If the split ratio is 100:1, this means that for every 1 part that is injected into the instrument 100 parts goes to waste.
3. Input the mobile phase parameters. The flow rate is controlled by the pressure set. Faster flow rates lead to faster separations, but there is less time for the analyte to interact with the column.
4. Enter the temperature programming. For an isothermal run, enter the temperature of the separation and then a time for the separation. For a gradient elution, enter the starting temperature and hold time, the ending temperature and hold time, and the ramp speed in °C/min. An equilibration time is also set that allows the column to cool back down the original temperature between runs.
5. Enter the detector parameters. A detector temperature and sampling rate will be entered. The detector must always be a higher temperature than the column temperature so that no analyte condenses on the detector.
6. Save the methods file. The parameters may also need to be downloaded so they are read by the GC.

3. Collection of GC Data

1. Turn on the hydrogen gas and make sure the pressure gauge is set correctly. Light the flame of the FID.
2. On the autosampler rack, fill the wash vial with wash solvent, like acetonitrile or methanol. Make sure the waste vial is empty.
3. Prepare the sample. If there is any chance of particulates in the sample, filter the sample. Because plastic residues can sometimes be seen with GC, use only glass syringes and glass vials to prepare your sample.
4. Fill the vial at least half way with sample so the autosampler syringe is ensured to pick up the sample. Autosampler vials are typically 2 mL, but if sample volume is limited, vial inserts are available to reduce the sample volume needed.
5. Load the sample vial(s) into the autosampler rack. Keep track of what position each sample is in.
6. Before the run, zero the baseline of the chart recorder on the computer software.
7. Files can be collected either as a single run or using a batch table for multiple runs. Make sure to specify the correct vial number for the sample. Hit the "start" button and make a file.
8. Data is typically analyzed with a software program. Parameters that can be measured include retention time, peak height, peak area, and number of theoretical plates.

4. Results: GC Analysis of Coffee Samples

1. In this example, GC-FID analysis was performed for caffeine and palmitic acid, two compounds found in coffee. The caffeine is less polar than the palmitic acid, which has a long chain alkane tail. Thus, the caffeine is less retained and elutes first on the nonpolar column of 95% dimethylpolysiloxane and 5% phenyl-arylene (Figure 1).
2. From the chromatogram, the peak areas can be calculated. The peak areas are proportional to the mass of carbon that goes through the detector and they can be used to make a calibration curve of instrument response vs concentration. For Figure 1, the peak area is 27,315 for caffeine and 18,852 for palmitic acid.
3. A measure of column efficiency is N, the number of theoretical plates. N can be calculated from the chromatogram for each peak. For Figure 1, N is 283,000 for caffeine and 261,000 for palmitic acid.
4. Figure 2 shows the effect of temperature on isothermal separations. Two separations are overlaid of the same caffeine and palmitic acid sample. The first is at 180 °C and the second at 200 °C. The retention times are much smaller for the higher temperature run.

Figure 1. GC-FID analysis of caffeine and palmitic acid samples. The 5 mM caffeine standard elutes first, followed by the 1 mM palmitic acid sample. The temperature ramp was 0.1 min at 150 °C followed by a ramp at 10 °C/min to 220 °C where the temperature was held for 5 min.

Figure 2. GC-FID analysis of isothermal runs of a dark roast coffee sample. A comparison of GC-FID runs at 180 °C and 200 °C for a dark roast coffee sample. The peaks elute much quicker with the 200 °C temperature.