Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Gas chromatography (GC) is used to separate and detect small molecular weight compounds in the gas phase. The sample is either a gas or a liquid that is vaporized in the injection port. Typically, the compounds analyzed are less than 1,000 Da, because it is difficult to vaporize larger compounds. GC is popular for environmental monitoring and industrial applications because it is very reliable and can be run nearly continuously. GC is typically used in applications where small, volatile molecules are detected and with non-aqueous solutions. Liquid chromatography is more popular for measurements in aqueous samples and can be used to study larger molecules, because the molecules do not need to vaporize. GC is favored for nonpolar molecules while LC is more common for separating polar analytes.
The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert. Pressure is applied and the mobile phase moves the analyte through the column. The separation is accomplished using a column coated with a stationary phase. Open tubular capillary columns are the most popular columns and have the stationary phase coated on the walls of the capillary. Stationary phases are often derivatives of polydimethylsiloxane, with 5–10% of the groups functionalized to tune the separation. Typical functional groups are phenyl, cyanopropyl, or trifluoropropyl groups. Capillary columns are usually 5–50 m long. Narrower columns have higher resolution but require higher pressures. Packed columns can also be used where the stationary phase is coated onto beads packed in the column. Packed columns are shorter, 1–5 m. Open tubular capillaries are generally preferred because they allow higher efficiencies, faster analyses, and have higher capacities.
Flame-ionization detection (FID) is a good general detector for organic compounds in GC that detects the amount of carbon in a sample. After the column, samples are burned in a hot, hydrogen-air flame. Carbon ions are produced by the combustion. While the overall efficiency of the process is low (only 1 in 105 carbon ions produce an ion in the flame) the total amount of ions is directly proportional to the amount of carbon in the sample. Electrodes are used to measure the current from the ions. FID is a destructive detector, as the entire sample is pyrolyzed. FID is unaffected by noncombustible gases and water.
1. Initialization of the GC
2. Making a Methods File
3. Collection of GC Data
4. Results: GC Analysis of Coffee Samples
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.
Gas Chromatography, or GC, is a technique that is used to separate, detect, and quantify small volatile compounds in the gas phase.
In GC, liquid samples are vaporized, then carried by an inert gas through a long, thin column. Analytes are separated based on their chemical affinity with a coating on the inside of the column.
Because GC requires that analytes are vaporized to the gas phase, the instrument is ideally suited for volatile, nonpolar chemicals less than 1,000 daltons in mass. For larger, aqueous, or polar molecules that are difficult to vaporize, liquid chromatography is a useful alternative. This video will introduce the basics of gas chromatography, and illustrate the steps required to analyze the chemical species in a non-aqueous mixture sample using a gas chromatograph.
The GC instrument has five essential components. First, an injection port is used to introduce the sample into the instrument. Next, a heating chamber vaporizes the sample and mixes it with an inert gas. The inert gas, such as helium or nitrogen, carries the vaporized sample through the system. Combined, the carrier gas and sample make up the mobile phase. Next, the mobile phase enters the heated column, separating the analytes as they flow through. Lastly, a detector records the gases as they exit the column, or elute, and sends data to a computer for analysis. The most critical component of the instrument is the column. The column is a capillary with a stationary phase matrix coating the inner walls. Alternatively, columns can be packed with matrix-coated beads. The stationary phase is usually modified polydimethylsiloxane, which is ideal for resolving nonpolar molecules. Its separation properties are refined by adding 5–10% phenyl, cyanopropyl, or trifluoropropyl groups.
Analytes with low chemical affinity for the stationary phase move quickly through the column, while molecules with high affinity are slowed as they adsorb to the column walls.The length of time a compound spends inside the column is called its retention time, or Rt, and allows compounds to be identified. The detector sits at the end of the column and records gases as they elute. Flame-ionization detection, or FID, is widely used because it senses carbon ions, allowing it to detect virtually any organic compound. In FID, analytes combust in a hydrogen-air flame as they exit the column, producing carbon ions that induce a current in nearby electrodes. The current is directly proportional to the carbon mass, thus, the concentration of the compound can be determined. The final result is a chromatogram, which is a plot of FID signal vs time, showing each eluted component as they exit the column. Ideally, each peak will have a symmetrical, Gaussian shape. Asymmetrical features, such as peak tailing and peak fronting, can be due to overloading, injection problems, or the presence of functional groups that stick to the column, such as carboxylic acids.
Now that the principles of gas chromatography have been discussed, let's take a look at how to carry out and analyze a gas chromatography analysis in the laboratory.
Before running an experiment, turn on the helium gas tank. Open the software on the computer, then bake out the column to remove any potential contaminants. Set the oven to a high temperature, typically 250 °C or above, and bake the column for at least 30 min.
Next, adjust the autosampler settings. Set the number of pre- and post-run rinses to clean the column between samples.
Use a sample volume of 1 μL and set the split ratio setting to program the instrument to accept only a fraction of the input. Adjust the flow rate of the carrier gas, and use established settings or trial and error to find the ideal pressure.
Now enter the temperature settings for the experiment. For an isothermal run, enter the temperature and the time for the separation. Alternatively, for a temperature gradient, enter the starting temperature and hold time, the ending temperature and hold time, and the ramp speed in °C per min.
Set the time for the column to cool between runs for either a gradient or isothermal run.
Finally, set the sampling rate and the detector temperature. The detector must always be hotter than the column to prevent condensation. After all the settings are programmed, save the methods file.
Activate the detector by opening the hydrogen tank valve and ignite the flame of the FID. The instrument is now ready for sample analysis.
To run the sample on the GC, first fill a vial with a wash solvent, such as acetonitrile or methanol. Prepare the sample, being certain to use glass syringes and glass vials as plastic residues can contaminate the GC.
Now add the prepared sample to a vial with a pipette. Fill at least half way, so that the autosampler syringe will be fully submerged. Then, load the wash and sample vials into the autosampler rack. Before running the sample, zero the baseline of the chromatogram on the computer software. Data can be collected either as a single run or using a batch table for multiple runs. Press "start" to run the sample.
In this example, caffeine and palmitic acid levels in coffee were analyzed using GC with FID. Caffeine is smaller and less polar, so it is less attracted to the column, and elutes first. Palmitic acid, which has a long alkane chain tail, elutes later due to a higher affinity with the stationary phase.
Because peak dimensions are proportional to carbon mass, the concentration of each component can be determined from its respective peak area on the chromatograph and compared to standards of known concentration.
The effect of column temperature was also explored. At 200 °C, samples moved through the column twice as fast as the sample run at 180 °C. Note that while peak heights change, the area under the curve remains constant.
GC is an important technique for chemical analysis, and is widely used in scientific, commercial, and industrial applications.
Due to the simplicity of GC, chemists routinely use it to monitor chemical reactions and product purity. Reactions can be sampled over time to show product formation and reactant depletion. The chromatograph reveals product concentrations and also the presence of unintended or side products.
GC is commonly used in tandem with mass spectrometry, called GS-MS, to unambiguously identify chemicals in samples or air. Mass spectrometry, or MS, separates molecules based on their mass to charge ratio, and enables the determination of compound identities. GC-MS is a powerful tool, as GC first separates complex mixtures into individual components, and MS gives precise mass information and chemical identity.
GC is routinely used in air monitoring to detect volatile organic compounds, or VOCs, which may arise from environmental pollution, pesticides and explosives. GC can be used to track and identify VOCs both indoors for headspace analysis and outdoors, for health, safety, and security.
You've just watched JoVE's introduction to gas chromatography with FID. You should now understand the basic principles of gas chromatography and FID detection.
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