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.
The equilibrium for gas chromatography is partitioning, and the components of the sample will partition (i.e. distribute) between the two phases: the stationary phase and the mobile phase. Compounds that have greater affinity for the stationary phase spend more time in the column and thus elute later and have a longer retention time (tR) than samples that have higher affinity for the mobile phase. Affinity for the stationary phase is driven mainly by intermolecular interactions and the polarity of the stationary phase can be chosen to maximize interactions and thus the separation. Ideal peaks are Gaussian distributions and symmetrical, because of the random nature of the analyte interactions with the column. Asymmetrical peak features, such as peak fronting or tailing, can be due to overloading the column, injection problems, or the presence of adsorptive functional groups such as carboxylic acids.
In GC, the temperature is adjusted to change the equilibrium and thus the elution times. Separations in GC are based on volatility because higher boiling point substances may condense on a column if the temperature is low, thus they are not eluted or take a long time to elute. Isothermal separations are performed at one temperature or gradient separations are performed where the temperature is ramped up during the separation. Temperature ramps allow both low- and high-boiling point compounds to be separated in the same separation.
The readout produced by GC is a chromatogram that gives the signal over time. Peaks are observed for each compound in the sample. For each peak, a peak height and a peak area can be calculated. Peak area is generally used to make calibration curves and to calculate concentrations of samples in unknowns. The number of theoretical plates (N) is calculated from each peak to give a measure of column efficiency. A practical equation for measuring N is N=16(tR/W)2 where tR is the retention time of the analyte and W is the width of the bottom of the peak. N is used to compare separations on different columns.
The flame-ionization detector is mass sensitive. Thus, the amount of signal is proportional to the mass of carbon in the sample, not the number of moles. Compounds with more carbon give greater signals. The burning of carbon produces ions which are detected as a current. FID is one of the most sensitive general detectors for GC with a limit of detection in the picogram range. The response is linear over seven orders of magnitude, giving it a large linear range.
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1. Initialization of the GC
- Turn on the helium carrier gas and air and adjust the pressure gauges on the instrument.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- Save the methods file. The parameters may also need to be downloaded so they are read by the GC.
3. Collection of GC Data
- Turn on the hydrogen gas and make sure the pressure gauge is set correctly. Light the flame of the FID.
- On the autosampler rack, fill the wash vial with wash solvent, like acetonitrile or methanol. Make sure the waste vial is empty.
- 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.
- 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.
- Load the sample vial(s) into the autosampler rack. Keep track of what position each sample is in.
- Before the run, zero the baseline of the chart recorder on the computer software.
- 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.
- 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
- 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).
- 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.
- 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.
- 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.
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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Applications and Summary
GC is used for a variety of industrial applications. For example, it is used to test the purity of a synthesized chemical product. GC is also popular in environmental applications. GC is used to detect pesticides, polyaromatic hydrocarbons, and phthalates. Most air quality applications use GC-FID to monitor environmental pollutants. GC is also used for headspace analysis, where the volatiles that are evaporated from a liquid are collected and measured. This is useful for the cosmetic and food and beverage industries. GC is used for forensic applications as well, such as detecting drugs of abuse or explosives. In addition, GC is useful in the petroleum industry for measuring hydrocarbons. The extensive applications makes GC a billion dollar per year worldwide market.
Figure 3 shows an example of how GC could be used in the food industry. Figure 3 shows a chromatograph of artificial vanilla (black) and real vanilla (red). GC can be used to identify the real sample, which contains a large peak for vanillin but does not contain a second peak for ethylvanillin.
Figure 3. GC-FID chromatogram of vanilla samples. Both imitation and real vanilla show large peaks at 4.7 min due to vanillin, the principle component of vanilla. However, imitation vanilla also has a large peak at 5.3 min, which is due to ethylvanillin, a compound not present in large quantities in real vanilla.
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