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In the biological sciences, to go-to method for molecular separation is liquid chromatography (LC). But those engaged in forensics, petrochemical research, food and flavor development, environmental science, and metabolomics take another approach: gas chromatography (GC).
What all these fields have in common is that they typically involve small, heat-stable, low-to-mid polarity organic molecules, says Mark Taylor, GC and GC/MS product manager at Shimadzu Scientific Instruments. "As a general rule of thumb, if it's organic, or is a gas, and can withstand high heat, it is probably a compound that can be run by GC," he says. "In contrast, liquid chromatography uses very polar compounds, such as things in the life sciences, which are soluble in water, and which would be chemically changed by heat."
Essentially a column in a precisely controlled oven, a gas chromatograph flash volatilizes compounds into the gas phase and separates them as they pass through a long, thin tube. But, because the oven can only get so hot, gas chromatography imposes an inherent size limit.
"Typically the highest mass you can get through a GC is around 800 or 900 mass units, because it has to volatilize and flow through at a temperature no hotter than about 450C or so. And that's too low to make some things volatile," Taylor explains. Bigger molecules "just don't evaporate at that temperature."
Though directly analogous to liquid chromatography, gas chromatography does differ substantially. Instead of using a running buffer as in LC, GC separations occur with the sample to be separated embedded in a flowing stream of carrier gas, such as helium. In place of the packed beads that comprise the LC column's stationary phase, the inside wall of the GC capillary column is coated with a thin film of material such as dimethyl or diphenyl polysiloxanes, which can offer a boiling point or polarity separation depending upon their respective percentages. And the gas chromatography column itself is much longer than in LC, sometimes up to 120m in length.
Gas Chromatography Injectors
For gas chromatographs, several injector types are available. The simplest is direct injection, in which a sample is injected, flash vaporized, and passed directly to the column. However, because it can be difficult to reliably inject small sample volumes, other injection methods have been developed. One is called "split injection," in which only a fraction of the injected sample passes to the column; the rest goes to waste. A related method is called "split-splitless injection," in which the sample inlet port is heated, but not the column, causing the sample to recondense and concentrate prior to chromatography.
Gas Chromatography Detectors
A number of different detectors exist to analyze the eluent that emerges from GC columns. The oldest, according to Taylor, is the gas chromatograph thermal conductivity detector (GC TCD), a "universal detector."
"It will detect pretty much anything that can go though a GC column," Taylor says, "but at the same time it is limited in its sensitivity."
One of the most popular detectors, and the one that's directly analogous to the UV detectors that typically frequent the tail end of LC columns, is the gas chromatograph flame ionization detector (GC FID), says Taylor.
Lucas Smith, Product Manager at LECO Separation Sciences, calls the GC FID "a non-selective detector. Essentially it just burns, so as a general rule the more carbon you have, the more signal you obtain."
Another popular choice is the GC ECD, or gas chromatograph electron capture detector. Specific for halogenated compounds, ECD measures the impact of electronegative compounds on the current generated by an otherwise constant electron beam. "Essentially, all we are doing is we are allowing electronegative compounds to enter an area, and then we measure how many electrons they suck up," says Taylor.
Other detectors include flame photometric detectors (GC FPD), which measure sulfur- and phosphorus-containing compounds; thermionic or nitrogen phosphorus detectors (NPD), which measure compounds containing nitrogen or phosphorus; the photoionization detector (PID); and the pulsed discharge helium ionization detector (PD-HID), used to assess the purity of carrier gases.
Each of these detectors has its own application areas, Taylor says. A forensics lab, for instance, might use a flame ionization detector to look for accelerants or to measure blood alcohol levels, and a nitrogen phosphorous detector for drugs. Environmental scientists looking for pesticide contamination might use an electron capture detector, while petrochemicals researchers "almost always use an FID or a thermal conductivity detector, mainly just to see if there's anything there."
Unfortunately, that's pretty much precisely what these detectors provide: a sense of whether anything is there. "If you have a sulfur detector and there are no sulfur compounds in the sample, you get a flat line," says Taylor. "However, on the TCD, using the same mix, you'll see hundreds of compounds, because it's universal."
What you won't know, however, is what those compounds are. That's where mass spectrometry comes in. MS provides "speciation" data, says Smith. "We provide not only the total ion count, but also what ions comprise the signal."
The most common mass analyzer, "by far," says Taylor, is the single quadrupole mass analyzer. "I think it's something like 85% of all GC/MS out there are single-quad instruments," he says.
Other available mass analyzers include ion traps, magnetic sector analyzers, and time-of-flights (TOF). LECO specializes in GC-ready TOFs.
According to Smith, though TOFs are more expensive than quadrupoles, they offer several advantages, including sensitivity, speed (with scan rates of up to 500/sec vs. 10/sec for quadrupoles, for instance, TOFs are more compatible with faster separations), a lack of spectral skewing, and the ability to detect unknown compounds.
"If you are going to be looking for known compounds, and time is not an issue, then you're probably better off with a quadrupole or a magnetic sector," says Smith. "If you're looking for time-compressed chromatography, or if you're looking for significantly increased throughput, or very complicated matrices, or unknown analytes, a TOF is very hard to beat."
For those who require more efficient separation than a single column can provide, multidimensional gas chromatographs have also been devised. One, called "heart cutting," takes selected sections of the sample as it emerges from one column and introduces it into a second, orthogonal column (that is, one whose separation medium differs from the first column).
Another approach, commercialized by LECO, is "comprehensive two-dimensional chromatography," or GCxGC. The technique, says Smith is "essentially sequential heart cuts." Basically, as each "fraction" emerges from the first GC column, it is cryofocused—condensed and concentrated with a burst of very cold air—and then applied to a second, orthogonal column, with a narrow internal diameter and fast flow rate. The result, says Smith, is both higher, sharper peaks, and "massively increased chromatographic separation."
Ultimately, when making a purchase decision, which detector or detectors you choose will depend largely on your applications, and your anticipated sample throughput, says Taylor. Between gas chromatograph autosamplers, faster oven ramp speeds, narrower capillaries, and higher pressures, chromatography can be dramatically accelerated, he notes.
"Every sample is money, so you want to run as many as you can on as few boxes as possible."
Please check out our Gas Chromatography section for more information or to find manufacturers that sell these products.