by Jeffrey M. Perkel
On the television drama CSI, there’s almost nothing a lab tech cannot do. Need to determine which make and model car left an incriminating fleck of paint at the scene? Done. Want to identify that suspicious oily substance on the victim’s hands? No problem.
The tool of choice for pulling off these forensic feats? GC/MS. And though much that happens on CSI may be scientifically dubious, gas chromatography-coupled mass spectrometry is indeed a critical tool of modern criminology, not to mention a host of other application areas.
According to Terry Sheehan, GC/MS product manager at Agilent Technologies, GC/MS fills a different niche than does liquid chromatography- or MALDI-based MS. Where LC and MALDI MS have become de facto instruments of proteomics research, for instance, GC/MS users are largely involved in food safety, environmental monitoring, forensics and toxicology, petrochemical analysis, and the development of flavors and fragrances, Sheehan says.
The common thread uniting these various applications? They all involve relatively small molecules. "The rule of thumb that's applied to LC versus GC is that GC lends itself to smaller molecules," says Sheehan. "There are very few examples of GC where the mass of the molecule exceeds one thousand, and the majority of applications are for molecules that are smaller than 600."
More specifically, those small molecules must be neutral and relatively non-polar. It's not that charged molecules cannot be analyzed by GC/MS, they just have to be derivatized first, by oximation or trimethylsilylation, for example.
That's because of the defining difference between LC- and GC-based methods; while LC/MS separates liquid phase materials, GC/MS separates compounds in the gaseous phase. Volatile compounds are, by definition, neutral (that is, uncharged), and relatively non-polar—properties distinctly at odds with most biological macromolecules.
Yet GC and LC/MS still operate via the same fundamental principle. LC/MS relies on chromatographic separation of a liquid-phase sample in a flowing mobile phase as it passes over a packed bed of stationary (solid) phase material. The differential affinity of the sample for these two phases provides the separation.
Similarly, in GC/MS, volatile compounds are flash vaporized and injected into a flowing stream of hydrogen or helium gas as it passes through a hollow capillary. The walls of this capillary—which may measure 30 to 100 meters in length, as compared to the typical LC column of perhaps 10 cm—are coated with some sort of separation matrix, and chromatographic resolution occurs as the highly energetic, heated material passes rapidly through the column.
According to Sheehan, that chromatographic difference provides a real edge in separation: "LC guys talk about plates per [column] meter, for instance 100,000 plates per meter, but they have a 10-cm column," he says. "That's where you get the difference in the techniques. The GC achieves these very long columns that are not possible in LC, because the pressure constraints of a long packed-bed column would exceed the pressure of even UPLC."
Nevertheless, says Robert Synovec, a professor of chemistry at the University of Washington, the two techniques are basically complementary. In a recent metabolic profiling study in which Synovec's team used two-dimensional GC-MS and his collaborator used LC/MS to profile metabolite changes in yeast, he says, "We found lists of metabolites that were interesting. There was some overlap, but then there was also quite a few found only by one technique or the other."
By far the most common mass analyzer for GC/MS applications is the single quadrupole. Agilent sells three such systems. All use the company's 5975C mass analyzer, coupled with one of three GC systems of varying flexibility (the 6850, 6890, and 7890).
According to Sheehan, a single-quad is sufficient for most GC/MS applications because the ionization process typically used to inject the sample into the MS fragments molecules in a very predictable fashion, providing structural detail that would otherwise require tandem capabilities to obtain.
That process, called electron ionization (EI), involves bombarding the sample with a high-energy electron beam to knock an electron out of orbit and impart a charge of +1 to the molecule. Weak bonds occasionally break during EI, but do so predictably, regardless of which manufacturer’s instrument is used to collect the data. As a result, researchers have compiled massive databases of EI fragmentation patterns, which GC/MS users can mine for compound identification. (No equivalent database exists for LC/MS-based fragmentation patterns.)
Large as those databases are, researchers will inevitably run into compounds they have never seen before. Or, they may be forced to try to detect their compound against a very dirty background. For those occasions, a single-quad may not cut it, says Gary Harland, product manager for tandem-quadrupole mass spectrometry at Waters, which offers a line of GC/MS instruments based on alternative analyzers, including a triple-quadrupole and a time-of-flight.
"Our instruments come into play for very complex or dirty sample matrices, where they want to get to very low sample quantities. Triple-quads work better under those conditions," says Harland. "If they want to identify compounds not in a spectral library, the GC-TOF gives exact mass to give greater confidence in compound identification, particularly in unknowns. There are many cases where there are no library data."
The advantages these instruments offer, says Harland, are tandem capabilities (in the case of the triple-quad) and high mass accuracy (for the TOF). The former provides greater confidence in sample analysis through both multiple reaction monitoring and the availability of structural detail; the latter can distinguish compounds sharing the same nominal mass.
"You have seen over the last decade a movement from LC-quadrupole to LC-triple-quads and LC-TOFs, as samples have become more complex," says Harland. "Exactly the same thing has happened in the GC/MS market. A single quad can do a lot of work, but it may take additional work to get the correct answer, and by adding MS/MS or exact mass [capabilities], they can get the answer the first time."
Also offering a GC-compatible TOF is JEOL, whose AccuTOF™-GC is one of three high-resolution, high-performance instruments the company provides for GC work, according to Robert "Chip" Cody, product manager for JEOL USA; the other two are double-focusing magnetic sector mass analyzers, which separate ions by passing them through magnetic and electric fields.
Other GC/MS providers include Shimadzu Scientific Instruments, with its QP2010 quad-based systems; LECO, whose product line includes the Pegasus 4D GCxGC-TOF, a two-dimensional GC-TOF MS; Thermo Fisher Scientific, offering single-quads, ion traps and magnetic sector analyzers; Varian, with its triple-quadrupole and ion trap-based systems; and Bruker Daltonics, which has a mobile GC/MS system called the E2M for on-site environmental analysis.
With so many choices available, how should you make a decision? As always, says Cody, the answer is simple: Know your application.
Do you need high resolution or will low resolution suffice? What type of samples do you plan to run? And, do you anticipate encountering things you've never seen before?
"The bottom line," he says: "Choose the instrument that answers your question."
