Product Focus: Chromatography-Mass Spectrometry
Among the hyphenated mass spectrometry (MS) techniques, gas chromatography-MS (GC-MS) and high-performance liquid chromatography- MS (LC-MS) are the most notable. These methods combine the resolving power of high-resolution chromatography with the specificity and sensitivity of MS.
Best of Both Worlds
Among the “hyphenated” mass spectrometry (MS) techniques, gas chromatography-MS (GC-MS) and high-performance liquid chromatography-MS (LC-MS) are the most notable. These methods combine the resolving power of high-resolution chromatography with the specificity and sensitivity of MS.
GC-MS has been around since the 1950s, although the technique was impractical for routine analysis until the advent of small, inexpensive mass detectors. LC-MS came into its own later, with the introduction of “soft” ionization methods such as electrospray (ESI) and atmospheric pressure chemical ionization (APCI).
The power of LC-MS and GC-MS derives from the orthogonality of their components. Chromatography’s principal drawback is the co-elution of different chemicals, which complicates their definitive identification by ultraviolet or flame ionization detection, while mass spectrometry may not distinguish between isomers. Together, chromatography and MS practically eliminate these shortcomings.
LC-MS and GC-MS are complementary with respect to the types of molecule they work best with. GC is most appropriate for small, apolar, “traditional” organic compounds that volatilize easily, while LC shines with larger, polar compounds such as peptides, proteins, genes, and pharmaceuticals. In their native forms, LC treats molecules more gently than GC, which is even more true when comparing LC-MS and GC-MS.
Because LC-MS employs soft ionization, it generates a molecular ion peak corresponding to the molecule’s molecular weight, but few fragment peaks. GC-MS, by comparison, tears the molecule apart to provide information- rich fragmentation patterns. Users may apply tandem MS (referred to as MS/MS) to induce fragmentation in LC-MS, but tandem detectors are costly. Software is used to “piece” fragments together and compute the molecular weight.
Comparing the methods
LC-MS is extremely effective for polar pharmaceutical molecules, metabolites, and smaller biomolecules. “But analysis of intact proteins is still a difficult, complex problem,” observes Lester Taylor, Ph.D., LC-MS platforms and program manager at Agilent (Santa Clara, CA). Factors such as aggregation, folding, large size, and the presence of disulfide bonds or bound membranes don’t interfere with LC, but they do confound mass analysis. Analysts can circumvent these challenges through chemical treatments or by using enzymes to break the molecules into manageable pieces.
“Most customers acquire LC-MS instruments as complete systems, but piecemeal purchases are possible if a particular LC or MS instrument is preferred. Turnkey systems,” says Dr. Taylor, “are much less likely to suffer from LC-MS interface or communications issues.”
GC-MS is more predictable. “Pretty much anything that goes through a GC can be analyzed by GC-MS,” observes Ron Snelling, Ph.D., senior GC/GC-MS product specialist at Shimadzu Scientific Instruments (Columbia, MD). “The bad news is that GC is innately molecular-weight limited (to about 500-1000, depending on volatility). In the past, very large, polar molecules were derivatized (often by silylation), but this practice is dying out since LC-MS is so accessible.”
As many as 25 percent of GC systems today ship with MS instead of flame ionization, conductivity, or electron capture detectors. This is a relatively new development resulting from the miniaturization of and falling prices for MS instruments. Customers most often purchase complete GC-MS systems rather than components for the same reasons as for LC-MS. “In situations where labs decide to upgrade to MS detection for an existing GC,” Snelling says, “they generally purchase a new GC-MS system instead because the cost of the GC component is fairly small. Customers considering high-end GC systems with two columns and two detectors should also consider a GC-MS instead. For an additional $15,000, you can have GCMS and only have one column and one detector to worry about.”
Common ground
Mass detectors have revolutionized LC and GC by providing unequivocal identification and exquisite detail to every analytical run. Iain Mylchreest, Ph.D., vice president and general manager for life sciences MS at Thermo Fisher Scientific (San Jose, CA), calls MS the “ultimate detection tool” that provides qualitative and quantitative information. “UV, FID, and PD tell you something is there but not what it is. MS tells you something is there and provides absolute identification.”
GC and LC labs interested in mass detectors have a range of choices. Single- stage quadrupole MS instruments return a molecule’s nominal mass for LC, and more detailed mass plus fragmentation in GC mode. More advanced MS detectors utilizing tandem MS/MS are significantly more expensive, but provide very accurate mass to one or two parts per million, and accurate elemental ratios.
According to Dr. Mylchreest, dissatisfaction with derivatization is driving customers from GC-MS to LC-MS. An exception is for EPA methods, which are based on GC-MS for historical reasons. “It would take a lot of work to revalidate those methods,” he says. Dr. Taylor of Agilent disagrees, attributing the perception that LC is “taking over” to the explosion of life sciences LC-MS applications, particularly in genomics, proteomics, and metabolomics.
Regardless, both GC and LC have staunch supporters whose loyalty is magnified with the addition of MS. “There has been a huge shift toward MS being the detector of choice for LC and GC,” Dr. Mylchreest tells Lab Manager Magazine. “Mass detectors are slowly making inroads into the midpriced instrument market. MS won’t replace UV detection for $30,000 LC systems, not for a while, but maybe some day.”