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The Fast, the 2-D and the Sticky
GC has a strong presence at Pittcon 2008

Angelo DePalma

Although gas chromatography has been crowded from the limelight by its life sciences counterpart, HPLC, innovation in GC methods continues at a furious pace. Since this month’s issue coincides with preparation for Pittcon 2008, we present a preview of noteworthy gas chromatography posters and talks from the conference.

Life in the fast lane

Figure 1: DANI Instruments’ MasterGC and TOFMS will be on display at Pittcon.

High-throughput chromatography methods are much sought after by analytical laboratories. But traditionally, “fast GC” has come at the expense of resolution. Manuela Bergna, R&D Manager at DANI Instruments (Cologno Monzese, Italy) will demonstrate in her presentation how this need not be so.

No hard and fast performance criteria circumscribes fast GC. A reasonable definition is any method that reduces analysis time by at least 75 to 80 percent compared with conventional instrumentation. Bergna and colleagues achieve these efficiencies through very rapid column heating, higher carrier gas pressure and faster signal acquisition. Where normal GC oven heating rates are around 20 to 30°C/min, DANI typically uses 50 to 60°C/min, with a maximum of about 140°C/min. Maximum carrier gas inlet pressure weighs in at around 120 psi. Under these conditions, rapid signal acquisition becomes more a necessity than a luxury, even with a relatively fast mass detector.

Column and injection hardware for fast GC also differ from those for conventional GC. Analytical scientists used to 0.25-mm diameter columns will note that “fast” columns are significantly narrower. DANI uses short, narrow-bore columns with internal diameters on the range of 50 to 100 mm. Since capacity is so much less than for conventional columns, and the column is smaller than a typical injection needle, DANI has redesigned the injector. Instead of the needle entering the column for on-column injection, the column enters the needle through an automated sampler. Because of the minuscule volumes involved, injection volume is determined not by syringe gradations, but by the length of time the column and needle remain in contact (usually on the order of milliseconds).

Together, these improvements allow neat, cold injections of complex mixtures of petrochemicals, foods, fragrances and other samples for which discrimination effects and solvent additions are undesirable. And it all happens with no loss of resolution compared with conventional GC. “You don’t lose any theoretical plates by moving to fast GC,” Bergna comments. “Notwithstanding the injection of a smaller amount of sample, sensitivity is almost comparable to conventional GC because of the narrower peak width.”

At Pittcon 2008, DANI Instruments is also presenting two posters on detection systems for fast GC. One illustrates the analysis of pesticide residues, and the other, a new small-footprint, competitively priced benchtop time-of-flight (TOF) mass detector.

Drink your wine

Figure 2: A demonstration of fast gas chromatography. Click to enlarge.

Along a similar vein, Lingshuang Cai, Ph.D. of Iowa State University (Ames, IA) will report on rapid GC-MS methods for wine analysis, specifically in six Iowa vintages. Cai's method, based on microextraction and on-fiber silylation-derivatization, targeted resveratrol (3,5,4’-trihydroxystilbene), is the latest “miracle” phenolic dietary supplement. Resveratrol exists principally in red wine, but one would need to drink a tank car full of merlot to ingest a reasonable dose. The compound’s low concentration and polar structure, combined with a host of other polar materials in wine, presents a challenge to quantitative analysis.

Cai and coworkers used multidimensional GC (polar, non-polar columns) connected in series, a valve for isolating heart cuts from the first column and a cryogenic focusing device—a cold finger—to reconcentrate the cut before re-injection.

LECO Instruments (St. Joseph, MI) will unveil an approach to fast GC-MS that involves higher carrier gas flows and very rapid out-of-oven column heating and cooling. Co-eluting peaks are differentiated through rapid spectral acquisition, automated peak-find, and deconvolution algorithms resident in the LECO ChromaTOF software.

LECO’s modular, low-thermal mass-fused silica column is co-wrapped as a toroid with a heating element and thermocouple, allowing ultra-fast heating and cooling—up to 1800°C/min. “This is possible because only the column needs to be heated,” says applications chemist Pete Steves. “There is no oven.” Where a conventional GC column can take several minutes to return to equilibrium following temperature ramping, a low-thermal mass column, so configured, recovers its initial temperature in as little as 20 to 30 seconds.

2-D methods: alive and well

Figure 3: Ultra-fast GC: A scientist at LECO analyzing data collected on the company’s Comprehensive Two-dimensional Gas Chromatograph – Time of Flight Mass Spectrometer (GCxGC-TOFMS) utilizing GERSTEL’s MACH-LTM Low Thermal Mass Column mounted on the oven door

Shimadzu (Columbia, MD) will present three posters on two-dimensional GC, a rapidly growing technique that is set to break into mainstream industrial use. According to GC and GC-MS product manager Mark Taylor, 2-D GC methods are just coming into their own in academic laboratories. “Companies are still deciding where it fits in.”

Two-dimensional GC uses two sequential columns with orthogonal separation capabilities (usually one polar and the other non-polar). The technique is particularly effective with complex mixtures of polar and apolar compounds, as are found in petrochemicals and foods.

Mr. Taylor would not say when Shimadzu would introduce a commercial 2-D system. “We’re still putting the prototypes through their R&D paces.” End users interested in beta testing a Shimadzu 2-D system should get in touch with the company.

Shimadzu’s anticipated commercial 2-D GC system will take heart cuts of co-eluting compounds from the first column, re-concentrate them and divert them to the second column, where the peaks will be further resolved.

Taylor estimates that eventually a 2-D GC system will cost about 1.5 times as much as a single-dimension system. The price tag depends on whether one or two ovens are used. Savings will accrue, though, from the use of one set of controls and a single information system.

Figure 4: David Clarke, a graduate student in Robyn Hannigan’s group at Arkansas State, at work on a GC-fluorescence instrument

Of Shimadzu’s three 2-D GC presentations at Pittcon, two describe measurement of oxygenated compounds (alcohols and ethers) in gasoline, which is important for product characterization as well as for meeting mandated emissions targets. Single-column analysis is complicated by the difficulty of separating oxygenated (polar) species and hydrocarbons (apolar) on one stationary phase. One of the posters describes a study in which 13 oxygenated compounds were baseline resolved and quantified. The other reports on coupling an MS detector to the 2-D GC to overcome sensitivity issues with flame ionization detection.

Shimadzu’s third poster illustrates the sensitivity of 2-D methods for the water contaminants 2-methylisoborneol (2-MIB) and geosmin, whose musty odor humans can detect at ng/liter levels (parts per trillion). Solid-phase extraction (SPE) followed by single-column analysis does not work because SPE also concentrates matrix compounds, which co-elute with the analytes. The two-column approach separated the contaminants easily, with “excellent repeatability and linearity.”

Stop the flow
One potential problem with 2-D GC is that the separation on the second column depends on the modulation period, which determines how frequently eluent from the first column is sampled and introduced into the second column. Shorter modulations better preserve the resolving power of the first dimension because they sample less eluent, while longer modulations provide more efficient secondary separations. Approaches that seek to optimize the modulation period include mathematical treatments of first-column peak width, and the compromise approach of inducing broad peaks in the first dimension, which reduces overall separation power. Chemistry professor Tadeusz Gorecki, Ph.D., at the University of Waterloo (Waterloo, ON), will discuss a new method, stop-flow with pneumatic switching, which incorporates novel hardware.

Stop-flow involves halting flow in the first column while analyzing continuously in the second dimension. The technique essentially allows more-frequent sampling of the first column without swamping the second column. Stop-flow has been used in GC in selectivity tuning (i.e., Richard Sacks, U. Michigan), but typically flow is halted only intermittently, and then only briefly. Gorecki stops flow at regular (short) intervals throughout the run.

Conventionally, stop-flow is accomplished through high-temperature valves, but Dr. Gorecki found that valves were too complex and not sufficiently rugged for routine use. His innovation is pneumatic switching, which uses pressure applied between the columns to stop or attenuate flow, but only in the first-dimension column. The second column operates normally, but without the time constraint of a rapidly eluting first column.

One would expect this technique to reduce first-column efficiency, but it does not. Professor Gorecki will present results that demonstrate, that for GC-GC methods, first-column resolution is actually better in stop-flow mode than in normal GC-GC mode using the identical modulation period. Two additional benefits: the second column may be longer than in conventional 2-D GC, and modulation periods may be lengthened as well.

Another group from University of Waterloo will present data on a flavor profile analysis of coffee using GC-time-of-flight MS. Coffee analysis has become crucial for quality assurance, since a fair amount of the world’s coffees sold as “premium” blends do not come from their alleged country of origin. For this study and one on “eiswein” (ice wine), another favorite of counterfeiters because of its high price, Sanja Ristevic used Leco’s ChromaTOF data processing software to deconvolute the thousands of flavor components into those which, together, create the unique flavor signatures of Brazilian coffee and authentic ice wine. For example, Ristevic identified 102 volatile and semi-volatile flavor components whose concentrations define the unique flavor of authentic Brazilian java.

High on the environment
Of the significant inorganic environmental pollutants, mercury belongs to a limited number that are stable as covalently bonded organometallics. Organomercury compounds are toxic because they bear the heavy metal in an unusually bioavailable form.

Mercury toxicity and bioavailability depend not just on total mercury, but its speciation—what the metal is bound to. Techniques for organomercury speciation include the hyphenated methods of liquid chromatography-inductively coupled plasma(ICP)-mass spectrometry (MS) and GC-ICP-MS; plus hydride-generation atomic absorption (AA) and cold-vapor AA; and the more traditional GC-MS, ICP-MS and LC-MS. The most common environmentally problematic organomercury compound, methyl-mercury, is analyzed by cold vapor methods.

According to Robyn Hannigan, Ph.D., who directs the Environmental Sciences Program at Arkansas State University (State University, AR), a dedicated GC-ICP setup is desirable for mercury speciation due to the instrument’s high sensitivity and the potential for cross contamination with other samples. “Should a lab routinely run water, blood, urine or soil/sediment digests, the instrument will require significant cleaning and maintenance to ensure that GC-ICP-MS detection limits are meaningful.” Unfortunately, dedicating a system for organomercury analysis is beyond the capabilities of most environmental labs.

This is why Hannigan’s presentation about novel GC methodology for quantifying mercury and determining its speciation will attract a good many environmental scientists. According to Professor Hannigan, her GC technique provides organomercury quantification and speciation on par with results from GC-ICP. The method uses a laboratory-grade GC coupled to a fluorescence flow cell inside a pyrolyzer. The GC identifies derivatized organomercury species by retention time and comparison with a reference database. As the compounds emerge, the pyrolyzer converts them to elemental mercury, whose concentrations and fluorescence times are read by the detector. Hannigan has developed a related method that does not require forming derivatives, and uses headspace injection, but has only tested it on biological samples.

The GC-fluorescence method detects methylmercury down to ng/L concentrations, which is not considered toxic. But MeHg bioaccumulates in organisms, increasing its concentration tens of thousands of times. “So concentrations of methylmercury in environmental samples is environmentally relevant from a monitoring perspective, but not a toxicological perspective.”

And now for something completely different…this may sting
Norman Schmidt, Ph.D., of Georgia Southern University (Statesboro, GA) will present data on a novel approach to environmental chemical monitoring using GC. Instead of sampling soil, air, water or crops, Professor Schmidt gets a handle on environmental chemical levels by analyzing honeybee hives. Bees, it turns out, collect a lot more than nectar.

Schmidt samples hives with solid-phase microextraction fibers (carboxen-polydimethylsiloxane or polydimethylsiloxane alone) exposed to hives for up to 64 hours, followed by GC-MS. Most of what he observed is wax components (long-chain fatty acids and hydrocarbons), chemicals added to wax by bees during the course of their activities and compounds released from the polymer supports on which commercially raised bees build their hives (styrene, benzaldehyde, benzoic acid). Based on the small molecule composition, he can tell where the bees spend most of their time. “Hives containing a lot of terpenes are probably occupied by bees that spend time in pine forests. “ Sugars are not normally extracted from healthy hives because they are usually bound up inside wax structures. When sugars do turn up in the analysis, it is probably due to an infestation of hive beetles that eat bee eggs and honey, and in so doing, disrupt the hive’s delicate architecture. “The combination of polar and non-polar compounds makes for very messy chromatography,” he notes. Schmidt is now looking into the possible role bee pheromones might play in attracting harmful organisms like the hive beetle. Could these predators intercept chemical messages from bees, to the latter’s detriment? We will probably have to wait until Pittcon 2009 to find out.

Angelo DePalma is a chemist- turned-freelance writer based in Newton, NJ. He may be contacted at ChromatographyTechniques@advantagemedia.com.


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