SEPARATIONS SCIENCE MOLECULAR-BEAM METHODS and novel electrodes simplify analysis of mixtures MITCH JAC0BY, C&EN CHICAGO
LONG AFTER technical innovations have been turned into successful commercial products, clever people continue to dream up ways to improve and refine the inventions. Automobiles, computers, and telephones, for example, have been redesigned and enhanced for decades, yet every new model boasts advantages relative to its predecessors. The situation is much the same with laboratory tools for chemical separations. For years, analytical chemists have benefited from instrument makers' efforts to extend the capabilities of gas and liquid chromatography systems. From modest beginnings as simple analyzers that probe a relatively limited number of compounds, chromatography instruments have evolved to today's high level of sophistication, sensitivity, and automation. Nonetheless, the drive to advance the equipment's usefulness continues.
In gas chromatography, for example, researchers are developing molecular-beam methods that enhance separation and analysis of mixtures. And in the area of liquid chromatography, novel types of carbon electrodes are being developed to improve sensitivity and device durability. For more than 50 years, researchers have used gas chromatographs coupled to mass spectrometers to detect and analyze the chemical components of mixtures. Commercial GG-MS systems are practically standard laboratory equipment in industry and academia as well as in many government and smallbusiness labs. Despite broad popularity and generations of improvements, the
powerful lab combo is still fraught with shortcomings. "GC-MS suffers from a major Achilles heel in terms of the relatively small range of volatile and thermally stable compounds that are amenable to analysis." That assessment comes from Aviv Amirav, a chemistry professor at Tel Aviv University. Amirav, a specialist in analytical instrumentation development, has commercialized novel sample introduction devices and detectors for use in gas chromatography (G&EN, March 28,2005, page 53). Another limitation common to GC-MS analysis, according to Amirav, arises from the electron bombardment process typically used to ionize analyte molecules.
"The science of separations is the art of compromise." WWW.CEN-0NLINE.ORG
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In that widely used procedure, electrons emitted from a hot filament collide energetically with sample molecules and ionize those species by dislodging electrons. Mass spectrometry measurements based on that ionization method are straightforward and have been used to generate enormous spectral reference libraries. The trouble, however, is that the electronbombardment technique often yields mass spectra in which the molecular ion— the unfragmented parent molecule—is indiscernible. "Without knowledge of the molecular ion, we can never be completely confident that an unknown compound has been identified correctly," even if extensive spectral libraries are available for searching, Amirav argues. The reason for the uncertainty, he explains, is that the analyte molecule and its homologs, derivatives, and degradation products may all lead to the same or nearly the same mass spectrum. More than 10 years ago, Amirav and his research group began addressing these issues by drawing upon the benefits of molecular-beam methodology. This is an area of experimental science that originated in chemical physics and spectroscopy and eventually came to be applied to analytical chemistry. Following a number of investigations based on the group's earlier instrument designs, Amirav and Tel Aviv senior scientist Alexander B. Fialkov teamed up with researchers Urs Steiner and Larry Jones of Varian, an instrument manufacturer in
Walnut Greek, Calif. Recently, the team developed and tested an advanced-generation GC-MS interface device that transports sample compounds eluting from a GC column to a mass spectrometer by way of a supersonic molecular beam. THROUGH VARIOUS comparative stud ies, the team showed that due to the properties of molecular beams and the nature of the apparatus that generates them, their beam-based technique broadens the range of compounds suitable for GC-MS analysis relative to conventional GC-MS methods. In addition, it enhances molecular-ion signals and sensitivity and offers a number
of other advantages AT THE SHOW At Pittcon, Pohl compared with (left) and Cheng standard GC-MS of Dionex discuss methodology. the company's Basically, a sunew carbon personic molecular electrodes, which are designed for use beam can be generas electrochemical ated by seeding a detectors for HPLC carrier gas such as systems, such as the one shown here. helium with a low concentration of sample molecules and then directing the gas mixture through a tiny orifice into a vacuum chamber. As the gas stream passes through the small opening (a nozzle) to the evacuated chamber,
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Yamazen the Leader in Flash Chromatography the lightweight helium atoms collide with the sample molecules and strip away some of the molecules' vibrational energy. The helium atoms, in turn, acquire additional translational energy. Eventually, that process "cools" the analyte molecules, leaving them with very little vibrational energy. The upshot is that if the molecules are ionized in a state of reduced vibrational energy, they have a lower probability of fragmenting upon ionization. That increases the likelihood that molecular ions will survive the trip through the mass analyzer intact. The molecular-beam advantage is further bolstered by ionizing sample molecules using a so-called fly-through ionizer. As its name implies, this type of electronimpact device is designed to ionize compounds in molecular beams as they fly
evaluate the performance of the new system, Amirav and coworkers began by focusing on aliphatic hydrocarbons. In this class of compounds, the fragmentation pattern of one member often resembles those of other members, making identification difficult. In a proof-of-concept demonstration using hexadecane, the Tel Aviv group showed that with the molecular beam method, the analyte's molecular ion, at 226 amu, is far and away the most prominent feature of the mass spectrum. In classic electron-impact mass spectra, the molecular ion shows up as a minuscule, hard-to-discern signal. But other than the large boost in the abundance of the molecular ion, the group's mass spectrum for that G^ compound closely matches reference-library spectra, such as the one published by the National Institute of Standards & Technology. The researchers also showed that even better matches with reference spectra can be obtained (if needed for certain applications) by altering experiment conditions on the fly—for example, by briefly reducing the carrier-gas flow rate to temporarily reduce the vibrational cooling effect. After the C l6 demonstration, Amirav's group moved on to larger hydrocarbons. Compounds of that type are tough to analyze via standard through the ionizer while preventing GO WITH THE FLOW Analytes separated GC-MS methods not the molecules from coming in contact by LC are detected only because of their with the device's hot surfaces. Such as they flow tendency to yield noncontact, according to Amirav, can octhrough this distinct fragmentation cur as many as 50 times as a molecule electrochemical cell, which features patterns but also due to bounces around or scatters from the a novel (hidden) their low volatilities. Unsurface of a conventional ionizer. This carbon electrode. derscoring those points, contact would warm the molecule and Amirav notes that for restore its vibrational energy. compounds with carbon The scattering process also norchains in the range of C28 and larger, momally broadens chromatography peaks, lecular-ion signals are absent or nearly causing peak tailing, and promotes sample absent under typical GC-MS conditions decomposition on the hot surfaces—prob(carrier-gas flow rate of 1 mL per minute). lems that are avoided in the fly-through The largest hydrocarbons that can be elutionizer design. ed from a chromatography column under At present, the Tel Aviv group uses a those conditions are around C4Q. commercially available triple-quadrupole Now, the Tel Aviv's group's method has GC-MS system (Varian 1200 series) that extended that upper limit significantly. has been modified to accommodate the Using a commercial mixture of large linmolecular-beam instrumentation. To WWW.CEN-0NLINE.ORG
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the MS-MS mode of operation, molecules ear-chain hydrocarbons in test studies, flow rates, low column temperatures, and eluting from a GG column are ionized Amirav and coworkers find that with their short run times can reduce the separating in the molecular-beam interface and molecular-beam-based GC-MS technique, power of the GG portion of the experiment directed to the first quadrupole mass anathey can separate the components of the for some types of analytes. "There is always lyzer, which is set to transmit ions of just mixture readily, and they see dominant a trade-off between speed, resolution, a single mass (often the molecular ions molecular-ion signals for compounds as sample capacity, and other properties," he of an analyte). Those ions are fragmented large as C 84 H 170 (1,179.3 amu) (Int. J. Mass says. "It's just a matter of choice because in a second quadrupole by collision with Spectrom. 2006,2$i, 47). the science of separations is the art of coma gas such as argon. The daughter ions promise." Reducing run time, for example, As Amirav explains, the principal feature are then steered to a third quadrupole for may cause some analytes to co-elute from of the new GG-MS method that overmass analysis. a GC column. But if the co-eluting species comes the low-volatility challenge is the can be detected adequately and confidently high carrier-gas flow rate, which can be up In a demonstration study, Amirav's with a mass spectrometer—for example, by to 90 times greater than flow rates used group found that the molecular-beam identifying their molecular ions—then the in conventional GC-MS methodology. GC-MS-MS technique can be used to time benefit can be exploited without losEssentially, the fast gas flow pushes slugseparate and detect the organophosphate ing separating power. gish, nonvolatile molecules through the insecticide diazinon in a fruit and vegetable GG column. Ordinary GC-MS extract at the nanogram-persystems cannot tolerate the gram-of-produce level in less HIGH MASS onslaught of gas, which, in than 10 seconds. That separaUsing a molecular-beam-based GC-MS m e t h o d , the researchaddition to other problems, tion is several minutes shorter ers separate components of complex mixtures of large hywould bog down the mass than the time required by condrocarbons and identify t h e m with the aid of their prominent spectrometer's vacuum sysventional techniques. For even molecular-ion signals, as shown here for C72H146. tem. But being that high gas more demanding applications, flow rates are essential for the team is exploring molecuRelative intensity, % producing molecular beams, lar-beam-based GC-GC-MS100 the GC-MS interface in the MS separations. Tel Aviv lab is designed to acVarian continues to collaboMolecular ion 75 commodate (and continuously rate with Amirav to explore pump) all that gas. and develop the technique, but for now, the instrument 50 THE HIGH FLOW RATE of maker is not announcing comfers another key advantage: It mercialization plans. John drives thermally labile comD. Mills, Varian's scientific 25 pounds through the GC colinstruments marketing vice umn at relatively low temperapresident, notes that the mo1 ltlJiLiiu....... 1 .. . ..... 0 tures. That ability extends the lecular-beam method appears range of compounds amenable to offer a number of advanc) 250 500 750 1,000 1,2 50 m/z to GC-MS analysis by including tages that maybe especially analytes that would ordinarily beneficial for analyzing hydrodecompose at temperatures encountered carbons by the petrochemical industry. The separating power of an analytical in typical GC experiments. "As with all projects at this stage of develtechnique can be boosted by including opment, the ultimate commercialization As a case in point, the Tel Aviv group multiple separation phases—for example, of the technology will depend upon the showed that mixtures of aldicarb, oxamyl, by coupling two GC separations (GG-GC) results achieved by the research team," and other carbamate pesticides can be septo resolve analytes that elute together from Mills says. arated easily and quickly via their method. a single column. Alternatively, two mass Those compounds are typically separated spectrometers can be coupled to work in Meanwhile, on the solution-phase side and analyzed by customized liquid chrotandem (MS-MS) to select certain ions for of separations, chemists have used electromatography procedures. The team reports focused analysis. chemical cells as detectors for LC systems similar success with steroids, explosives, since the 1970s. The cells detect a variety of Recently, the Tel Aviv team members and other compounds that are not ordinaranalyte molecules whose functional groups combined their molecular-beam method ily analyzed via GC-MS methods due to are electro-oxidized or electro-reduced with the MS-MS capabilities of their comtheir sensitivity to heat. at characteristic potentials. As with other mercial triple-quadrupole instrument Amirav points out that high carrier-gas analytical lab tools, LG detectors have unto explore fast separation techniques. In
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dergone numerous modifications over the years to improve their sensitivity, reliability, and durability. AT THE HEART of an electrochemical cell is the electrode upon which the reactions take place. Carbon electrodes are widely used to analyze various types of analytes including aromatic compounds with amine and hydroxyl groups, aliphatic thiols, and other species. Nonetheless, the electrodes suffer some shortcomings. According to Jun Cheng, a staff chemist at Dionex, Sunnyvale, Calif., today's carbon electrodes exhibit poor electrode-to-electrode reproducibility. Furthermore, the sensitivity of individual electrodes drops markedly after just a small number of chromatography runs, forcing lab workers to try to restore electrode performance through time-consuming polishing and reconditioning of the device. To tackle these problems, Cheng and coworker Petr Jandik have developed an inexpensive, disposable type of carbon electrode that's designed to be replaced after a few weeks of use. Unlike conventional glassy-type carbon electrodes, which are prepared commercially via high-temperature pyrolysis, the new disposable electrodes are prepared by a vacuum technique in which a thin film of carbon is deposited onto a thin polymeric material. In an evaluation study, the Dionex team incorporated the new electrodes in flowthrough electrochemical cells that were used to detect a mixture of neurotransmitter compounds, including dopamine, epinephrine, and norepinephrine, as they eluted from an LC column. The study showed that those analytes are readily detected by the new electrodes at concentrations below the nanomolar range—a twoto threefold improvement in detection sensitivity compared with standard carbon electrodes. The team also tested the electrodes on mixtures of amino acids, metabolites, and other compounds. They report that the devices are stable and provide reproducible results from electrode to electrode. Dionex Chief Science Officer and Vice President for R&D Christopher A. Pohl points out that, compared with conventional carbon electrodes, the disposable ones offer improved response linearity. "We believe that the improved performance is a result of the deposition process, which controls the purity of the electrode material," Pohl suggests. He adds that the
new products should be generally available in two to three months. Rather than making electrodes from thin films of carbon, researchers at Eksigent, Dublin, Calif., are developing methods for fabricating inexpensive highsurface-area electrodes based on packed beds of tiny carbon particles. Eksigent researcher Nicole E. Hebert explains that the motivation for the highsurface-area feature is the need to construct a robust device that withstands fouling from electrogenerated products that stick to electrode surfaces. Made via microfabrication, the new electrodes, which feature picolitersized packed-bed volumes, Hebert can be connected in series—but controlled independently—to add resolving power to chromatographic separations. The idea is to set the potentials on the electrodes in a series to succes-
sively higher values—for example, to 100, 200, and 300 mV. In that way, if analytes co-elute from a chromatography column, then as they flow to the electrochemical cell, they can be detected selectively when they reach an electrode that's set to a suitable potential. Hebert notes that tests based on mixtures of neurotransmitters and other compounds demonstrate the new electrodes' robustness and selectivity. She says the company is now working to reduce cell volume and improve sensitivity. Inventions and innovations that survive the test of time eventually come to be referred to as "mature technologies." Some technology users prefer to abide by the old adage, "If it ain't broke, don't fix it." But not chemists. They know that even if technologies in separations science are mature, there's always room for improvement. •
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