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SCIENCE/TECHNOLOGY
Analytical instrument future highlighted Pittcon—the Pittsburgh Conference & Exposition on Analytical Chemistry & Applied Spectroscopy—can be a tease. A jumbo showcase for current leading-edge technology in instrumental analysis, it can also provide a forum for tantalizing tidbits about the future.
Pittcon '93, which recently concluded in Atlanta, did just that, as several analytical chemistry professors used speaking opportunities to hazard predictions about what is in store. Some fairly substantive developments were among those envisioned: further miniaturization of devices, massively parallel arrays of samples, and imaging of complex sample geometries, for example.
The pathway to development will not always be easy, however. After predicting capillary electrophoresis on a microchip, Milos V. Novotny of Indiana University, Bloomington, quipped that the inventor would probably surround the tiny system with a huge block of detection gear. Novotny spoke at a symposium honoring the late analytical chemistry professor Lockhart B. (Buck) Rogers.
And in accepting the Pittsburgh Analytical Chemistry Award, Edward S. Yeung of Iowa State University, postulated an array of 4096 simultaneously laser-scanned capillaries that might sequence the human genome in 35 days. But Yeung admitted that preparing the device with DNA and reagents might be a daunting task.
In another vision of massively parallel sample arrays, David M. Hercules of the University of Pittsburgh told the Buck Rogers symposium that matrix-assisted desorption of samples from silver surfaces might eliminate the need for chromatography prior to mass spectrometry. For example, a dime has a 250-mm surface area. If 150-μιη-diameter sample spots are deposited on it, the capacity would be 7000 samples. These spots could be desorbed in
rapid succession and the ions fed into a time-of-flight mass spectrometer.
And Gary M. Hieftje of Indiana University told the same audience that imaginative visualization of data will be commonplace. Humans are great pattern recognizers, he explained. Combinations of three-dimensional space, false colors, multiple images, and animation, can convey up to seven dimensions to the chemist.
Also on the subject of miniaturization, Hieftje predicted a nanospectrophotom-eter in a "flat" configuration, that would use Fresnel lenses, holograms, and silicon nitride wave guides, with a multi-chromophore sensor. He described this sensor by analogy to the human eye, which uses the same 11-c/s-retinal to see three colors because of three different protein environments.
Among other predictions, M. Bonner Denton of the University of Arizona, Tempe, said that thin-layer chromatography will get new attention, thanks to automation and detection by two-dimensional charge-coupled device detectors. Denton envisioned 100 plates per minute spotted uniformly with a "spray paintlike" device. After an eight- to 10-minute ride on a conveyor belt for development, they would flop off the end under a charge-coupled device or two-dimensional indium antimonide infrared detector for quantitative imaging.
In accepting the Bomem-Michelson Award, Jack L. Koenig of Case Western Reserve University, Cleveland, also spoke of imaging. He has used animated, two-dimensional microspectropho-tometry to study orientation of domains of liquid crystals embedded in polymers. His goal is to develop switchable building windows that can be darkened or lightened by application of electric fields.
Stephen Stinson
Methods seek to find mycotoxins in food, feed Progress is being made toward faster, cheaper analyses of human food and animal feed for fungus-produced toxins called mycotoxins. As described at Pittcon '93 by several research groups, such tests may not only lead to easy and economical detection of mycotoxin contaminants in food and feed but aid research in this field as well.
Some examples of mycotoxins are ergot alkaloids, aflatoxins, fumonisins, ochratoxins, and trichothecenes. In addition to being acutely poisonous, aflatoxins are known to be human carcinogens.
Representative of work being done is that of veterinary medicine professor George E. Rottinghaus of the University of Missouri, Columbia, who described the development of a technique employing high-performance liquid chromatography (HPLC) to determine ergot alkaloids. The method detects alkaloids to a low concentration of 50 μg per kg and may thus eliminate the need for costly tandem mass spectrometry (MS-MS) to analyze feeds at those levels. The method may also aid research into production of ergot alkaloids by hitherto unsuspected fungi.
Lynn Jordan of Zymark Corp., Hop-kinton, Mass., discussed work in which immobilized antibody-packed HPLC columns were combined with a robotic workstation to detect aflatoxins and fumonisins. The methods are fast, automated, reproducible, and free of such objectionable solvents as benzene and chloroform.
Ergot is usually found in rye, oats, wheat, and barley, and in such grasses as bromegrass, bluegrass, and ryegrass, when they are infected with the fungus Claviceps purpurea. The fungus invades the flowers and replaces the seed with hard masses called sclerotia.
Sclerotia are as much as 1% ergot alkaloids, chiefly ergotamine. Milling of grains tends to concentrate sclerotia, which are slightly larger and heavier than seeds, in leavings that are then ground or pelleted into animal feed.
Chronic symptoms of ergot poisoning at low levels in cattle are lameness and dry gangrene that results in loss of hooves, ears, tips of tails, and lower parts of limbs. At high levels, horses and sheep may develop acute symptoms of nervousness and convulsions.
In recent years, another fungus, Acre-monium coenophialum, has been found to infest a grass called tall fescue, where it produces ergot alkaloids, mainly ergo-valine. Cattle who feed on such grass develop a dry gangrene called fescue foot. The link of fescue foot to ergot alkaloids was disputed for a long time and has only recently been confirmed.
Until now, testing for ergot infestation has been by microscopic examination of ground or pelleted animal feeds for
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signs of sclerotia. MS-MS has been adapted to such analysis, but the equipment is costly and not widely available. Thin-layer chromatography (TLC) is also useful, but is limited to milli-gram-per-kilogram levels of ergot alkaloids.
Rottinghaus extracted ergot alkaloids from feed with chloroform. He passed the extract through a "cleanup" column, which bound alkaloids tightly, and used acetone-chloroform to elute plant pigments. Then he eluted the ergot alkaloids with methanol and subjected part of the eluate to HPLC with fluorescence detection.
Ergot alkaloids showed up in a characteristic pattern of five peaks for ergosine, ergotamine, ergocor-nine, ergokryptine, and ergocris-tine. He confirmed identification of ergot alkaloids by heating the rest of the eluate with dilute acetic acid, which isomerized ergotamine to ergotaminine and the other four ergot alkaloids to a mixture of their "-inine" isomers. The five -inine isomers likewise gave a diagnostic pattern of five peaks. He further confirmed identifications with MS-MS, though that technique will not be used in rapid screenings of feeds with his HPLC method.
Rottinghaus has also adapted this method to screening of tall fescue for ergovaline. The A. coenophialum fungus that produces ergovaline lives in a symbiotic relationship with tall fescue, and infestation is endemic. Thus, the technique may be useful not only in screening tall fescue as feed, but also in crop breeding research to produce a tall fescue that will not tolerate symbiosis with A. coenophialum.
Both Rottinghaus and Jordan have worked to develop analyses for fumonisins Βλ and B2. These mycotoxins are produced by Fusarium moniliforme, which infests corn. They are linked to leukoen-cephalomalacia (softening of white brain matter) in horses, pulmonary edema (fluid in the lungs) in swine, and organ damage and failure to thrive in poultry. And they are also suspected of causing human esophageal cancer.
Fumonisins have been found in up to microgram-per-gram levels in corn muffin mixes, popcorn, cornmeal, cereal, and
Some mycotoxins are toxic
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grits, bought in supermarkets. The human risks posed by these amounts of mycotoxins are unknown.
Fumonisin Bi is the 4,5-bis(tricarbal-lylate) ester of 19-amino-3,7-dimethyl-eicosane-4,5,9,16,18-pentaol. The lack of chromophores in such a compound makes detection in chromatographic methods difficult, but the amino group makes derivatization possible.
Rottinghaus developed a rapid TLC method, capable of detecting a lower limit of 1 μg per gram, to screen animal feeds routinely for fumonisins. He extracted corn or cornmeal with aqueous acetonitrile, then passed the extract through a cleanup column to retain fumonisins while eluting interfering substances. Then fumonisins were eluted with aqueous acetonitrile and a TLC plate spotted with the eluate. Spots of fumonisin B7 and B2 were detected by spraying the spots with boric acid and fluorescamine, which caused them to
fluoresce under ultraviolet irradiation.
Identification of the mycotoxins was confirmed by hydrolyzing the bis(tricarballylate) esters and chromatographing the resulting aminoalcohols. All experiments were checked with authentic samples of fumonisin B^ and B2.
Zymark's Jordan used immu-noaffinity technology to develop automated detection methods for both fumonisins and aflatox-ins. Aflatoxins are produced by Aspergillus flavus- and A. parasiti-cws-contaminated peanuts, soybeans, cottonseeds, rice, sorghum, and corn.
The five types are aflatoxin Bv B2, Gv G2, and M. In these names, Β and G come from the blue or green fluorescence the compounds show in TLC. Aflatoxin M is named for its appearance in the milk of animals feeding on Aspergillus-infested grains, though it also appears in their urine. The Food & Drug Administration has set upper limits of 20 ppb for total aflatoxins Bv B2, Gv and G2 in feed grains for interstate shipment and 0.5 ppb for aflatoxin M in milk.
Animal poisoning is called aflatoxicosis and may be chronic or acute. In acute toxicity, the LD50 (the dose that kills 50% of a group of animals tested) is 2 mg
per kg of body weight for calves, sheep, and chickens. Chronic effects include tissue death in liver and other organs, blood coagulation defects that result in internal hemorrhaging, depressed immunity to infectious diseases, and failure to gain weight.
Jordan's analyses for fumonisins and/ or aflatoxins use columns of monoclonal antibodies against either of the two mycotoxin classes immobilized on agarose. Antibodies hold up the mycotoxins temporarily while other substances in the sample are separated. Then aflatoxins are derivatized by reaction with iodine and determined by HPLC. Fumonisins are reacted with phthalal-dehyde and mercaptoethanol, which produces a fluorescent derivative for HPLC detection. Programming a robotic workstation with the method reduces analysis times to 15 minutes per sample.
Stephen Stinson
MARCH 29,1993 C&EN 29
SCIENCE/TECHNOLOGY
Use of scanning probe microscopy expanding A technique that in one of its variations, just a few short years ago, was a device for doing R&D in physics is now beginning to emerge as an analytical tool. Scanning probe microscopy (SPM) is gaining applications at a rapid clip, judging from the booth exhibits of two manufacturers at Pittcon '93—TopoMet-rix, Santa Clara, Calif., and Digital Instruments Inc., Santa Barbara, Calif.
It's all part of the "age of the nano revolution/' as Gary D. Aden, president and chief executive officer of TopoMet-rix, describes the 1990s. The decade, he notes, marks the convergence of solid-state science and chemistry.
Atomic force microscopy images of polyethylene show (top) individual polymer chains, including the individual methylene groups (CH2) of the chains and (bottom) stacked lamellar single crystals of the polymer
That convergence is underscored as well by the experience of Digital Instruments. A spokesman for the company says, for example, that the vast majority of the company's units are being sold to chemistry departments at universities— to surface scientists mostly—for studies of polymer behavior and for electrochemical work in studies on corrosion. And a lot of analytical companies, he says, are buying units to use as another tool in their repertoire, mostly for topographic work.
SPM, especially in its atomic force microscopy (AFM) variation, has been spinning off so-called initialed techniques seemingly as fast as new imaging modes can be conceived. Such modes now include scanning tunneling microscopy (STM), scanning force microscopy (SFM), scanning magnetic microscopy (SMM), scanning near-field optical microscopy (SNOM), scanning thermal microscopy (SThM), scanning electrochemical microscopy (SEcM), scanning capacitance microscopy (SCM), scanning Kelvin probe microscopy (SKPM), scanning chemical potential microscopy (SCPM), and sœnning ion-conductance microscopy (SICM).
The continuum of imaging modes is based on differing interactions between probe tip and sample, as well as the detection scheme used. AFM operates by measuring the forces between a probe and the sample, such forces depending in part on the nature of the sample, the distance between probe and sample, probe geometry, and any surface contamination on the sample.
The list of chemical applications of SPM techniques is growing rapidly, TopoMetrix's Aden notes. Among them are: investigating electrochemical processes such as corrosion on a nanometer scale, looking at temperature variations on a nanometer scale, studying microhardness in the investigation of blended r e cycled materials, probing the mineralization of bone tissue, mapping the location of surface functional group differences on a nanometer scale, and studying Lang-muir-Blodgett films. Still another application, Aden points out, is the imaging of
Pittcon '93 display booths by TopoMetnx (top) and Digital Instruments feature scanning probe microscopes
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polymeric materials such as polyethylene and even individual polyethylene chains.
A Digital Instruments approach—the company's lift mode—illustrates another way SPM can be used. In one example, the topography of a sample is traced and displayed, and the tip is then //lifted,, a few nanometers above the sample and the data used to hold the probe at a constant separation while a magnetic force gradient is mapped.
Further applications lie ahead. In the development of SNOM, for example, researchers are using an extremely fine optical fiber that is guided by a probe to where laser light can be used to observe molecular clusters. Other researchers are investigating the possibility that a usable force may exist between an antibody affixed to a probe and an antigen on the surface of a biological sample.
James Krieger
Analysis yields clues to coffee, wine flavors Over and above the more esoteric research presentations at Pittcon '93, two groups of chemists reported on the application of analytical chemistry to everyday concerns such as how to get a good cup of coffee or an enjoyable glass of wine.
Chemistry professor James K. Hardy along with Farid R. Zaggout of the University of Akron, Ohio, studied the effects of particle size and roasting temperature of coffee beans on development of flavor and odor components and caffeine content in the brew. And Santford V. Overton and John J. Manura of Scientific Instrument Services, Ringoes, N.J., studied changes in volatile organic compounds (VOCs) in wines and wine coolers at one month and six months after opening.
To gauge the effects of coffee roasting temperatures, the Akron workers roasted coarsely ground (20-mesh) coffee beans at various temperatures between 300 and 400 °F, brewed each batch in the same way, extracted the fresh coffees with methylene chloride, and analyzed the extracts by gas chromatography-mass spectrometry. They reported each flavor/odor component as a percentage of the maximum content attained over the temperature range.
For example, 2-methoxy-6-vinylphenol decreased steadily from 100% to 50% as roasting temperature increased from 300 to 400 °F. Furfural and furfuryl alcohol in
creased from 10% to 100% over the range. And cyclopentenolone rose from 0% at 300 °F to 100% at 350 °F, then feU back to 0% at 400 °F. Caffeine concentrations were unaffected by roasting temperatures. The Akron chemists concluded that optimum roasting temperatures are 380 to 390 °F.
Similarly, the researchers analyzed coffees brewed from 20- (coarse) to 80-mesh beans (fine), roasted at the same temperature. Among the most notable changes, guaiacol decreased uniformly from 100% to 40% as grind size decreased. Meth-ylpyrazine rose from 85% to 95% between 20 and 40 mesh, then sank to 55% at 80 mesh. The team concluded that 20 to 40 mesh was the optimum grind.
In their studies of wines and wine coolers, Overton and Manura realized that drinkers' impressions of overall taste and bouquet came from VOCs in the air just above the liquid (headspace). Off-odors and tastes would appear over time because of changes in these compounds.
The two workers selected two commercial wines, identified as burgundy and red. They also included two homemade
wines, a 30-year-old grape and a two-year-old dandelion. The four commercial wine coolers examined were called strawberry, lime, berry, and cooler.
The Ringoes investigators placed 2.5-mL samples of each in test tubes, bubbled helium through the liquids, passed the helium-borne vapors through a d e sorption tube, and expelled the volatiles in a Scientific Instrument Services short-path thermal desorption system into a gas chromatograph interfaced with a mass spectrometer.
Among overall conclusions: Gradual formation of acetal in opened wines, possibly catalyzed by a parallel decreasing pH, caused off-odors and "unusual" taste. Though there was little volatilization of components in resealed wines, volatilization was a significant contribution to development of off-tastes and off-odors in wine coolers. Further contributions to deterioration of all products seemed to come from oxidation of terpenes and increases in concentrations of acetic acid and branched-chain alcohols.
Stephen Stinson
Hoechst offers out of R+D:
1,3-Difluoro-
benzene
now available in lab quantities
in pilot plant quantities
another example of Hoechst High Chem
Hoechst Celanese Corp. Fine Chemicals Division P.O. Box 1026 Charlotte, NC 28 201-1026 USA Fax 704-559-6153' Tel 800-242-6222
Hoechst AG Marketing Feinchemikalien Postfach 80 03 20 6230 Frankfurt am Main 80 Germany Fax: (69) 31 20 21/31 66 77
Hoechst Celanese
Hoechst DB
CIRCLE 3 ON READER SERVICE CARD
MARCH 29,1993 C&EN 31
