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New CE applications deliver sweet separations.

Capillary electrophoresis is making great strides in solving longstanding problems, from identifying food additives to detecting herbicides.

For the past decade, capillary electrophoresis (CE) applications have focused primarily on protein, peptide, and amino acid analysis, with little work being conducted on smaller molecules outside the biochemical area.

Now, however, thanks to lively scientific conferences, such as HPCE '92 held recently in Amsterdam, Netherlands, a wide variety of additional uses for CE are becoming known. As a result, this convenient analytical technique is beginning to find its way into many new industrial surroundings.

At HPCE '92, scientists from the Hershey Foods Technical Center, Hershey, PA, presented many examples of their use of CE in the analysis of food. Several of these have the potential of becoming routine quality control procedures.

Among the CE-based test methods they discussed were the determination of glycyrrhizic acid in licorice root extract, analysis of cocoa for caffeine and theobromine, the determination of vitamin C in citrus and other fruit beverages, and the measurement of niacin in peanut butter extract.

Using a prepared standard, they also demonstrated the feasibility of separating the mycotoxin sterigmatocystin, a potential carcinogenic precursor. In a similar way, they readily separated a mixture of several water-soluble vitamins, including niacin, thiamine, riboflavin, vitamins C, B-6, B-12, and folic acid.

Further studies led to more exciting breakthroughs. "The separation of seven commonly occurring food ingredients--caffeine, theobromine, vanillin, ethyl vanillin, aspartame, sorbate, and benzoate--in a single run is truly remarkable," says W. Jeffrey Hurst, Hershey Foods' senior staff scientist.

"CE is especially advantageous because it allows a rapid changeover from one component type to another," continues Hurst. "Not seen with many other analytical techniques, this feature allows the serial determination of different components in different samples without the need to switch columns or buffer systems.

"These CE methods are faster, easier, and at least as sensitive as what we have in place now. This capability allows the analyst to respond to production and consumer concerns in a timely and efficient manner. CE has excellent potential for the food analyst," notes Hurst.

On a different front, agrochemical scientists are beginning to use CE for the analysis of pesticides. Although many methods currently exist for the analysis of pesticides in raw agricultural commodities and environmental samples, most of these use gas chromatography or HPLC coupled with a variety of detectors.

Recognizing the need for rapid and sensitive methods for herbicide analysis, researchers at the Laboratory of Instrumental Analysis, Eindhoven Univ. of Technology, Netherlands, have developed a technique to separate the toxic chlorophenoxy acid herbicides by micellar electrokinetic capillary chromatogrpahy (MECC).

This research team, led by Quanji Wu, was able to easily separate a mixture of 10 chlorophenoxy acids within 10 min. They also found that selectivity could be fine-tuned by using different combinations of surfactants to achieve even greater separations.

The MECC approach avoids the extensive preparation of surface water, urine, soil, or sediment samples which HPLC analysis of herbicides usually requires.

U.S. federal and state regulatory agencies now require industry and agrochemical manufacturers to develop suitable methods to monitor their pesticide materials as they are commercialized.

Relatively new among these products is an environmentally safe class of herbicides known as imidazolinones.

Scientists at American Cyanamid Co., Princeton, NJ, have recently begun using CE in the analysis of agricultural and environmental samples for these imidazolinone herbicides, and have obtained results that compare closely with traditional analytical methods such as GC and HPLC.

Moreover, the CE technique is especially attractive since it avoids the use of organic solvents and their associated disposal problems.

These imidazolinones kill plants by inhibiting acetohydroxyacid synthase (AHAS), an enzyme common to the biosynthetic pathway of the branched-chain amino acids valine, leucine, and isoleucine. This inhibition disrupts protein synthesis, which, in turn, interferes with DNA synthesis and cell growth.

Animals do not have any AHAS and do not synthesize these three amino acids. This partially explains the low toxicity of the herbicide to mammals and other non-target animal species in the environment.

Since imidazolinones and their metabolites may remain in soil, water, and crop commodities, manufacturers are required to have assay methods available for residue detection.

"CE yields high resolution separations of these herbicides in crop samples within minutes, using only nanoliter sample volumes," says Max Safarpour, a research chemist at American Cyanamid. "Because extensive sample cleanup procedures are minimized as compared to other techniques, I expect CE to become a valuable tool for residue chemists in the near future."

Capillary electrophoresis has developed into a powerful tool for separating ions as well as neutral analytes because of its remarkable separation efficiency.

In an effort to augment these capabilities, researchers at Millipore, Milford, MA, have recently developed a new technique that combines HPCE and membrane technology. The system recovers the separated species for further analysis. This is in contrast to normal practice, which is to simply discharge the analytes into a reservoir.

Previous attempts to collect these CE fractions have included using small vials to sequentially capture the eluted species, but this technique proved unsuccessful. It generally resulted in significant sample dilution or was too difficult to obtain closely spaced sample bands in separate vials.

Another approach to collecting eluted fractions is to carefully construct an on-column frit near the exit end of the capillary. The electric circuit is then completed through this frit.

This method minimizes the dilution effect. However, it is difficult and time consuming to make the frit, and it functions only when electroosmotic flow is in the right direction.

Millipore's experimental CE fraction collector consists of a membrane assembly placed in contact with the exit end of the capillary. The assembly comprises a polyvinylidene difluoride (PVDF) membrane cover, a buffer reservoir consisting of two layers of 3MM chromatography filter paper, and a stainless steel plate. The plate completes the electrical circuit for electrophoretic separation by acting as the ground electrode.

The entire collection assembly is rotated by a stepping motor at 2.2 revolutions per hour while electrophoretic separation takes place. Analytes are deposited onto the membrane at discreet locations as they emerge. The collected fractions can be either stained in place or removed from the membrane for further treatment.

Since the analytes are deposited onto the porous membrane substrate, they are in a form that permits further analysis by a variety of presently available techniques, including staining, immunoassay, ELISA, amino acid sequencing, DNA probes, and chemiluminescence.

In principle, the membrane can be engineered to trap or bind virtually any species that can be separated by CE to allow these species to be subjected to further analysis.

To demonstrate the feasibility of this technique, the research team performed direct protein sequencing of two recovered proteins following CE separation.

Capillary electrophoresis is moving from a research technology to becoming an accepted analytical tool with a seemingly unlimited range of applications. Methods development is expanding rapidly also as scientists try the technique and quickly discover the many benefits it possesses over existing systems.
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Title Annotation:capillary electrophoresis
Author:Goldner, Howard J.
Publication:R & D
Date:Aug 1, 1992
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