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CHEMICAL ANALYSIS
SEPARATIONS IN A FLASH Microsecond electrophoresis technique separates transient species A capillary electrophoresis (CE) technique devised to explore the ultimate speed limits of chemical separations has come closer to reaching those limits than ever before. The technique greatly improves the speed of electrophoretic separation, making it possible to separate and analyze reaction intermediates and other transient chemical species previously inaccessible with CE. The technique was developed by graduate student Matthew L. Plenert and associate professor of chemistry and biochemistry Jason B. Shear of the University of Texas, Austin [Proc. Natl. Acad. Sci. USA, published online at http:// dx.doi.org/10.1073/pnas.0637211100]. The researchers report having achieved separations in less than 10 microseconds--about 100-fold faster than any previous CE procedure. The work "represents a significant advance in ultrafast separation and detection of biologically relevant compounds," write graduate student Jeffrey N. Stuart and chemistry professor Jonathan V. Sweedler of the University of Illinois, Urbana-Champaign, in a PNAS commentary. "We certainly expect a number of exciting applications to present themselves for what is now the ultimate in fast separation."
In their initial demonstration of the technique, Plenert and Shear used a laser pulse to rapidly generate a binary mixture of fluorescent hydroxyindole photoproducts in a glass capillary. They obtained baseline resolution of the photoproducts in less than 10 microseconds in a specially designed capillary tube and used laser fluorescence spectroscopy to detect the separated species. A critical element of the technique is use of a pulled, hourglass-shaped capillary, which makes it possible to attain higher electric field strengths and correspondingly higher separation efficiencies. The electric fields used to carry out CE separations are amplified in the pulled part of the capillary because of higher electrical resistance there. Prior to their study, the fastest CE separations had been achieved by Stephen C. Jacobson, J. Michael Ramsey, and coworkers at Oak Ridge National Laboratory. They separated a binary mixture on a microchip in under 1 millisecond, using an electrophoretic field strength of about 50 kV/cm. Plenert and Shear have now employed field strengths up to three times as high to separate a binary mixture about 100 times faster in their hourglass capillary. Potential applications of microsecond CE include studies of other reaction intermediates, rapid biomolecular events (such as neurotransmitter release in synapses), and protein-folding mechanisms. The researchers believe it may ultimately be possible to reduce separation times to the submicrosecond range by optimizing field strengths, sample viscosities, and separation distance, perhaps extending the technique's applicability still further. |
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