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Guiding light LC with UV-vis absorbance detection is typically not sensitive enough for environmental analysis in which concentrations tend to be in the sub-ug/L range. Improving sensitivity is as simple as increasing the optical pathlength. In this case, however, sensitivity comes only at the expense of losing chromatographic resolution. With conventional-size LC columns (3.0 or 4.6 mm I.D.), the cell volume should not exceed 10 uL—larger cells cause a decrease in resolution. Cees Gooijer and co-workers at Free University (The Netherlands) have found a way to increase the path length of the detector cell without compromising resolution. Their approach relies on the use of liquid-core waveguides (LCWs)—capillary cells that guide light by total internal reflection to the liquid-cell boundary.
Low-affinity ligands Recently, there has been an explosion of analytical methods that rapidly detect and measure ligand binding to large biomolecules. With systems in which the interactions are between proteins and carbohydrates, however, the binding can be relatively weak and difficult to assess. Understanding this
LCWs work on the same prerequisite as of LCWs instead of conventional detector optical fibers—the refractive index of the cells is shown to improve the sensitivity 30liquid inside the cell must be greater than to 40-fold for three pesticides—atrazine, the refractive index of the cell material for diuron, and linuron. Detection limits in the total internal reflection to occur. To extend 0.3-0.5-ug/L range are reported. (J. .hrothe applicability of LCWs to low-refractivematogr..A 1998,824,1-5) index liquids, including aqueous solutions, the researchers turned to plastic LCWs made of a recently developed perfluorinated polymeric material (Teflon AF2400) with a refractive index (n = 1129) )owee rhan that of water (nD = 1.33). The LCW (90-cm length, 280-um I.D.) is fitted inside a conventional UV-vis absorption detector and a quartz optical fiber (1.0 m length) is installed in the reference channel, resulting in a detector cell that has an optical path length of —90 cm and LCW detection system. (Adapted with permission. an internal volume of 55 uL The use Copyright 1998 Elsevier Science.)
type of binding is important because it plays key roles in cellular events such as fertilization, inflammation response, cell signaling, and pathogen response. Laura L. Kiessling and colleagues at the University of Wisconsin-Madison demonstrate an assay based on surface plasmon resonance (SPR) that rapidly screens carbohydrates that bind proteins.
Formation of lipid bilayers on the SPR surface by mixing phosphatidylcholine with a synthetic glycolipid.
The researchers studied the jack bean protein (or lectin) concanavalin A (Con A). Varying molar ratios of a synthetic mannose-derivatized glycolipid were mixed with phosphatidylcholine to form lipid bilayers with an alkane thiol monolayer bound to the optical sensor chip surface. Con A in solution could then bind to the surface and be detected by SPR. Quantitative data were obtained by running competition experiments between the surface-bound ligands and other ligands in solution with Con A. Relative affinity values of carbohydrate-Con A binding as measured by the SPR approach are comparable with values determined by titration microcalorimetry. Thus, this assay provides a rapid means to screen low molecular weight compounds that inhibit proteincarbohydrate interactions. The Wisconsin researchers also used the SPR approach to identify multivalent ligands capable of attaching simultaneously to the two saccharide binding sites on Con A, which are located approximately 65 A apartt .J. Am. Chem. Soc. 1998,120,10575-82)
Analytical Chemistry News & Features, January 1, 1999 15 A
