Sugar-switching bacteria
Schematic representation of partial bilayers formed by exposing gold substrate to stearic acid (X = CHfiH^) and hexadecanedioic anid (X = COJ-I).
height; however, the phase contrast image exhibited negligible phase shift. These data indicate that the phase images observed in the initial experiment were caused by differences in the chemical compositions of the functional groups rather than the height variations. The authors caution that samples with more complicated surfaces than those used in this study may exhibit more complex images because of mechanical differences, thus making interpretation more difficult. (J. Am. Chem. Soc. 1997,119,8564-65)
Separating living cells
Carbohydrate profiling of whole bacterial cell hydrolysates provides structural information that can aid in differentiating among species. Neutral and amino sugars are often analyzed by GC/MS, following a derivatization process that incorporates alditol acetates; however, acidic sugars require additional derivatization steps before they can be analyzed by the same method. Alvin Fox and co-workers at the University of South Carolina, Kansas State University, and the Naval Research Laboratory avoid these extra steps with high-performance anion-exchange chromatography (HPAEC) which does not require prior derivatization for the separation of neutral amino and acidic sugars The procedure involves the hydrolysis of bacterial cells with sulfuric acid, followed by neutralization in an organic base. Hydrophobic and posi-
be complete, rapid, and efficient. The effect of lifting forces on the retention time is investigated for both nucleated and red Although the separation of cellular material blood cells. Flow rates and field intensity by sedimentationfield-flowfractionation are manipulated to determine the opti(SdFFF) was first reported more than 15 mum conditions for separating human red years ago, specific instrumentation for bioblood cells. Injection protocol is also exmedical applications has been slow to deamined to determine if data obtained with velop. Philippe J. P. Cardot and co-workers a stop-flow procedure or with direct injecat Universite de Paris Sud, Hopital Cochin, tion into the flowing stream differ. and Hopital Necker (all in France) have The materials used in constructing the designed a new SdFFF apparatus that sepa- channel (two Lexan plates separated by a rates nucleated and living red blood cells. Mylar spacer) and the carrier phase (albu The apparatus is tested by eluting cells min-supplemented) were found to have that differ in size, rigidity, density, shape, little interaction with the cellular material. and other characteristics. The separation In addition, over 90% of the injected cells of cells by this method is demonstrated to survived the separation process. The authors warn that the mobile phase used in this apparatus is a good medium for bacterial growth, and therefore decontamination procedures must be carried out regularly (Anal. Biochem. 1997, 251, Schematic of an SdFFF apparatus for the separation of living cellular 178-86) material. (Adapted with permission. Copyright 1997 Academic Press)
tively charged interferences (including amino acids) are removed from solution with C18 and SCX columns. The sugars react with sodium hydroxide to form anions with the general form [M-H]". Electrospray ionization MS provides selective detection of these anions. Because of the high ionic strength of the mobile phase, an online suppressor and decreased postcolumn flow rates are required before electrospray to achieve adequate sensitivity. Absolute identification of the sugars is determined by MS/MS after collision-induced dissociation. The method was used to monitor sugar production in certain bacteria that were deprived of phosphate during growth. Fox and co-workers found that some bacteria cease producing teichoic acid, which contains ribitol phosphate, and instead switch to teichuronic acid, which contains glucuronic acid, when grown under phosphate-limited conditions. (J. Chromatogr. A 1997, 776, 205-19)
CD with thermal gratings Although chirality can greatly affect biological properties, the signal measured by conventional circular dichroism (CD) is small. David W. Chandler and co-workers at Sandia National Laboratories (Iivermore, CA) and San Diego State University describe a new technique for measuring CD that enhances the normally weak signal. They generate polarization and intensity thermal gratings in liquid samples with four-wave mixing. The dissymmetry ratio (Ae/e) can be determined from the ratio of the difference of scattered laser light from two polarizations to the average amount of scattered light, or AS/Save. The theoretical limit of AS/Save, which is 2, can be reached when destructive interference causes the signal from one of the polarizations to go to 0, a phenomenon achieved by controlling the ellipticity of the polarized laser light. CD is easier to measure with AS/S
because it is sev-
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eral orders of magnitude larger than the dissymmetry ratio. They use camphorquinone as a model because its AS/S approaches 2 at the 266-nm excitation wavelength used in the study By adjusting the polarization of the laser pump beams with a photoelastic
Analytical Chemistry News & Features, November 1, 1997 6 5 1 A
