Evaluation of the impact of peak description on the quantitative capabilities of comprehensive two-dimensional liquid chromatography.

Comprehensive, two-dimensional liquid chromatography (LC × LC) is a powerful technique for the separation of complex mixtures. Most studies using LC × LC are focused on qualitative efforts, such as increasing peak capacity. The present study examined the use of LC × LC-UV/vis for the separation and quantitation of polycyclic aromatic hydrocarbons (PAHs). More specifically, this study evaluated the impact of different peak integration approaches on the quantitative performance of the LC × LC method. For well-resolved three-dimensional peaks, parameters such as baseline definition, peak base shape, and peak width determination did not have a significant impact on accuracy and precision. For less-resolved peaks, a dropped baseline and the summation of all slices in the peak improved the accuracy and precision of the integration methods. The computational approaches to three-dimensional peak integration are provided, including fully descriptive, select slice, and summed heights integration methods, each with its own strengths and weaknesses. Overall, the integration methods presented quantify each of the PAHs within acceptable precision and accuracy ranges and have comparable performance to that of single dimension liquid chromatography.

Influence of glycosidic linkage on the solution conformational entropy of gluco- and mannobioses.

Employing size-exclusion chromatography, an entropically-controlled separation technique, we have determined the solution conformational entropy (-ΔS) of (1→2)-, (1→3)-, (1→4)-, and (1→6)-linked gluco- and mannobioses with an α anomeric configuration, at quasi-physiological conditions of solvent, temperature, and pH. The experiments allowed for comparison both among and between each series of disaccharides. Results included quantitative information on how the additional degrees of freedom of the (1→6) linkage influence -ΔS, as well as on the influence on solution conformational entropy of a single axial hydroxyl (OH) group and of the relative positioning of the glycosidic linkage and the anomeric hydroxyl group. We also contrasted the (1→4)-α-D-linked gluco- and mannobioses to their counterparts with a ß anomeric configuration. Comparison between (1→4)-ß-D-linked glucobiose (cellobiose) and (1→4)-ß-D-linked mannobiose showed that the restrictive effect on solution flexibility of the axial OH in the latter disaccharide is offset by the combined effect of hydroxyl group orientation and anomeric configuration on intramolecular hydrogen bonding.

Determining the solution conformational entropy of oligosaccharides by SEC with on-line viscometry detection.

Introduced here is a method for determining the solution conformational entropy of oligosaccharides (-ΔS) that relies on the on-line coupling of size-exclusion chromatography (SEC), an entropically-controlled separation technique, and differential viscometry (VISC). Results from this SEC/VISC method were compared, for the same injections of the same sample dissolutions and under identical solvent/temperature conditions, to results from a benchmark SEC/differential refractometry (SEC/DRI) method which has been applied successfully over the last decade to determining -ΔS of a variety of mono-, di-, and oligosaccharides. The accuracy (as compared to SEC/DRI) and precision of SEC/VISC were found to be excellent, as was the sensitivity of the viscometer in the oligomeric region. The experiments presented here contrast three sets of (1→4)-ß-d-oligosaccharides, namely manno-, cello-, and N-acetylchitooligosaccharides of degree of polymerization (DP) 2 through 6. For each series, the dependence of -ΔS on DP was found to be monotonic while, between series, differences at each DP could be ascribed to either the additional degrees of freedom imparted by large, multi-atomic substituent groups, or to the presence or absence of additional intramolecular hydrogen bonds, depending on the axial versus equatorial arrangement of particular hydroxyl groups. An hypothesis is advanced to explain the unexpectedly high sensitivity of viscometric detection for low-molar-mass analytes. The method presented can be extended to the analysis of oligosaccharides other than those studied here.

Does the "C3 effect" offset the Δ2 effect, as regards the solution flexibility of aldoses?

The solution flexibility of carbohydrates influences a variety of molecular recognition and regulatory processes. For aldoses and other monosaccharides, this flexibility is dictated by the orientations of the various hydroxyl (OH) groups present, which influences conformer and anomer ratios, interactions among these OH groups, and interactions between them and the surrounding solvent. Depending on the number and position of axial OH groups, a variety of structures can coexist in solutions at equilibrium. In 1950, as part of his pioneering studies on the shape of pyranoside rings, Reeves described the Δ2 effect, the greater destabilization of the pyranose ring conformation when the OH group on carbon 2 (C2 ) is in the axial position. It was later proposed by Angyal that the Δ2 effect could be cancelled by the presence of an axial OH on C3 , termed here the "C3 effect." Employing size-exclusion chromatography, an entropically-controlled separation technique, we have investigated whether or not the C3 and Δ2 effects indeed do compensate for one another with respect to their influence on the solution flexibility of select aldohexoses and aldopentoses. As will be seen, while qualitatively and semiquantitatively this mutual cancellation of effects does occur in aldohexoses, it does not appear to do so in aldopentoses. An explanation for the latter appears to lie in the variety of anomers and conformers present in equilibrium solutions of those aldopentoses studied and in the relative entropic contribution of individual conformers or anomers to the total solution flexibility.