No abstract

A novel type of silica-based sulfonate-modified reversed phase ( − SO 3 -HC-C 8 ) has been synthesized; it is based on a newly developed acid stable "hyper-crosslinked" C 8 derivatized reversed phase, denoted HC-C 8 . The − SO 3 -HC-C 8 phases containing controlled amounts of sulfonyl groups were made by sulfonating the aromatic hyper-crosslinked network of the HC-C 8 phase at different temperatures. The − SO 3 -HC-C 8 phases are only slightly less hydrophobic than the parent HC-C 8 phase. The added sulfonyl groups provide a unique strong cation-exchange selectivity to the hydrophobic hyper-crosslinked substrate as indicated by the very large C coefficient as shown by Snyder's hydrophobic subtraction reversed-phase characterization method. This cation-exchange activity clearly distinguishes the sulfonated phase from all other reversed phases as confirmed by the extraordinary high values of Snyder's column comparison function F s . In addition, as was found in previous studies of silica-based and zirconia-based reversed phases, a strong correlation between the cation-exchange interaction and hydrophobic interaction was observed for these sulfonated phases in studies of the retention of cationic solutes. The overall chromatographic selectivity of these − SO 3 -HC-C 8 phases is greatly enhanced by its high hydrophobicity through a "hydrophobically assisted" ion-exchange retention process.

Efficient and comprehensive screening of enzyme activity was accomplished in a combinatorial array of 96 reaction microvials. Quantitation of the extent of the reaction at well-defined time intervals was achieved by using 96-capillary array electrophoresis coupled with a multiplexed absorption detector. Capillary electrophoresis provides high separation resolution to isolate the product from the reactants. Absorption detection provides universal applicability to combinatorial screening. For the conversion of NADH to NAD+, the catalytic activity of LDH was confirmed to be the highest at pH 7. This scheme should be useful for high-throughput drug discovery, clinical diagnosis, substrate binding, as well as combinatorial synthesis.

The separation and determination of hydrophilic basic compounds are of great importance in many fields including clinical and biological research, pharmaceutical development and forensic analysis. However, the most widely used analytical separation technique in these disciplines, reversed-phase liquid chromatography (RPLC), usually does not provide sufficient retention for several of the important classes of highly hydrophilic basic compounds including catecholamines, many drug metabolites and many drugs of abuse. Commonly eluents having little or no organic modifier and/ or strong ion pairing agents must be used to achieve sufficient retention and separation. Use of highly aqueous eluents can lead to column failure by dewetting, resulting in poor retention, selectivity, reproducibility and slow recovery of performance. The use of a strong ion pairing agent to increase retention renders the separation incompatible with mass spectrometric detection and complicates preparative separations. This paper describes the successful applications of a novel type of silica-based, hyper-crosslinked, sulfonate-modified reversed stationary phase, denoted as − SO 3 -HC-C 8 -L, for the separation of highly hydrophilic cations and related compounds by a hydrophobically assisted cation-exchange mechanism. Compared to conventional reversed-phases, the − SO 3 -HC-C 8 -L phase showed significantly improved retention and separation selectivity. Concurrently, due to the presence of both cation-exchange and reversed-phase retention mechanisms and the high acid stability of hypercrosslinked phases, the separation can be optimized by changing the type or concentration of ionic additive or organic modifier, and by varying the column temperature. In addition, gradients generated by programming the concentration of either the ionic additive or the organic modifier can be applied to reduce the analysis time without compromising resolution. Furthermore, remarkably different chromatographic selectivities, especially toward cationic solutes, were observed upon comparison of the − SO 3 -HC-C 8 -L phase with conventional reversed-phases. We believe that the combination of these two types of stationary phases will be very useful in two-dimensional liquid chromatography.