Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry.

A comprehensive two-dimensional liquid chromatographic separation system based on the combination of a CN column and an ODS column is developed for the separation of components in a traditional Chinese medicine (TCM) Rhizoma chuanxiong. Two columns are coupled by a two-position, eight-port valve equipped with two storage loops and controlled by a computer. The effluent is detected by both the diode array detector and atmospheric pressure chemical ionization (APCI) mass spectrometer. More than 52 components in the methanol extract of R. chuanxiong were resolved and 11 of them were preliminary identified according to their UV and mass spectra.

High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolith.

Reactive monoliths of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications.

Synthesis of chemically bonded cellulose trisphenylcarbamate chiral stationary phases for enantiomeric separation.

Cellulose trisphenylcarbamate is regioselectively bonded to 3-aminopropyl silica gel and underivatized silica gel, respectively, at the 6-position of the primary hydroxyl group on the glucose unit of cellulose with 4,4'-diphenylmethane diisocyanate (DPDI) as a spacer. Enantioseparations are evaluated on these prepared chiral stationary phases (CSPs) with several organic acids as the modifiers in the mobile phase by high-performance liquid chromatography. The influence of the amount of DPDI used on chiral resolution is investigated. Also, the corresponding coated-type phase is also prepared for the aim of comparison. It is observed that the bonded-type phase shows a lower chiral recognition power but a better column efficiency than the coated-type phase under the liquid chromatographic mobile phase with hexane-alcohol. However, the bonded-type CSPs are compatible with a wider number of solvents such as tetrahydrofuran (THF) or chloroform, which generally result in the solubility or swelling of the cellulose derivatives on the coated-type CSPs. The results obtained from this study indicate that the bonded-type CSP may provide complementary enantioselectivity over the coated-type phase by adopting THF as a component in the mobile phase.

A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry.

We report a novel method termed matrix suppressed laser desorption/ionization to improve the analysis of low-mass molecules by MALDI-TOF mass spectrometry. In this method, the surfactant of cetrimonium bromide (CTAB) is added to the conventional matrix of alpha-cyano-4-hydroxycinnamic acid solution to prepare the MALDI samples. During the MALDI process, the presence of CTAB could substantially or even completely suppress the matrix-related ion background. As a result, very clean mass spectra can be routinely obtained in the low-mass range. In addition, the presence of CTAB can significantly improve the mass resolution of low-mass molecules. It is seen that high-quality spectra were routinely obtained at a matrix/CTAB ratio of 1000:1. This method has been successfully used to analyze a variety of low-mass molecules.

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography.

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule.

[Application of genetic algorithm in optimization of mobile phase composition in high performance liquid chromatography].

Referring to traditional optimal methods, a method for the optimization of isocratic elution mobile phase composition in high performance liquid chromatography has been developed. In this method, the genetic algorithm based on line-crossover and plane-mutation is used. The principle of genetic algorithm and the process of optimization of mobile phase composition in reversed-phase ion-pair high performance liquid chromatography using genetic algorithm are introduced in details. With the concentrations of acetonitrile and ion-pair reagent sodium octane sulfonate chosen as the optimal parameters, the optimum was obtained by three times of optimization procedures. The mean relative error between the predicted and experimental values was 0.75% at the optimum and the optimization results were satisfactory.

Separation of Anionic Compounds byStrong Anion-exchange Capillary Electrochromatography / 分析化学

The separation of anionic compounds by strong anion-exchange capillary electrochromatography (SAX-CEC) was carried out. It was found that the analytes could be absorbed onto the stationary phase, and this would lessen the retention factors (k) of the analytes, thus the column separation capability decreased. For the acidic compounds, k increased with increase of applied voltage. And the change of the applied voltage could provide different separation selectivity for the solutes. The separation with different eluent was studied. It showed that the logarithm of the capacity factor linearly decreased with increase of the logarithm of the ionic strength. The different retention behavior of the anionic compounds in SAX-CEC and CE was also studied