Ruyu Gao

Separation of peptides by pressurized capillary electrochromatography

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresiscan be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated.

Fast and precise determination of phenthoate and its enantiomeric ratio in soil by the matrix solid-phase dispersion method and liquid chromatography.

A fast and precise method was developed for the determination of phenthoate and its enantiomeric ratio (ER) in three soil samples. A recently developed sample pretreatment technology--matrix solid-phase dispersion (MSPD) was used to extract phenthoate simply and effectively. MSPD conditions, i.e. solid-phase Florisil amount, water content of Florisil-soil mixture, the constituent and volume of the eluting solvent, were optimized stepwise. The MSPD extract was directly used for quantitative determination of phenthoate by silica-based high-performance liquid chromatography (HPLC) with UV detection. The recoveries of phenthoate from three different types of soils fortified at three levels of 0.1, 1, 10 microg/g ranged from 75 to 94% with RSDs of 1.5-6.5%. On this basis, phenthoate was further isolated from the remainder of MSPD extract by silica-based HPLC and then ER determined on HPLC with cellulose tris-3, 5-dimethylphenylcarbamate as chiral stationary phase. The ERs determined in the soils spiked with racemic or enantiomer-enriched phenthoate agreed sufficiently well with those in the corresponding standard solutions. Finally, the proposed method was successfully applied to the study of enantioselective degradation of phenthoate in the three soils under laboratory conditions. High enantioselectivity was observed in the two alkaline soils with (+)-enantiomer degrading faster than the (-)-enantiomer, while there was little to no enantioselectivity in the acidic soil. The methodology can be used to study the enantioselective environmental behavior of chiral pollutants.

Enantioseparation of fourteen O-ethyl O-phenyl N-isopropyl phosphoroamidothioates by high-performance liquid chromatography on a chiral stationary phase

Abstract A series of fourteen O-ethyl O-phenyl N-isopropyl phosphoroamidothioate enantiomers containing a phosphorus atom as a chiral center have been separated by high-performance liquid chromatography on a Pirkle model chiral stationary phase. Chromatography data, the capacity factor (k′) and the separation factor (α) of all solutes are presented. The influence of chromatographic conditions was investigated. The probable mechanism for chiral recognition is proposed.

Optimization stacking by transient pseudo-isotachophoresis for capillary electrophoresis: example analysis of plasma glutathione.

Low concentrations of both reduced form glutathione (GSH) and oxidized form glutathione (GSSG) in diabetic nephropathy (DN) patients plasma were measured with transient pseudo-isotachophoresis. The plasma samples were deproteined with acetonitrile and centrifuged. The method was performed at constant voltage of 5 kV using a 300 mM borate buffer (pH 8.0), with a fused-silica capillary of 21 cm x 75 microm. The sample length can reach 25% of the efficient length of the capillary, and the sensitivities of GSH and GSSG increased 15-20-fold. The method was also systematically optimized, and the results show that this type of stacking offers good repeatability for routine clinical assay of glutathione in DN plasmas.

High-performance capillary zone electrophoretic assay for markers of diabetic nephropathy in plasma and urine.

A new high-performance capillary zone electrophoretic assay for creatine (Cr), creatinine (Cn), urea (U) and uric acid (Ua), markers of human diabetic nephropathy, both in plasma and urine has been developed with UV detection at 200 nm. The plasma sample was deproteinized with trichloroacetic acid and centrifuged at 10 000 rpm for 10 min. The urine sample was diluted 20-fold with buffer before analysis. The optimum separation conditions for the markers was investigated with respect to the concentration of the buffer, the pH, the voltage and the capillary temperature. Baseline separation was achieved in 25 mmol/L phosphate buffer (pH 3.45) using a 21 cm x 75 microm I.D. fused-silica capillary at 40 degrees C with an electric field of 1190 V/cm. The calibration curves showed good linearity in the range 3.5-1000, 0.18-700, 500-5000 and 2-800 microM (r2 min > 0.998) for Cr, Cn, U and Ua, respectively. The proposed method also has a high reproducibility (peak area RSD max < 3%) and has been successfully applied to the determination of clinical samples.

Direct Chiral High Performance Liquid Chromatography Separation of O,O-Diethyl-(p-methyl-benzene-sulfonamido)- Aryl (Alkyl)-Methylphosphonate

Abstract The enantiomers of a series of twelve O,O-diethyl, (p-methyl-benzenesulfonamido), aryl(alkyl)-methylphosphonates have been separated by high performance liquid chromatography on a Pirkle model chiral stationary phase. Chromatographic data and a chiral recognition model are presented for the separation of these organic phosphorus enantiomers on the chiral phase. The influence of column temperature and flow rate of mobile phase has been described.

Influence of the Structure of Organic Phosphonate Compounds on Chiral Separation on a Pirkle Type Chiral Stationary Phase

SummaryThe enantiomers of eightO,O-dialkyl-2-benzyloxycarbonyl-aminoarylmethyl-phosphonates are directly separated on anN-(3,5-dinitrobenzoyl)leucine (DNBleu) column. The influence of the mobile phase composition and the column temperature on the retention and the enantioselectivity are investigated. The influence of the length and steric hindrance of alkoxyl groups of the phosphonate ester and the nature of the substituentp-Cl and pH on the benzene ring which is attached to the chiral carbon atom on chiral separation are discussed also.

PREDICTION OF RETENTION OF O-ETHYL, O-ARYL AND N-ISOPROPYL PHOSPHOROAMIDOTHIOATES IN RP-HPLC FROM MOLECULAR STRUCTURE PARAMETERS

SummaryMulti-variable regression analysis between lnkw, c (in retention equation lnk′=lnkw+cCb) and molecular structure parameters, including hydrophobicity, electric effect, field effect and position-specific effect constant, was carried out with O-ethyl, O-aryl and N-isopropyl phosphoroamidothioates as test solutes. With these quantitative relationships, the retention behaviour of these solutes for different mobile phase compositions was predicted. The results showed that there are only 26.7% of total, experimentally measured, capacity factors with relative deviations larger than 5% and only 2.2% with deviations larger than 10%, which means that it is possible to apply the method reported to predict retention values for qualitative purposes for different mobile phase compositions.

Enantiomer separation and preparation of five organophosphorus compounds and their biological activities.

SummaryThe direct enantiomeric resolution of five O-aryl O-alkyl N-alkyl-phosphoramidothioates was carried out and their pure enantiomers were prepared on a commercially available Pirkle model chiral column (OA-4700). Absolute configurations of the enantiomers were determined by circular dichroism (CD) spectroscopy. S-enantiomers were the first compounds to elute chromatographically and the chromatographic elution order was in accordance with the results of computer simulating calculation. The biological activity test suggested that all the S-enantiomers were more active than R-enantiomers in killing barnyard grass.

Statistical scanning method for the optimization of gradient elution high performance liquid chromatography

SummaryA computer-assisted method is presented for the optimization of separation in gradient elution reversed-phase HPLC. The method is based on a polynomial estimation from nine preliminary experiments according to a two-factor (initial solvent composition C and gradient time T) rectangular design. This is followed by a two-dimension computer scanning technique. Resolution is used as the selection criterion. Good agreement was obtained between predicted data and experimental results.