Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 turn x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges. (c) 2005 Elsevier B.V. All rights reserved.

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. in addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Interface of chip-based capillary electrophoresis-inductively coupled plasma-atomic emission spectrometry (CE-ICP-AES)

An interface of chip-based capillary electrophoresis (CE)-inductively coupled plasma-atomic emission spectrometry (ICP-AES) that is based on cross-flow nebulization has been developed. A polydimethylsiloxane (PDMS) CE-chip with conventional cross channel layout was used. A stainless steel tube was placed orthogonal to the exit of the CE separation channel for cross flow nebulization. A supplementary flow of buffer solution at the channel exit was used to improve nebulization efficiency. Two capillaries were inserted into the CE chip near the inlet of the separation channel for sample and buffer solution injection. Syringe pumps were used to manipulate the flow rate and flow direction of the sample, buffer, and supplementary buffer solution. Peak broadening due to the shape (bulb and tube-shaped) and size of the spray chambers was studied. The smaller tube-shaped spray chamber was used because of smaller peak broadening effect due to aerosol transport. The nebulization and transport efficiency of the CE-ICP interface was approximately 10%. Ba2+ and Mg2+ ions were eluted from the CE-chip within 30 s. Resolution of the Ba2+ and Mg2+ peaks was 0.7 using the chip-based CE-ICP-AES system.