Repeatedly usable immobilized pH gradient in a monolithic capillary column

Glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were used to synthesize a monolithic capillary column containing reactive epoxy groups. Glutaraldehyde was introduced and linked to the monolith after a process of amination. An aqueous solution of commercial carrier ampholytes (CAs, Ampholine) was focused in such a polymer column. The primary amino groups of CAs reacted with glutaraldehyde along the capillary. CAs were immobilized at different positions in the column according to their isoelectric points (pl), resulting in a monolithic immobilized pH gradient (M-IPG). Isoelectric focusing (IEF) was performed without CAs in such an M-IPG column. Due to the covalent attachment of the CAs this M-IPG can be repeatedly used after its preparation. Good stability, linearity, and reproducibility were obtained.

Study of an electroosmotic pump for liquid delivery and its application in capillary column liquid chromatography

A packed-bed electroosmotic pump (EOP) was constructed and evaluated. The EOP consisted of three capillary columns packed in parallel, a gas-releasing device, Pt electrodes and a high-voltage power supply. The EOP could generate output pressure above 5.0 MPa and constant flow rate in the range of nl/min to a few mul/min for pure water, pure methanol, 2 mM potassium dihydrogenphosphate buffer, the buffer-methanol mixture and the pure water-methanol mixture at applied potentials less than 20 W The composition of solvent before/after pumping was quantitatively determined by using a gas chromatograph equipped with both flame ionization detector and thermal conductivity detector. It was found that there were no apparent changes in composition and relative concentrations after pumping process for a methanol-ethanol-acetonitrile mixture and a methanol-water mixture. Theoretical aspect of the EOP was discussed in detail. An capillary HPLC system consisting of the EOP, an injection valve, a 15 cm x 320 mum i.d., 5 mum Spherigel C(18) stainless steel analytical column, and an on-column UV detector was connected to evaluate the performance of the EOP. A comparative study was also carried out with a mechanical capillary HPLC pump on the same system. The results demonstrated that the reproducibility of flow rate and the pulsation-free flow property of the EOP are superior to that of mechanical pump in capillary HPLC application. (C) 2004 Elsevier B.V. All rights reserved.

Protein A immobilized monolithic capillary column for affinity chromatography

Monolithic capillary columns for affinity chromatography were prepared by an in situ polymerization procedure using glycidyl methacrylate (GMA) as a monomer and trimethylolpropane trimethacrylate (TRIM) and ethylene dimethacrylate (EDMA) as cross-linkers, respectively. Scanning electron microscopy was applied to characterize the morphology of the end of monolithic capillary and mercury intrusion porosimetry to characterize the polymer rod prepared within the confines of a stainless steel column with 50 mm x 4.6 mm i.d. under the same polymerization condition. Obvious differences in the porous properties between the TRIM- and EDMA-based monoliths could be observed. Moreover, the mechanical stability of these two monolithic capillary columns was compared by testing the reproducibility of the column performance. The rod prepared with GMA and TRIM proved to be mechanically more stable than that prepared with GMA and EDMA. Protein A was immobilized on the monolithic rod for affinity chromatography and the experiments were performed on a capillary electrophoresis instrument, using its pressure system as the driving force. Non-specific adsorption was not observed on the TRIM-based affinity column, as proved with bovine serum albumin (BSA) as a test protein. The affinity column prepared with GMA and TRIM was then applied to determine the hIgG concentration in human serum. The correlative coefficient of the calibration curve reached 0.9942. The amount of adsorbed hIgG was unaffected by the flow rate of the loading buffer, which makes this method suitable for fast determination of biomacromolecules in microliter samples. (C) 2002 Elsevier Science B.V All rights reserved.

Electrochromatographic evaluation of a silica monolith capillary column for separation of basic pharmaceuticals

A silica-based monolithic capillary column was prepared via a sol-gel process. The continuous skeleton and large through-pore structure were characterized by scanning electron microscopy (SEM). The native silica monolith has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). Column efficiencies greater than 250000 plates/m for capillary electrochromatography (CEC) separation of basic compounds were obtained. It was observed that retention of basic pharmaceuticals on the silica monolith was mainly contributed by a cation-exchange mechanism. Other retention mechanisms including reversed-phase and normal-phase mechanisms and electrophoresis of basic compounds also played a role in separation. A comparison of the differences between CEC and capillary zone electrophoresis (CZE) separation was also discussed.

Monolithic column with zwitterionic stationary phase for capillary electrochromatography

A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycylic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.

Capillary electrochromatographic separation of ionizable compounds with a molecular imprinted monolithic cationic exchange column

A polymer-based monolithic capillary column imprinted with 4-aminopyridine (4-AP) was prepared by a thermally-initiated polymerization process; and its performance as a capillary electrochromatographic medium was evaluated in separating 4-AP and 2-AP isomers. The effects of experimental parameters, such as pH value and ionic strength of the buffer, the acetonitrile content in the mobile phase, and the applied voltage, on the resolution of these isomers had been carefully investigated. It was found that in the retention process there were interplays of multiple mechanisms of ion-exchange, molecular imprinting, and electrophoresis. These mechanisms allowed more sophisticated control of experimental parameters in the separation of ionizable compounds.

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong canon-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for to and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.

An electroosmotic pump for packed capillary liquid chromatography

An electroosmotic pump (EOP) capable of generating pressure above 3 MPa and mul/min flow rate with reverse phase mobile phases of HPLC was constructed and evaluated. The pump consisted of three parallel connected fused silica capillary columns (25 cm x 320 mum I.D.) packed with 2 mum silica materials, hollow electrodes, a high voltage DC power supply, and. a liquid pressure transducer. The EOP was applied in a capillary liquid chromatographic system for mobile phase delivery instead of a mechanical pump. Standard samples containing thiourea, naphthalene, anthracene, phenanthrene and acetonitrile were separated on a 15 cm x 320 mum I.D. 5 mum Chromasil C-18 packed capillary column with acetonitrile/water as mobile phase. (C) 2003 Elsevier Science B.V. All rights reserved.

Light-emitting-diode-induced fluorescence detector for capillary electrophoresis using optical fiber with spherical end

A simple fluorescence detector for capillary electrophoresis (CE) using a blue light-emitting-diode (LED) as excitation source is constructed and evaluated. An optical fiber was used to collect the fluorescence, and a flat end of the fiber was modified to spherical end, resulting in 50% increase of efficiency over the flat end. A simple device for optical alignment of the fibers and capillary column was designed. The concentration and mass detection limits for fluorescein were 1.8 x 10(-7) Mol l(-1) and 4.3 femol, respectively. (C) 2002 Elsevier Science B.V. All rights reserved.

Capillary electrophoresis of urinary normal and modified nucleosides of cancer patients

This paper gives a capillary electrophoretic method for the separation of 15 urinary normal and modified nucleosides from cancer patients in less than 40 min. A 500 mmx50 mu m uncoated capillary column (437.5 mm to window) was used. The effects of the voltage and the sodium dodecyl sulfate (SDS) concentration in the buffer on the separation were studied. With reproducibilities of migration times better than 1.2% (R.S.D.) and determined concentrations better than 5-25%, depending on the concentrations of nucleosides in the urine, the analytical characteristics of the method were food. Using this developed method, the concentrations of 13 normal and modified nucleosides, extracted on a phenyl boronic acid affinity chromatography column, in 25 urines from patients of 14 kinds of cancer were determined. The levels (nmol/mol creatinine) of modified nucleosides in urines from cancer patients were increased as compared with those in normal urines. (C) 1998 Elsevier Science B.V.

Capillary liquid chromatographic-high-resolution mass spectrometric analysis of ribonucleotides

A method of capillary HPLC-high-resolution MS was developed for the trace analysis of ATP, GTP, dATP and dGTP Dimethylhexylamine (DMHA) was used as ion-pairing agent for the HPLC retention and separation of the nucleotides and positive ion electrospray time-of-flight MS was used for the detection. The application of capillary HPLC allowed minimal usage of DMHA while providing excellent peak retention and resolution, which significantly reduced the ion suppression in electrospray ionization-MS analysis and thus increased the sensitivity. Adduct ions of nucleotides and DMHA were used as quantitative ions in order to achieve the best sensitivity. DMHA concentration at 5 mM in the aqueous mobile phase at pH 7 was found to be the optimal conditions for the C Is capillary column. The method was applied to determine ATP level in cultured C6 glioma cells that were treated with toxic concentrations of Zn. The results showed that the cellular ATP level decreased from 2.7 pmol/cell (<10% cell death) in average control cell samples to 0.36 pmol/cell as the concentration of Zn increased to 120 mg/l (>35% cell death) in culture medium.

Separation and determination of flavone and xanthone glycosides in Tibetan folk medicinal species Swertia mussotii and S-franchetiana by capillary electrophoresis

The contents of five pharmacologically active flavone and xanthone glycosides, namely, swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[alpha-L-rhamnopyranosyl-(1 -> 2)-beta-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode-array detection. The separation of five components has been optimized with a capillary column with a total length of 48.5 cm and effective length of 40 cm (50 mu m i.d). The influence of the running buffer, the sodium dodecyl sulfonate (SDS) concentration, organic modifier, etc. on the resolution was evaluated. The background electrolyte contained 30 mM borate buffer, 28 mM SDS, 1.0% (v/v) acetonitrile, and was adjusted to pH 9.0 with 0.1 M NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with the working voltage of 24 kV and a column temperature of 25 degrees C. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and franchetiana plant samples.

Separation of neutral compounds by high speed capillary electrochromatography

With using short capillary column packed with porous and non-porous ODS stationary phases, high speed separation of 6 neutral aromatic compounds within 36 s by capillary electrochromatography (CEC) has been performed. Good reproducibility of the migration times for those solutes in high speed CEC was observed with RSD less than 1%. Both the linear velocity of EOF and the current linearly increases with the applied voltage, which means that the thermal effect by Joule heating was small. However, the capacity factor of solutes was found to decrease with the increase of the applied voltage, which was caused by the fact that about several seconds needed for the increase of voltage from 0 to applied value on a commercial CE instrument made larger contributions to the migration times of the early eluted compounds than those of lately eluted ones during high speed CEC, and voltage effect would increase with the higher applied voltage used. The linear relationship between the logarithm of capacity factor and the number of carbon for homologous compounds was observed, and positive value of slope means that the hydrophobicity of solutes is one of the main contribution factors to retention in high speed CEC packed with ODS stationary phases.