Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix

A novel protocol has been established to separate dsDNA fragments with high efficiency on glass chips by using an ultralow viscosity sieving matrix with added glucose. Low-molecular-weight hydroxypropylmethylcellulose (HPMC), with a viscosity nearly equivalent to that of water, was used to electrophoretically separate fluorescent inter-calator-labeled double-stranded DNA (dsDNA) fragments on microfluidic glass chips. In comparison with conventional sieving protocols, low-molecular-weight HPMC as sieving matrix could result in reduced running cost and analysis time, in addition to a comparable separation efficiency of dsDNA fragments. In this paper, the addition of glucose was investigated to enhance the separation of DNA in the lowest viscosity polymer evaluated. The effect of staining dye and field strength were also evaluated. At an applied electric field strength of 200 V/cm, satisfactory resolution of the PBR322/HaeIII DNA marker could be achieved within 4 min by using 2% HPMC-5 with 6% glucose added. Coelectrophoresing PCR product along with phiX174/HaeIII DNA sizing marker was also demonstrated by using the ultralow viscosity HPMC-5 solution on a glass chip.

Capillary electrochromatographic separation of enantiomers on chemically bonded type of cellulose derivative chiral stationary phases with a positively charged spacer

Positively charged chiral stationary phases (CSPs) were prepared for capillary electrochromatography (CEC) separation of enantiomers by chemically immobilizing cellulose derivatives onto diethylenetriaminopropylated silica (DEAPS) with tolylene-2,4-diisocyanate (TDI) as a spacer reagent. Anodic electroosmotic mobility was observed in both nonaqueous and aqueous mobile phases due to the positively charged amines on the surface of the prepared CSPs. For comparison, the traditionally used 3-aminopropyl silica (APS) was also adopted as the base material instead of DEAPS to prepare CSP. It was observed that the EOF on the DEAPS-based CSP was 18%-60% higher than that on the APS-based CSP under nonaqueous mobile phase conditions. Separation of enantiomers in CEC was performed on the positively charged CSPs with the nonaqueous mobile phases of pure ethanol or mixture of hexane-alcohol and the aqueous phases of acetonitrile-water or 95% ethanol. Fast separation of enantiomers was achieved on the newly prepared CSPs.

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers

The method for preparation of molecularly imprinted monolithic stationary phase has been improved to achieve liquid chromatographic separation of enantiomers and diastereomers. By adopting low polar porogenic solvents of toluene and dodecanol and optimal polymerization conditions, the molecularly imprinted monolithic stationary phases with good flow-through properties and high resolution were prepared. Enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids were completely resolved using the monolithic stationary phases. The influence of porogenic composition, monomer-template ratio and polymerization conditions on the chromatographic performance was investigated. Some chromatographic conditions such as the composition of the mobile phase and the temperature were characterized. Scanning electron microscopy showed that the molecularly imprinted monolithic stationary phase has a large through-pore structure to allow the mobile phase to flow through the column at very low backpressure. Accelerated separations of enantiomers and diastereomers were therefore achieved at elevated flow rates. Finally, the chiral recognition performance of the prepared stationary phase in aqueous media was investigated. Hydrophobic interaction, and ionic and/or hydrogen bonding interactions were proposed to be responsible for the recognition mechanism. (C) 2002 Elsevier Science B.V. All rights reserved.

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 electrophoresis can 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. (C) 2002 Elsevier Science B.V. All rights reserved.

Pressurized capillary electrochromatography separation of peptides with strong cation exchange and hydrophilic interaction

A pressurized capillary electrochromatography (pCEC) instrument with solvent gradient capability has been used for the separation of a peptide mixture. Retention mechanism and selectivity of the peptides were studied by pCEC using a strong cation exchange (SCX) column. The effects of applied voltage, supplementary pressure, organic modifier concentration, ionic strength,, and pH value on pCEC separation were investigated. It was found that the retention mechanism of the peptides in this system is based on a mixed mode of hydrophilic interaction, strong cation exchange, and electrophoresis. Compared with the separation results obtained by reverse phase pCEC and capillary electrophoresis (CE), this mixed-mode pCEC is more powerful for the separation of hydrophilic peptides with similar charge-to-mass ratio.

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C-12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150 000 plates/meter) and good reproducibility (migration time with RSD < 0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.