Nelson Cooke

Comparison of short and ultrashort-chain alkylsilane-bonded silicas for the high-performance liquid chromatography of proteins by hydrophobic interaction methods

Abstract The optimization of the reversed-phase high-performance liquid chromatography of proteins has beene xamined using a series of maximum-coverage alkylsilane-bonded silica packings with different carbon chain-lengths (C1–C18). The greatest range of individual compounds that could be successfully chromatographed was obtained with 200 h) of these columns under acid (pH 2.1) conditions. This system has been used to separate prolactin and growth hormone (22 kD) from biological samples by employing a trace-enrichment step on 10-μm tap-packed short alkyl-chain columns prior to gradient-elution chromatography on a 5-μm particle-size (8 nm pore-size) Ultrasphere SAC column.

Factors influencing chromatography of proteins on short alkylsilane-bonded large pore-size silicas☆

Abstract The separation of proteins by high-performance gradient chromatography on short alkylchain bonded silica was studied with respect to pore size, mobile-phase composition, and temperature. Selectivity could be increased in particular cases by varying temperature or eluate compositon. Recovery of late-eluting, hydrophobic proteins was found to increase with flow rate and gradient slope. Recovery was also shown to be dependent on eluate composition—decreasing with added salt. The applicability of reverse-phase chromatography to proteins as large as 150 kD was demonstrated by the separation of monoclonal immunoglobulin.

High-performance protein separations with novel strong ion exchangers

Abstract In this paper, properties of new ion exchangers specifically designed for protein separations are reported. These sorbents are constituted of two main parts: a rigid, porous polystyrene-silica composite material which forms a rigid skeleton and a soft hydrogel bringing strong ionic groups. The later is regularly distributed inside the pores of the skeleton. Characterization of these materials was performed by measuring dynamic sorption capacity, resolving power, separation efficiency and protein recovery. These studies were done using various known proteins and protein mixtures. Some comparisons have been made with commercially available ion exchangers also designed for protein separations.

Effects of chain length and carbon load on the performance of alkyl-bonded silicas for protein separations

Abstract The retention and recovery of proteins in reversed-phase liquid chromatography with gradient elution using aqueous-organic mobile phases were found to be similar from large pore (30 nm) silica bonded to maximal extents with either propylor octyldimethylsilanes. Proteins were less retained and less completely recovered from silica partially bonded with octyldimethylsilane when used with an acetonitrile-containing mobile phase but not with 1-propanol as modifier. The elution position of proteins under gradient conditions was shown to be independent of flow-rate for fixed gradient slope, but to move later in the gradient with increases in gradient slope. The effects of gradient slope and flow-rate on resolution, separation time, and sensitivity are illustrated.

Capillary sodium dodecyl sulfate gel electrophoresis of proteins

Abstract In recent years, there has been considerable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interest in using this technique to separate on the basis of size and to estimate molecular mass. In this paper we report a new improved and automated electrophoretic method in the form of high-performance capillary SDS gel electrophoresis. Rapid separations of protein molecules in the molecular mass range of 20 000–200 000 daltons were demonstrated with excellent linearity and intra- and inter-day reproducibility of the migration time. Monomer—dimer forms of the recombinant human ciliary neurotrophic factor were examined by the use of this method under reducing and non-reducing conditions.

Enhanced separation of DNA restriction fragments by capillary gel electrophoresis using field strength gradients

The effect of electric field strength gradients on the separation of DNA restriction fragments was investigated. As reported in our earlier work, the mobility of different size double-stranded DNA molecules is a function of the applied electric field which suggests that the use of a nonuniform (time varying) electric field may increase the resolving power. We demonstrate that in capillary gel electrophoresis enhanced separation of DNA restriction fragments up to 1353 bases pairs (bp) in size can be achieved by employing the field strength gradient method

Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis

The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.

Practical aspects of chiral separations of pharmaceuticals by capillary electrophoresis. I: Separation optimization

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. In our model system hydroxypropyl-β-cyclodextrin was used as chiral selector for the separation of racemic propranolol. Results are presented regarding the effect of different operational variables such as buffer pH, concentration of chiral selector, applied electric field and temperature on the chiral separation. Based on the experimental data, the operational variables were optimized to attain maximum resolving power with minimal analysis time.

Isoelectric focusing of proteins in capillary electrophoresis with pressure-driven mobilization

SummaryAn isoelectric focusing (IEF) method in the capillary format with wide linear pH range (pH 3–10) and high resolution has been developed for separations of proteins. The methodology involves applying pressure and voltage simultaneously during the mobilization step of the IEF process, to elute the focused protein zones for detection. Capillary isoelectric focusing (CIEF) is performed in neutral, hydrophilic, coated capillaries of 50 μm I.D., using various concentrations of methyl cellulose to reduce the distortion of focused zone by parabolic laminar flow. A linear relationship between the retention time and isoelectric point (pI) of protein standards was observed. The high resolution of this technique was demonstrated by the separation of hemoglobin variants F and A with pI difference of 0.05 pH units, and by the resolution of the isoforms of an anti-carcinoembryonic antigen monoclonal antibody.

Capillary sodium dodecyl sulfate gel electrophoresis of proteins I. Reproducibility and stability

Abstract This paper investigates the use of a non-cross-linked polymer gel as a separation matrix for sodium dodecyl sulfate (SDS) capillary gel electrophoresis of proteins. The method employs a polyacrylamide coated capillary filled with a polyethylene oxide-gel buffer solution. A standard seven-protein mixture was chosen for the evaluation of coating stability and reproducibility. It was found that the coating is stable for more than 400 runs with a 1 M HCl wash between each run. The hydrophilic nature of polyethylene oxide also allows high resolution and high efficiency for all protein peaks. The linear plot of log Mr vs. mobility demonstrates a pure sieving mechanism of polyethylene oxide matrices. The molecular masses of twenty-nine standard proteins determined by SDS capillary gel electrophoresis are in good agreement with those obtained from the SDS polyacrylamide gel electrophoresis (PAGE) slab gel method. The capability to replace the gel after each run allows improved run-to-run and batch-to-batch reproducibility. The relative standard deviation (R.S.D.) in migration time of 19 injections of the seven-protein standard mixture, with the 190 injections of crude fetal calf serum in between, falls in the range of 0.351 to 0.453%. The application of this technique for the separation of proteins in chicken egg white and bovine milk is demonstrated.