Borate-containing background electrolytes to improve CE separation in bare capillaries.

The paper describes how borate-containing BGEs modify ζ-potential and so EOF in bare fused silica capillaries. This surface modification can be used to suppress EOF and improve the separation performance of CZE including capillary sieving electrophoresis (CSE). Boric acid forms complexes with polysaccharides used as sieving matrices in CSE and other compounds containing hydroxyl groups, including polyol bases such as Tris, triethanolamine, and Bis-Tris propane. High concentration of boric acid in BGEs leads to a strong interaction of boric acid with the silica surface of the capillary wall and this suppresses or even completely eliminates ζ-potential and EOF. Using a polyol base with several charge-carrying amino groups, such as Bis-Tris propane, can actually reverse EOF. We demonstrate the use of various borate-containing BGEs in bare fused silica capillaries for size-separation of DNA fragments, size-separation of proteins by SDS CSE, and also by CZE in the absence of any sieving matrix.

A novel microchip-based imaged CIEF-MS system for comprehensive characterization and identification of biopharmaceutical charge variants.

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS-compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF-MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real-time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N-linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF-MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

Chemical modification of proteins to improve the accuracy of their relative molecular mass determination by electrophoresis.

We studied the electrophoretic behavior of basic proteins (cytochrome c and histone III) and developed a carbamylation method that normalizes their electrophoretic size separation and improves the accuracy of their relative molecular mass determined electrophoretically. In capillary zone electrophoresis with cationic hitchhiking, native cytochrome c does not sufficiently bind cationic surfactants due to electrostatic repulsion between the basic protein and cationic surfactant. Carbamylation suppresses the strong positive charge of the basic proteins and results in more accurate relative molecular masses.

Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking.

The paper describes a method of size separation of proteins by capillary sieving electrophoresis with cationic surfactant. Proteins are separated within 12 min with repeatability of migration times better than 0.2%. Some proteins achieve the separation efficiency of 200,000 theoretical plates. The method can be used for determination of protein relative molecular masses. The accuracy of the determined relative molecular masses and the limitation of the method were investigated by the analysis of more than 60 proteins. The method also allows separation of protein oligomers. Proteins can be quantitated after the electrokinetic injection in the concentration range 0.07-0.43 g/L. The average detection limit is about 2 mg/L.

Capillary electrophoresis of proteins 2005-2007.

This review article with 239 references describes recent developments in capillary electrophoresis of proteins, and covers the two years since the previous review (V. Dolník, Electrophoresis 2006, 27, 126-141) through spring 2007. It includes topics related to CE of proteins, such as sample pretreatment, wall coatings, improving separation, various forms of detection, and special electrophoretic techniques including ACE, CIEF, capillary ITP, and CEC. The paper describes applications of CE to analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals and recombinant protein preparations.

Capillary electrophoresis of proteins 2003-2005.

This review article with 304 references describes recent developments in CE of proteins, and covers the two years since the previous review (Hutterer, K., Dolník, V., Electrophoresis 2003, 24, 3998-4012) through Spring 2005. It covers topics related to CE of proteins, including modeling of the electrophoretic migration of proteins, sample pretreatment, wall coatings, improving separation, various forms of detection, special electrophoretic techniques such as affinity CE, CIEF, and applications of CE to the analysis of proteins in real-world samples including human body fluids, food and agricultural samples, protein pharmaceuticals, and recombinant protein preparations.

Applications of capillary electrophoresis on microchip.

CE on microchip is an emerging separation technique that has attracted wide attention and gained considerable popularity. Because of miniaturization of the separation format, CE on chip typically offers shorter analysis time and lower reagent consumption with potential development of portable analytical instrumentation. This review with 143 references is focused on proteins and peptides analysis, DNA separation including fragment sizing, genotyping, mutation detection and sequencing, and also the analysis of low-molecular-weight compounds, namely explosive residues and warfare agents, pharmaceuticals and drugs of abuse, and various small molecules in body fluids.

Wall coating for capillary electrophoresis on microchips.

This review article with 116 references describes recent developments in the preparation of wall coatings for capillary electrophoresis (CE) on a microchip. It deals with both dynamic and permanent coatings and concentrates on the most frequently used microchip materials including glass, poly(methyl methacrylate), poly(dimethyl siloxane), polycarbonate, and poly(ethylene terephthalate glycol). Characterization of the channel surface by measuring electroosmotic mobility and water contact angle of the surface is included as well. The utility of the microchips with coated channels is demonstrated by examples of CE separations on these chips.

Capillary electrophoresis of proteins 2001-2003.

This review article with 244 references describes recent developments in capillary electrophoresis of proteins and covers the two years since the previous review (Dolník, V. Hutterer, K. Electrophoresis 2001, 22, 4163-4178) through Spring 2003. It covers topics related to CE of proteins including modeling of the electrophoretic properties of proteins, sample pretreatment, wall coatings, improving selectivity, detection, special electrophoretic techniques, and applications.