Journal of capillary electrophoresis and microchip technology最新文献

Electroosmotic flow (EOF) is one of the fundamental processes affecting both resolution and separation times in capillary zone electrophoresis (CZE). EOF is a function of pH and buffer composition (zeta potential) in bare-silica capillaries at any pH above 3 and runs in the cathodal direction, decreasing the effective separation length for cationic species. On the other hand, the absence of EOF at low pH can significantly increase separation times, particularly for low pl zwitterionic species. This interdependence of pH and EOF limits the ability to design effective separations by capillary zone electrophoresis. The EOTrol family of dynamic coatings (Target Discovery, Inc., Palo Alto, CA, U.S.A.) allows both the magnitude and direction of EOF to be adjusted independently of the buffer composition and pH. Here we report the use of EOTrol in two challenging CZE separations: (1) inorganic cations with small mobility differences, and (2) the rapid separation of organic zwitterions that are only resolvable at low pH.

A capillary electrophoresis method for the simultaneous separation of caseins, whey proteins, and para-kappa-casein that appears during the manufacture of cheese was optimized using the response surface methodology. The parameters selected for this study were pH, voltage, and temperature. Under the optimized conditions (30 kV at 20 degrees C with 10 mM phosphate buffer at pH 3) casein proteins alpha(s)-casein; beta-casein, including genetic variants A1, A2, and B, kappa-CN, and para-kappa-CN; and whey proteins alpha-lactalbumin and beta-lactoglobulin (A and B variants) were separated. The method was applied successfully to skim milk, to casein precipitated at pH 4.6, and to a model sample containing rennet casein and milk. The milk used was of three types: cow, ewe, and goat. The present procedure can be easily applied to the separation of the major bovine, ovine, and caprine milk proteins in binary and ternary milk mixtures.

A capillary electrophoretic method for the analysis and separation of ortho- and para-phthalic acid isomers was developed. The best separation was achieved using micellar electrokinetic capillary chromatography (MECC) performed in a 110 mM borate buffer pH 8.3 also containing 40 mM sodium dodecyl sulfate (SDS) and 20% methanol. The effect of the addition of methanol, acetonitrile, and beta-cyclodextrin on the separation of the isomers was investigated. The buffer system developed was shown to provide a dependable method of analysis from which the concentration of each analyte could be reliably determined.

The methodological aspects for the separation, fractionation, and peptide mapping by free zone capillary electrophoresis (CZE) of beta-lactoglobulin (beta-Lg) variants A and B were established. First, beta-Lg variants A or B were separated and fractionated by CZE. Then, the collected protein fraction was subjected to off-line tryptic digestion. Second, peptide mapping of the tryptic hydrolysates and peptide fraction collection were carried out by CZE. beta-Lg variants were separated and collected using an uncoated capillary (72 cm x 75 microm i.d.) in 0.05 M borate buffer containing 0.1% Tween 20 at pH 8.0 by applying 20 kV. By subjecting the capillary under pressure after a delay time of 15%, the protein was collected in a microvial containing digestion buffer. The most suitable conditions for the tryptic digestion of beta-Lg were established by monitoring the reaction products with CZE. A tryptic hydrolysis with an enzyme-to-substrate ratio (E/S) of 1/20 and incubation for 20 hr at 37 degrees C was found to result in the most suitable conditions. Peptides were separated and collected using an uncoated capillary (120 cm x 75 microm i.d.) in 0.15 M formic acid at pH 2.3 by applying 28 kV. Peptide maps were highly reproducible as shown by coefficients of variation of less than 0.89 and 5.42% for migration times and peak areas, respectively. Moreover, very good resolution of the peptide maps revealed the region in which the aberrant peptides of the beta-Lg variants may be located.

Cereal grains are important both nutritionally and economically in virtually every country in the world. Cereal grains can be made into a wide range of human foods and are also important animal feed components. Although all of the biochemical components of cereal grains are important, cereal proteins play major functional, as well as nutritional, roles in foods. Cereal proteins are complex mixtures of proteins that are often difficult to solubilize and separate. Because of this, a wide range of analytical techniques have been used to separate and characterize cereal proteins. One of the new techniques used to separate these challenging proteins is high-performance capillary electrophoresis (HPCE). This review covers methods developed to separate cereal proteins by HPCE.

During early-phase pharmaceutical development, it is important to find an initial separation of enantiomeric compounds quickly in order to determine the enantiomeric purity of chiral drug substances. Highly selective screening methods are necessary to analyze the products to discover a satisfactory separation of the enantiomeric compounds. A screening approach based on the use of mixtures of multiple cyclodextrins in chiral capillary electrophoresis was employed to find the initial separation of chiral compounds. In a later phase of development, these complex methods need to be simplified for transferability. This study describes the simplification of the complex mixture of cyclodextrins into a single or dual system of only the enantioselective cyclodextrins. This was achieved by applying fractional factorial experimental designs to select the cyclodextrins that were responsible for the enantiomeric separation and response surface modeling designs for the optimization of the separation. In order to obtain robust methods, the concentration of the chiral selector, together with other important electrophoretic method parameters such as the concentration of the background electrolyte, pH, and run voltage, were optimized by employing a Box-Behnken experimental design.

Sensitivity is a major obstacle to biochemists studying complex biological mixtures. Laser-induced fluorescence (LIF) is a very sensitive method that can be used with micro-HPLC (microHPLC) or capillary electrophoresis studies and provides an effective solution to these problems. Derivatization is a very useful tool in LIF detection because it allows for the detection of many compounds and is selective, i.e., one dye will label one chemical function specifically. The major difficulty is choosing a derivatization reaction among the huge number of reactions described in the literature and making it work at a very low level of concentration. Another limitation is the use of a laser wavelength, which can excite the derivatized molecules. This article discusses selected derivatization reactions for labeling molecules in the subnanomolar or nanomolar range for CE and microHPLC. Derivatization for amines, sugars, aldehydes, carboxylic acids, thiols, diols, and nucleotides are described, and derivatization procedures are presented. In many cases, the subnanomolar and nanomolar concentration of these compounds can be detected using these chemical reactions.

High-performance capillary electrophoresis (HPCE) has emerged over the past 20 years as a powerful multidimensional separation tool that is orthogonal to HPLC and comparable to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gel methods. HPCE is most frequently applied in the QC release testing of recombinant DNA-derived protein and monoclonal antibody (MAb) biopharmaceuticals. HPCE is a rugged and robust separation tool that can be used like HPLC to monitor the purification process, as well as to analyze bulk drug and drug substances. Examples of the practical applications of the predominant free-solution capillary electrophoresis (FSCE) and capillary gel electrophoresis (CGE) formats of HPCE, applied for process monitoring and product monitoring of recombinant protein and MAb biotherapeutics, are presented. HPCE has been applied in FSCE mode to monitor the purification of the rDNA-derived protein, recombinant human interleukin-4 (rhIL4). FSCE is demonstrated to be a robust method that can be used to monitor multiple column chromatographic purification processes, such as immobiilized metal-ion affinity chromatography (IMAC), ion exchange chromatography (IEC), and size exclusion chromatography (SEC) columns. The FSCE data are used to pool fractions to carry forward for further purification. The FSCE method is compared to the corresponding RP-HPLC method for rhIL4. HPCE has been applied in the CGE mode to monitor the purification of an rDNA-derived IgG4 MAb. CGE is demonstrated to be a convenient and rapid method to profile the purification process, compare purification processes, and provide a fingerprint of the MAb bulk drug that is useful for determining purity and lot-to-lot consistency. The practical advantages and limitations of CGE for process monitoring and product monitoring of MAbs are presented. The CGE method is compared to the high-performance SEC separation of the MAb under nondenaturing (HP-SEC) and denaturing (HP-DSEC) conditions and to SDS-PAGE gels for the analysis and characterization of MAb bulk drugs.