Proteins electrokinetic methods

The major advantage of protein adsorption studies on high surface area materials is that changes of some extensive properties which accompany the process of adsorption are large enough to be directly measured heat of adsorption through microcalorimetry 141), uptake or release of small ions by a combination of electrokinetic methods and titration 142), thickness of adsorbed layer or an increase of the volume fraction of solid phase by a hydrodynamic method like viscometry 143). Chromatographiclike analysis can also be applied to protein adsorption 144). 

Analysis of Grape and Wine Proteins and Peptides 7.3.1. Electrokinetic Methods... 

Electrokinetic methods certainly comprise some of the most powerful techniques for protein analysis. They are based on the property of charged molecules to migrate in an electric field according to their net charge (isoelectric focusing, IEF), net charge and size (native electrophoresis) or only size (SDS-PAGE). These techniques include both analytical and preparative systems. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

Electrokinetics play Important roles in colloid and surface science but also beyond these domains. The purpose of the present section is to consider some principles behind their measurements, emphasizing methods relevant for characterizing colloids and interfaces. The large number of semlquantitative methods, including paper or gel electrophoresis for diagnostic purposes and isoelectric focusing in protein chemistry, are considered "applications (sec. 4.10). For more details, see the literature of sec. 4.11, especially the books by Hunter, and Rlghettl et al. Unless where explicitly mentioned otherwise, the considerations apply to aqueous systems. 

Separation of kavalactones was also achieved by supercritical fluid chromatography using methanol-modified carbon dioxide as the mobile phase. An optimal separation was achieved on either an amino or protein C4 column at 125 atm and 80 °C. Semipreparative separation of kavalactones was also obtained with two columns connected in series (Ashraf-Khorassani etal., 1999). A micellar electrokinetic chromatographic method with diode-array detection has also been developed for the identification and quantitative determination of the major kavalactones from extracts of P. methysticum... 

A number of methods for the determination of electrophoretic velocity and electrokinetic potential of particles have been developed. These methods include the moving boundary method (a direct study of motion of the boundary between the disperse system and the free dispersion medium due to the applied potential difference), microelectrophoresis (a direct observation of moving particles using a microscope or ultramicroscope), electrophoresis in gels, paper electrophoresis, etc [ 13]. These methods are broadly used to study disperse systems formed with low molecular weight substances, as well as polymers, especially those of natural origin. Electrophoretic methods allow one to separate and analyze mixtures of proteins, and thus are effectively used in scientific research and medical diagnostic applications. 

To prevent biomolecular adsorption and water stiction, the surface materials for a microfluidic device should be carefully chosen with consideration of the actuation method (hydrodynamic or electrokinetic) and analytes to be manipulated (small solutes, DNA, proteins, and/or cells). Tables 2 and 3 summarize the surface materials that have been used in microfiuidic applications. 

Sample concentration is another way to increase the sensitivity of a method. Besides liquid-liquid and solid-phase extraction protocols, which result in more concentrated solutions in addition to sample cleanup, several electrokinetic concentration techniques can be applied including sample stacking and field-amplified sample injection/stacking. Over 1000-fold sensitivity enhancement compared to hydrodynamic sample injection without stacking has been reported. When acetonitrile has been used for the removal of proteins from plasma, sample injections of up to 50% of the capillary volume become possible. Stacking techniques are also available for MEKC. 

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