Electrophoresis in columns

A number of apparatuses were constructed for this purpose [257-264], some of which are commercially available. Here we describe the apparatus marketed by LKB (Bromma, Sweden) (see also Ref. 264) (Fig. 6.26). The apparatus consists of a inverted U-shaped tube that is composed of two parts connected by a joint in the upper part. One limb accommodates the electrophoresis column that is surrounded by a covering jacket, the other limb filled with buffer serves to make connection with the electrode vessel. At the lower end of the column a collecting funnel is attached. Liquid connection or disconnection between the two limbs can be done through a ground female joint. The stopcock above this joint connects the system to a buffer reservoir when the column is washed or when the zones are eluted. In the male joint of the top piece there is a hole that allows liquid to pass to the other limb. By turning the liquid stream can be disconnected at this point. The column size is 55 X 2 cm Sephadex or cellulose serve as the supporting material. Separation is run at 500-1000 V and 25-50 mA. 

Electro-osmosis, which is a troublesome effect in this system, can be counteracted by carefully adjusting buffer flow in the direction of electrophoretic migration. The separation is usually started in such a way that the sample is placed in the column (together with some dye to visualize the movement) by making use of the reservoir buffer. Only then is the electricity switched on. 

Electrophoresis in supports that themselves exhibit sieving effect improves the resolution. Diluted agarose (0.12-0.2%) was used as support by Hjerten [265]  

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P3. Porath, J., Zone Electrophoresis in Columns and Adsorption Chromatography on Ionic Cellulose Derivatives, as Methods for Peptide and Protein Fractionations. Almqvist and Wiksells, Uppsala, 1957. 

The relative mass difference of isotopes in ionic liquids is higher than in aqueous solution. This is because solvation of ions by water tends to diminish mass differences. Consequently, greater differences in electrophoretic mobility are possible in ionic liquids. Countercurrent electrophoresis in columns allowed isolation of highly enriched isotope mixtures without great difficulty. Some examples of the mass difference effect are summarized in Table 1. These results depend on the temperature as well as the composition of the ionic liquid. 

An injection technique in capillary electrophoresis in which pressure is used to inject sample into the capillary column. 

A form of capillary electrophoresis in which the capillary column contains a gel enabling separations based on size. 

Detection of the PSP toxins has proven to be one of the largest hurdles in the development of analytical methods. The traditional means, and still in wide use today, is determination of mouse death times for a 1 mL injection of the test solution. There are a variety of drawbacks to utilization of this technique in routine analytical methods, that have prompted the search for replacements. In 1975 Bates and Rapoport (3) reported the development of a fluorescence technique that has proven to be highly selective for the PSP toxins, and very sensitive for many of them. This detection technique has formed the basis for analytical methods involving TLC (77), electrophoresis (72), column chromatography (7J), autoanalyzers (7 ), and HPLC (5,6,7). 

Thin-layer chromatography (TLC) is a type of liquid chromatography in which the stationary pease is in the form of a thin layer on a flat surface rather than packed into a tube (column). It is a member of a family cf techniques that include some types of electrophoresis and paper chromatography, more generally referred to as planar chromatccraphy. Since we will not discuss electrophoresis in this section, and since TLC has virtually superseded paper chromategr pby in most analytical... 

A number of developments have increased the importance of capillary electrophoretic methods relative to pumped column methods in analysis. Interactions of analytes with the capillary wall are better understood, inspiring the development of means to minimize wall effects. Capillary electrophoresis (CE) has been standardized to the point of being useful as a routine technique. Incremental improvements in column coating techniques, buffer preparation, and injection techniques, combined with substantive advances in miniaturization and detection have potentiated rugged operation and high capacity massive parallelism in analysis. 

The pectinesterase produced by Sclerotinia libertiana78 was purified on columns of Duolite A-2, Amberlite CG-50, and CM-cellulose. The final product was purified 266-fold, its sedimentation coefficient was calculated to be 4.41 S, and zone electrophoresis in starch gel showed a slight contamination of this product. 

Capillary electrochromatography (CEC) is a miniaturized separation technique that combines aspects of both interactive chromatography and capillary electrophoresis. In this chapter, the theory of CEC and the factors affecting separation such as the stationary phase and mobile phase parameters have been discussed. The chapter focuses on the types and preparation of columns for CEC and describes the progress made in the development of open-tubular, particle-packed, and monolithic columns. The detection techniques in CEC such as the traditional UV detection and improvements made in coupling with more sensitive detectors such as mass spectrometry are also described. The chapter provides a summary of some applications of CEC in the analysis of pharmaceuticals and biotechnology products. 

Lin, C.-C., and Liu, C.-Y. (2004). Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derlvatlzatlon. Electrophoresis 25, 3216-3223. 

Y Ishihama, Y Oda, N Asakawa, M Iwakura. Nanoscale monitoring of the thermally induced unfolding of proteins using capillary electrophoresis with in-column incubation. Anal. Sci. 13 931-938 (1997). 

We have examined whether the sulfur that was bound to the proteins of a reconstituted system from the liver of phenobarbital-treated rats was bound to both the reductase and cytochrome P-450. I this experiment, the reconstitued system was incubated with [ s] parathion. The reaction mixture was dialyzed and applied to a Sephadex G-25 column to remove the last traces of unreacted parathion and its noncovalently bound metabolites. The protein fraction from the Sephadex column was reduced in volume and subjected to SDS-polyacrylamide gel electrophoresis in the absence of either dithiothreitol or mercaptoethanol. The results are shown in Figure 5. There was considerable protein and radioactivity at the origin. This material at the origin represents an aggregate of reductase... 

Cyanogen bromide cleavage of the polypeptide subunit should yield 4 peptides if the subunit contains 3 methionines (Table III). Cleavage products were isolated by gel filtration through Sephadex G-75 columns and analyzed for purity with polyacrylamide gel electrophoresis in 8 M urea containing 0.1 M thioglycolate. Four peptide products were obtained (34). 

To test this hypothesis, very low density lipoprotein (VLDL, d<1.0 gm/ml), low density lipoprotein (LDL, d=l.02-1.063) and high density lipoprotein (HDL, d=l.09-1.21) were isolated from outdated human plasma by ultracentrifugation according to established procedures (27,28), using potassium bromide for density adjustments and stored at -20° C in the presence of 20% sucrose before use. The purity of individual lipoprotein fractions thus obtained was established by polyacrylamide gel electrophoresis in sodium dodecyl buffer system (2 ) and filtration through a Sepha-rose 6B column, equilibrated with 0.2 M potassium bromide in 0.1 M sodium phosphate buffer, pH 7.2. Protein (30) and cholesterol... 

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