Electrophoresis, zone

In zone electrophoresis, the analytes and other sample components migrate as zones or bands with different velocity. 

The migration length is proportional to the applied field strength and time. The bands are approximately Gaussian shaped. The resolution in zone electrophoresis is given by 

Zone electrophoresis has traditionally been categorized as high-voltage electrophoresis (50-200 Vcm ) or low-voltage electrophoresis (2-10Vcm ). 

The pore size of the gels, and hence their fractionation range, can easily be varied. The fractionation range indicates the molecular masses that the gel is able to restrict. Ions with molecular mass smaller than the low cutoff can move freely in the gel, while ions with molecular mass larger than the high cutoff cannot move through the gel. 

Capillary zone electrophoresis [9,10,47-54] (CZE) is a separation technique where components of the sample are separated using 10-30 kV potential difference between the two ends of a 50-100 jxm diameter capillary filled with a buffer solution. The basic instrumental setup is demonstrated in Fig. 10. 

The capillary column is immersed into two buffer-filled reservoirs. High voltage is applied to these reservoirs via platinum electrodes. The sample is stored in 

The world of electromigration separations is sharply divided into two areas. Zone electrophoresis on paper and related procedures have (in spite of their wide applicability to diverse organic compounds) already passed their period of favour. The other branch is represented by the more recent techniques some of which have already became widely accepted (such as isoelectric focusing or separations in polyacrylamide gel) and the others that are at the moment in the centre of a rapid development like displacement electrophoresis (isotachophoresis). This chapter is devoted mainly to analytical procedures such as these which are governing the area of electromigration separations at the moment with a single exception flow deviation (curtain) electrophoresis which will be discussed in more detail because it offers several new dimensions in the separation field. The other preparative procedures are summarized only briefly. 

The approach to any separation (including electromigratory) is governed by the possibilities of the laboratory some of the electromigration techniques suffer from the fact that appropriate commercial equipment is not available. Therefore some rather simple but efficient procedures are included in this survey and examples of some home made equipment are presented. 

Prepare solution A (gel buffer) by dissolving 7.8 g NaH2P04-H20, 18.6 g Na2HP04 (or 38.6 g Na2HP04 7H20), and 2.0 g sodium dodecyl 

Prepare solution B (acrylamide) by dissolving 22.2 g recrystallized acrylamide and 0.6 g N,N -methylene-bis(acrylamide) in sufficient distilled water to yield a final solution volume of 100 ml. This solution should be stored in a brown bottle at 4°C. Solutions more than a month old yield inferior gels and should therefore be replaced. Acrylamide is a strong, cumulative neurotoxin that is easily absorbed through the lungs or directly through the skin. Therefore, extreme care should be taken not to breathe unpolymerized acrylamide or permit it to come in contact with the skin. 

Recrystallized preparations of acrylamide are available commercially or may be prepared as follows. Dissolve acrylamide (70 g) in 1 liter chloroform at 50°C. Filter the solution hot and cool it to —20°C to bring about crystallization. Collect the crystalline acrylamide in a chilled Buchner funnel, wash with chilled (—20°C) chloroform and/or heptane, and dry. Additional discussion of the purification of reagents used for electrophoresis may be found elsewhere (35,36). 

Prepare solution C (ammonium persulfate) just before it is to be used by dissolving 35 mg ammonium persulfate in 10 ml distilled water. 

Prepare upper and lower reservoir buffers by diluting solution A of step 6-1 with distilled water in a ratio of 1 1. 

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Capillary Zone Electrophoresis The simplest form of capillary electrophoresis is capillary zone electrophoresis (CZE). In CZE the capillary tube is filled with a buffer solution and, after loading the sample, the ends of the capillary tube are placed in reservoirs containing additional buffer solution. Under normal conditions, the end of the capillary containing the sample is the anode, and solutes migrate toward... 

Capillary zone electrophoresis also can be accomplished without an electroosmotic flow by coating the capillary s walls with a nonionic reagent. In the absence of electroosmotic flow only cations migrate from the anode to the cathode. Anions elute into the source reservoir while neutral species remain stationary. 

Capillary zone electrophoresis provides effective separations of any charged species, including inorganic anions and cations, organic acids and amines, and large biomolecules such as proteins. For example, CZE has been used to separate a mixture of 36 inorganic and organic ions in less than 3 minutes.Neutral species, of course, cannot be separated. 

The last set of experiments provides examples of the application of capillary electrophoresis. These experiments encompass a variety of different types of samples and include examples of capillary zone electrophoresis and micellar electrokinetic chromatography. 

Conte, E. D. Barry, E. E. Rubinstein, H. Determination of Caffeine in Beverages by Capillary Zone Electrophoresis, ... 

Diet soft drinks contain appreciable quantities of aspartame, benzoic acid, and caffeine. What is the expected order of elution for these compounds in a capillary zone electrophoresis separation using a pH 9.4 buffer solution, given that aspartame has pJC values of 2.964 and 7.37, benzoic acid s pfQ is 4.2, and the pfQ for caffeine is less than 0. 

CE. (sometimes CZE), capillary electrophoresis (or capillary zone electrophoresis)... 

Zone electrophoresis Zone melting Zone refining... 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

Fig. 2. Zone electrophoresis separation where S, F, S, and F are different materials.

The use of standards with samples makes zone electrophoresis particulady usehil as an analytical tool. However, when samples caimot be analyzed on the same gel, differences in the experimental conditions from experiment to experiment make direct comparison more difficult. To make comparisons from experiment to experiment, a relative mobility, is often measured by measuring the distance a component travels down the gel compared to some reference or standard component. 

Disc Electrophoresis. Resolution in zone electrophoresis depends critically on getting sample components to migrate in a focused band, thus some techniques ate employed to concentrate the sample as it migrates through the gel. The most common technique is referred to as discontinuous pH or disc electrophoresis. Disc electrophoresis employs a two-gel system, where the properties of the two gels are different. 

Most electrophoretic methods have been tried in a free-flow format, including isoelectric focusing, native zone electrophoresis, and isotachophoresis. Most free-flow electrophoresis equipment has very low (ca 1 g/(L-h)) capacity, and resolution is reduced by heating and electroosmotic considerations. 

In continuous-flow zone electrophoresis the solute mixture to be separated is injec ted continuously as a narrow source within a body of carrier fluid flowing between two electrodes. As the solute mixture passes through the transverse field, individual components migrate sideways to produce zones which can then be taken off separately downstream as purified fractions. 

FIG. 22-26 Types of arrangement for zone electrophoresis or electrochromatography. (a) Rihhon unit, with d > w cooling at side faces, (h) Block unit, with w > d cooling at electrodes. 

DETERMINATION OF POLYPHENOLIC ENANTIOMERS IN GREEN TEA EXTRACT BY CAPILLARY ZONE ELECTROPHORESIS... 

Catechin and epicatechin are two flavanols of the catechin family. They are enantiomers. The capillary zone electrophoresis (CE) methods with UV-detection were developed for quantitative determination of this flavanols in green tea extracts. For this purpose following conditions were varied mnning buffers, pH and concentration of chiral additive (P-cyclodextrin was chosen as a chiral selector). Borate buffers improve selectivity of separation because borate can make complexes with ortho-dihydroxy groups on the flavanoid nucleus. 

J. W. Jorgenson and K. D. Lukacs, Zone electrophoresis in open-tubular glass capillaries, Awa/. Chem. 53 1298 (1981). 

Electrodriven Separation Techniques encompass a wide range of analytical procedures based on several distinct physical and chemical principles, usually acting together to perform the requh ed separation. Example of electrophoretic-based techniques includes capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP), and capillary gel electrophoresis (CGE) (45-47). Some other electrodriven separation techniques are based not only on electrophoretic principles but rather on chromatographic principles as well. Examples of the latter are micellar... 

Zone electrophoresis is defined as the differential migration of a molecule having a net charge through a medium under the influence of an electric field (1). This technique was first used in the 1930s, when it was discovered that moving boundary electrophoresis yielded incomplete separations of analytes (2). The separations were incomplete due to Joule heating within the system, which caused convection which was detrimental to the separation. 

Figure 9.5 The generic setup for two-dimensional liquid chromatography-capillary zone electrophoresis as used by Jorgenson s group. The LC separation was performed in hours, while the CZE runs were on a time scale of seconds.

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, with permission from the American Chemical Society.

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