Isoelectric focusing and isotachophoresis

Isoelectric Focusing Isoelectric focusing electrophoresis is based on a pH gradient firom anode to cathode. Such a gradient, however, is not created by the buffers of different pH. It is produced and maintained by the electric field on the synthesized materials, known as carrier ampholytes, such as Ampholines (a commerical product of LKB) or Biolytes (Bio-Rad Laboratories). Ampholine has the following stmcture  

FIGURE 13.14 U-tube apparatus for density isoelectric focusing. 

FIGURE 13.15 Disk electrophoresis apparatus for gel isoelectric focusing. Q sample. 

For protein, v should be high, while milliamperes should be low. If v is high, the velocity (mobility) is high. If miUiamperes are low, the temperature is low, and the protein will not be denatured (at 35°C, proteins are usually denatured). For nucleic acid, milliamperes should be high. At high temperatures, the strains of nucleic acid are kept separated. 

Two-Dimensional Electrophoresis Two-dimensional electrophoresis is a combination of two different electrophoretic separation procedures. The use of two separation techniques is based on the principle that they have independent parameters that is, they separate chemical species according to different properties. 

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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. 

In considering the applicability of preparative classical electrophoretic methods to chiral separations, it should be noted that practitioners in the art of classical electrophoresis have been particularly inventive in designing novel separation strategies. For instance, pH, ionic strength and density gradients have all been used. Isoelectric focusing and isotachophoresis are well-established separation modes in classical electrophoresis and are also being implemented in CE separations [7, 8]. These trends are also reflected in the preparative electrophoretic approaches discussed here. 

The separation scientist with experience gained from a LC background may tend to limit the modes of electrochromatography to reversed phase (RP), normal phase, ion-exchange and, maybe, size-exclusion. Analysts from an electrophoretic background typically use the term "CE" in a much broader sense to include the main modes of capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, isoelectric focusing and isotachophoresis. 

Bier, M. (1998). Recycling isoelectric focusing and isotachophoresis. Electrophoresis 19, 1057-1063. 

BI7. Hours, J., and Delmotte, P., Age-dependent variations in the composition of the crystalline and albuminoid of the mouse lens, studied by isoelectric focusing and isotachophoresis. In Biochemical and Biological Applications of Isotachophoresis (A. Adam and C. Schots, eds.), pp. 207-237. Elsevier, Amsterdam, 1980. 

L3. Latner, A. L., and Ernes, A. V., Further findings related to gel isoelectric focusing followed by electrophoresis. In Progress in Isoelectric Focusing and Isotachophoresis (P. G. Righetti, ed.), pp. 223-233. North-Holland Publ., Amsterdam, 1975. 

Galante, E., Caravaggio, T., and Righetti, P. G. (1975), in Progress in Isoelectric Focusing and Isotachophoresis, North-Holland Publishing Co., Amsterdam, pp. 3—12. 

Electrophoretic modes include zone, gel sieving, isoelectric focusing, and isotachophoresis. But electrophoresis has a distinct advantage over HPLC for analysis of biopolymers a vastly superior resolving power, especially in a two-dimensional format, where two separation mechanisms can be used in succession. 

Catsimpoolas N (ed.) (1973) Isoelectric Focusing and Isotachophoresis. New York New York Academy of Sciences. 

Since the techniques applied in the referred literature and in this report are somewhat onesided, they should be comple-mented in future by non-denaturing approaches, e.g. by HPLC and by more sensitive methods with a higher resolving power, like isoelectric focusing and isotachophoresis, to elucidate and explain all unidentified components of tear fluid in a satisfactory way. 

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