Isotachophoresis mixtures

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. 

A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcentration-capillary electiophoiesis-mass spectiometry (mPC-CE-MS) in conjunction with transient isotachophoresis for analysis of peptide mixtures, J. High Resolut. Chromatogr. 18 384-386(1995). 

Figure 11.18 Schematic diagram of an in-line SPE unit for CE using (a) polyester wool frits to hold the sorbent, or (b) a paiticle-loaded membrane. Reprinted from Journal of Capillary Electrophoresis, 2, A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcenti ation-capillary electrophoresis-mass spectiometi y (mPC-CE-MS) in conjunction with ti ansient isotachophoresis for analysis of peptide mixtures, pp 225-233, 1995, with permission from ISC Teclmical Publications Inc.

Capillary tube isotachophoresis using a potential gradient detector is another technique that has been applied to the analysis of alcohol sulfates, such as sodium and lithium alcohol sulfates [303]. The leading electrolyte solution is a mixture of methyl cyanate and aqueous histidine buffer containing calcium chloride. The terminating electrolyte solution is an aqueous solution of sodium octanoate. 

In addition to this qualitative aspect, isotachophoresis may also be used quantitatively. Once the sample zones have developed, the conductivity of each zone and hence the voltage drop across it will be related to the concentration of the ions and in order to maintain uniform conductivity the concentration (i.e. the volume occupied by each component) will alter. Hence at equilibrium, not only will the components of a mixture be separated from each... 

Lincomycin hydrochloride and aminoglycoside hydrochloride were determined in pharmaceuticals by Isotachophoresis. In this method, a mixture of 0.02 M potassium acetate and 0.3 % hydroxypropylmethyl cellulose (15,000) was used as an aqueous electrolyte system, whereas a mixture of 20um 1,4-aminobutyric acid in acetic acid (pH 4.72) or 0.02 M glycylglycine or B -alanine was used as the terminator. The analysis was performed at a constant current of 200 jjA at 5°C. it was found that the minimum amount that could be quantitated by this method was 1.6 nmol and the relative standard deviation was 2%. 

T2. Thorn, W., Weiland, E., and Wasmus, G., Investigation of a glycoprotein-protein mixture from urine of anaemic patients by analytical isotachophoresis. Res. Exp. Med. (Berlin) 175, 155-158 (1979). 

Clark and Kricka have reviewed High-Resolution Analytical Techniques for Proteins and Peptides and Their Applications in Clinical Chemistry and include consideration of isotachophoresis, high-performance liquid chromatography, and high-resolution two-dimensional electrophoretic techniques for separation and analysis of complex protein mixtures. These techniques are not now widely used in clinical chemistry laboratories but represent the tools of the future, when laboratories will be required to measure gene products and the myriad proteins present, as in complex biologic fluids of significance in health and diseases. 

Isotachophoresis is now a fairly advemced microsepeuratlon method predominantly used for small, ionic molecules. The use of ITP for the separation of macromolecules has been limited until recently to biopolymer applications such as the separation of peptides, the profiling of protein mixtures (1-3). and the analysis of enzymes. These applications have been reviewed by Bocek ( ) cuid Hjalmeursson and Baldesten ( ). These successful applications of ITP to biomacromolecules can be attributed in large peurt to the predominance of electroklnetic separation techniques In the chcuracterlzatlon of biochemical systems. 

Fig. 2.1. Schematic representation of the four electrophoretic methods (A) zone electrophoresis (B) moving boundary electrophoresis (C) isotachophoresis and (D) isoelectric focusing, (a) The beginning of the experiment (b) separation of a mixture of the substances.

From the analytical point of view, the concentrating effect of isotachophoresis is of great significance, it allows to analyze minute amounts of ionic components in both electrolytic and non-electrolytic sample mixtures. "The self-sharpening effect causes that the isotachophoretic zone boundaries, having once been formed remain very sharp and do not change with time. This enables even very small amounts of the substances that form very narrow zones to be analyzed. 

Dehnotte, R, Analysis of complex protein mixtures hy capillary isotachophoresis. Sci. Tools, 1977, 24 33. 

Figure 6 Longitudinal profiles of analytes in capillary isotachophoresis as calculated from eqn [8] by a computer. Leading electrolyte 20mmoll acetic acid/IOmmoll" potassium. Terminating electrolyte 20 mmol r acetic acid/IOmmoir am-mediol. Sample introduction 6 mm plug of mixture of 5 mmol U Na", 5 mmol I Li + ions, injected at position of 7 mm. Current density y=75Am . The picture shows resulting profiles at f = 650 s, when sodium and lithium are separated. They form isotachophoretic zones with virtually rectangular profiles stacked one by one.

Over the last two decades, capillary electrophoresis (CE) has been developed as a powerful separation technique for complex mixtures. Its advantages include a high separation efficiency, short analysis time, small sample requirement, and applicability to a wide range of analytes. The basic modes of CE that are presently being exploited include capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary isotachophoresis, capillary isoelectric focusing, and capillary gel electrophoresis. 

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