Isotachophoresis

Capillary isotachophoresis was well established before the introduction of capillary electrophoresis, but was quickly overshadowed by the rapid development of the latter. Current use is limited for analytical applications [387-389] with capillary electrophoresis being preferred in most cases. Renewed interest in capillary isotachophoresis as a sample concentration and preseparation technique for capillary electrophoresis is responsible for a somewhat limited revival. The self-sharpening and concentration characteristics of capillary isotachophoresis make it more suitable than capillary electrophoresis for preparative scale separations, where single run and continuous flow instalments have been described for the isolation of milligram to gram quantities of material [392-394]. Capillary isotachophoresis is also suitable for the determination of values for effective ion mobility [395,396]. 

The stepwise migration of ions under the influence of an electric field is an important feature of capillary isotachophoresis. Should an ion diffuse out of its zone, it will experience the electric field, which is driving the zone into which it has diffused. This will cause it to either accelerate or decelerate back into its own zone, depending on the magnitude of the electric field. This effect produces a self-sharpened zone, which serves to maintain sharp zone boundaries. 

Electrolyte solutions and operating conditions for capillary isotachophoresis are selected based on a knowledge of sample properties. Table 8.8 [12,386,397]. The 

Composition of some common capillary isotachophoresis buffers 

Ammediol = 2-amino-2-methyl-1,3-propanediol HPMC = hydroxypropylmethylcellulose and OAc = acetate 

The theoretical foundations of isotachophoresis (ITP) were laid in 1897 by Kohlraust h [115], who. showed that, at a migrating Iwtundary between two salt solutions, the concentrations of ions were related to their effective mobilities (Kohlrausch autoregulating function) [116]. The term isotachophoresis underlines the most important aspect of this technique, namely the identical velocities of the sample zones at equilibrium [117]. ITP will take place when an electric field is applied to a system of electrolytes, consisting of  

1) A leading electrolyte, which must contain only one ionic. species, the leading ion L , having the same sign as the sample ions to be separated, and an effective mobility higher than that of any sample ions 

3) An intermediate zone of sample ions, having the same sign as the leading and terminating ions and intermediate mobilities 

The three zones are Juxtaposed, with the proviso that sharp boundaries must be created at the start of the experiment. The polarity of the electric field must be such that the leading ion migrates in front 

Generally, EOF is not wanted in isotachophoresis. Capillary isotachophoresis is generally not used for analytical separations, but the isotachophoretic principle is utilized for special injection techniques in CE. 

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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. 

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. 

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... 

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 9.4 General schematic illustration of the apparatus used to combine cliromatography with capillary isotachophoresis.

Yamamoto et al. also coupled gel permeation HPLC and CE in an on-line fashion in 1990, where capillary isotachophoresis was again used in the second dimension. This technique was also not comprehensive due to the loss of resolution between the techniques. It was also not particularly fast, with a 23 min CE cycle, which was repeated 90 times throughout the HPLC run (14). Volume incompatibility between HPLC and CE was one problem not addressed in this study, in which a large HPLC column was coupled to an electrophoresis capillary. 

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.

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. 

Membrane osmometry. fCapillary isotachophoresis. gFluorescence quenching. 

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 the 1970s a new analysis method was developed, referred to as isota-chophoresis [247]. Capillary isotachophoresis with conductivity detector is very useful in detergent analysis for the determination of all kinds of ionic species [248]. It is also useful for the determination of MCA in ether carboxylates. 

Shimao, K, Mathematical Simulation of Isotachophoresis Boundary Between Protein and Weak Acid, Electrophoresis 7, 297, 1986. 

CE was recently used for anthocyanin analysis because of its excellent resolution. This technique has different modes capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEE), and capillary isotachophoresis (CITP)."° CZE is the most popular method for anthocyanin... 

Milk Isotachophoresis Conductivity 2ng 40% methanol, 10 mM sodium acetate pH 4.8, 0.2% hydroxy-ceUulose (leading electrolyte), 40% methanol, 20 mM acetic acid (terminating electrolyte) Prometryne, desmetryne, terbutryne, atrazine (OH metabolites), simazine (OH metabolites) 126... 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Gysler, J., Mazereeuw, M., Helk, B., Heitzmann, M., Jaehde, U., Schunack, W., Tjaden, U.R., and van der Greef, J., Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation, /. Chromatogr. A, 841, 63, 1999. 

Gebauer, P. and Thormann, W., Isotachophoresis of proteins in uncoated open-tubular fused-silica capillaries with a simple approach for column conditioning, J. Chromatogr., 558, 423, 1991. 

Experimental non-ideality at pH extremes in isotachophoresis has been compared with theoretical models.56 The model was able to predict phenomena that are usually regarded as artifactual, including system peaks, diffuse... 

There are three main electrophoretic methods of separation (i) zone electrophoresis, where the components are separated on a basis of relative mobilities (ii) isotachophoresis, where the separation is again based on relative mobilities but where the solutes are sandwiched between leading and terminating electrolytes ... 

There are a few other analytical methods in which electrochemistry plays an essential role, such as (paper) electrophoresis, isotachophoresis, electrography and electrochromatography (according to Fujinaga) as they belong to analytical separation techniques, they are beyond the scope of this book. 

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