Zone concentration isotachophoresis

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

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... 

Several strategies have been described for the preconcentration of sample components present at low concentrations. These techniques include zone sharpening,28-29 on-line packed columns,30 and transient capillary isotachophoresis (cITP).31-32 Other standard laboratory techniques are often used, including solid-phase extraction, protein precipitation, ultrafiltration, etc. Two important points to keep in mind when selecting a concentration protocol are the sample requirements of the method and the potential selectivity on relative concentrations of sample components. The latter point applies to purity and concentration analysis. 

The length of the analyte zone in cITP is a quantitative parameter and is related to the concentration of the analyte in that zone. The height of the step is a quantitative parameter that is characteristic of the analyte, and it is directly proportional to analyte mobility. Conductivity detection is the detection system generally used for isotachophoresis, although UV detection is employed occasionally in commercial instrumentation. Figure 5.19 shows a typical electropherogram obtained using cITP with conductivity detection. 

The use of specific detectors, such as UV-absorption or fluorescence detectors, has provided useful additional information in isotachophoresis, especially since at the steady-state the sepa-rand zone in mixed only with the counter ion. The concentration is... 

Capillary zone electrophoresis with transient isotachophoresis as the online concentration procedure was developed for the determination of iodide in seawater (Yokota et ai, 2003). The effective mobility of iodide was decreased by the addition of 10 mmol cetyltrimethylammonium... 

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. 

K. Fukushi, Y. Nakayama and J.-l. Tsujimoto, Highly sensitive capillary zone electrophoresis with artificial seawater as the background electrolyte and transient isotachophoresis as the on-line concentration procedure for simultaneous determination of nitrite and nitrate in seawater, J. Chromatogr. A, 1005, 197-205, 2003. 

Figure 10 ITP-CZE analysis of lO il of five times diluted untreated serum. (A) ITP step in the preseparation capillary, ID 0.8 mm, LE 10mmoll HCI + j3-alanine, pH 3.3, TE lOmmoll" propionic acid. Denoted components migrating behind lactic acid are driven to the second capillary. (B) CZE step in the analytical capillary, ID 0.2mm, BGE SOmmolP propionic acid + -alanine, pH 3.8. The combination of electrolytes corresponds to the T-S-T, system as shown in Figure 6. LOD 5x10 moll ascorbate. Estimated concentration of ascorbic acid in serum 1 5.74mg in serum 2 12.15mg l (Reprinted with permission from Prochazkova A, Kfivankova L, and Bocek P (1998) Quantitative trace analysis of L-ascorbic acid in human body fluids by on-line combination of capillary isotachophoresis and zone electrophoresis. Electrophoresis, vol. 10, p. 302 Wiley-VCH.)...

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 ... 

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