Capillary isotachophoresis

In the electrolyte system used in capillary isotachophoresis (cITP), the sample zone migrates between a leading electrolyte at the front and a different, trailing electrolyte at the end. The leading electrolyte contains a coion with mobility greater than that of any of the analyte ions. The trailing electrolyte contains a coion with mobility that is lower than that of any of the analyte ions. In isotachophoresis, it is possible to analyze for anions or cations, but not both simultaneously. Analyses are usually performed in the constant-current mode. 

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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... 

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. 

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. 

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... 

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). 

Isotachophoresis. In isotachophoresis (ITP), or displacement electrophoresis or multizonal electrophoresis, the sample is inserted between two different buffers (electrolytes) without electroosmotic flow. The electrolytes are chosen so that one (the leading electrolyte) has a higher mobility and the other (the trailing electrolyte) has a lower mobility than the sample ions. An electric field is applied and the ions start to migrate towards the anode (anions) or cathode (cations). The ions separate into zones (bands) determined by their mobilities, after which each band migrates at a steady-state velocity and steady-state stacking of bands is achieved. Note that in ITP, unlike ZE, there is no electroosmotic flow and cations and anions cannot be separated simultaneously. Reference 26 provides a recent example of capillary isotachophoresis/zone electrophoresis coupled with nanoflow ESI-MS. 

Y. An, J. W. Cooper, B. M. Balgley, and C. S. Lee. Selective Enrichment and Ultrasensitive Identification of Trace Peptides in Proteome Analysis Using Transient Capillary Isotachophoresis/Zone Electrophoresis Coupled with Nano-ESI-MS. Electrophoresis, 27(2006) 3599-3608. 

Stransky [255] investigated the determination in amounts down to lOpg kg-1 growth regulator Chlormequat in soil by capillary isotachophoresis. 

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 hyphenation of CE and NMR combines a powerful separation technique with an information-rich detection method. Although compared with LC-NMR, CE-NMR is still in its infancy it has the potential to impact a variety of applications in pharmaceutical, food chemistry, forensics, environmental, and natural products analysis because of the high information content and low sample requirements of this method [82-84]. In addition to standard capillary electrophoresis separations, two CE variants have become increasingly important in CE-NMR, capillary electrochromatography and capillary isotachophoresis, both of which will be described later in this section. 

Capillary Isotachophoresis (cITP) Chargeable water soluble molecules Ion chromatography... 

Stehle, P, Bahsitta, H. P., and Piirst, P. (1986). Analytical control of enzyme-catalyzed peptide-synthesis using capillary isotachophoresis.. Chromatogr. 370, 131—138. 

Khan, K., Van Schepdael, A., Saison-Behmoaras, T., Van Aerschot, A., and Hoogmartens, J. (1998). Analysis of antisense oligonucleotides by on-capillary isotachophoresis and capillary polymer sieving electrophoresis. Electrophoresis 19, 2163-2168. 

High-performance liquid chromatography-fluorometry, mass spectrometry and capillary isotachophoresis have been applied successfully to identify or quantitate tetrodotoxin in small volumes of toxin extract from pufferfish. 

Capillary isotachophoresis (CITP) is an electromigration technique, which is performed using a discontinnous buffer system, formed by a leading electrolyte (LE) and a terminating electrolyte... 

Capillary isotachophoresis is usually performed in constant current mode, which implies the invariable ratio between concentration and electrophoretic mobility of ions. Therefore, bands that are less concentrated than the LE are sharpened, whereas those that are more concentrated than the LE are broadened to adapt their concentration to the requested constant value between concentration and electrophoretic mobility. The consequence of this unique property of CITP is that each sample component can be concentrated to an extent that depends on its initial concentration and the concentration of the leading electrolyte. Therefore, the opportune selection of composition and concentration of the leading electrolyte allows the enrichment of diluted analytes. 

Klein reported the use of a quantitative and qualitative determination of procaine in pharmaceutical preparations by capillary isotachophoresis [149]. 

Fanali et al. have described a capillary isotachophoresis method for the determination of procaine in pharmaceuticals [ 150]. The drug was determined in a 6 pL sample of solution (Spofa product, obtained from Czechoslovakia, and diluted 180-fold) by cationic isotachophoresis in the single column mode. The system used a PTFE capillary column (20 cm x 0.3 mm) and a conductivity detector. The separation was carried out at room temperature, at 50 pA (but switched to 25 pA during detection). 

Kostelecka and Haller have determined procaine in mass-produced and extemporaneous pharmaceuticals by capillary isotachophoresis [152]. The method was carried out using pH 4.85 acetate buffer solution, and 0.01 M formic acid as leading and terminating electrolytes, respectively. 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

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