Electrochromatography

Capillary electrochromatography (CEC) combines some aspects of mobile phase control and separation by capillary electrophoresis with the wider range of separation mech- 

An apparent retention factor in capillary electrochromatography can be defined in the usual way as kcEC = (Ir - teo) / teo. In this case, the retention factor only retains the same physical interpretation as the chromatographic retention factor (section 1.4) for neutral 

Guide for slurry packing fused-silica capillary columns for capillary electrochromatography (i) Forming the bed-retaining frit 

The end of the capillary column is tamped into a paste containing 3- xm silica and 25 % sodium silicate solution (50 mg of silica to 60 p.1 of silicate solution). A plug of about 0.5 mm is formed by sintering the mixture with a ring heater (section 8.4.2.1). The newly formed frit is tested for pressure stability and permeability before proceeding to the packing step. 

Capillary and slurry reservoir are placed in an ultrasonic bath and packed under conditions similar to those described below  

It is not only possible to force the mobile phase through a capillary or column by means of a pump but also by electroendosmosis. Thereby one utilizes the fact that an electric double layer occurs on all boundary layers. Silica or quartz glass are surfaces covered with fixed negative excess charges and a solution in contact with it forms positive boundary charges. If a potential gradient of approximately 50 kV m is applied the solution flows in the direction of the negative electrode. 

Electrophoresis on paper using a single buffer was applied to bile acid separation by Biserte et al. (41). The buffer system was made up of 30 ml of pyridine and 100 ml of glacial acetic acid in 5 liters of water. The pW was 3.9. In this system, cholic, glycocholic, taurocholic, and taurochenodeoxycholic acids were attracted toward the anode at rates increasing in the order named. Curiously, free deoxycholic acid remained at the origin. The locations of the bile acids on the paper were found by spraying with phosphomolybdic acid. Results with various animal biles were reported. 

The electrophoretic mobilities of C-labeled cholic, deoxycholic, and chenodeoxycholic acid and their corresponding taurine and glycine conjugates were determined by Norman (42). The paper electrophoresis was performed in barbiturate buffer of ionic strength 0.1, pH 8.6, in an electric field of 7.5 V/cm for 3 hr. When 1 pg of each acid, as the sodium salt dissolved in 25 pi of water, was applied to the paper strips, the isotope determination after electrophoresis showed broad peaks all with a mean mobility similar to that of albumin or slightly lower. The electrophoretic mobilities of all of the bile acids were influenced by the concentration in the solution applied and presented difficulties in identifying bile acids in natural extracts. The migration of bile salt-lecithin micelles on paper electrophoresis has been reported by Shimura (43). The micelles were prepared by addition of lecithin to mixed bile salts, which may have also contained cholesterol. 

Although both the gradient and the constant-pH approaches to the electrophoretic separation of bile acids could be further improved, at the present time these techniques do not seem to offer any advantages over the simpler thin-layer chromatographic methods which handle comparable amounts of bile acid. 

Because of its speed and simplicity thin-layer chromatography on silica gel is often used to follow the course of elution and separation of solutes from ion-exchange columns. It provides an excellent substitute for the paper-chromatographic monitoring of bile acids described by Gordon et al. (2). 

The detection of components on thin layers of anion exchanger is relatively simple. In case of the polyethylenimine-cellulose layers, the still-wet plate can be examined in the daylight for bright spots of hydrophobic material. By this method 5-10 ftgicm- of bile acid can be detected. Spraying with 0.05% 2,7-dichlorofluorescein in 50% methanol allows the detection of 1-2 / g/cm- of bile acid, provided care is taken to remove the water or solvents prior to the examination under the ultraviolet light (39). 

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Capillary Electrochromatography Another approach to separating neutral species is capillary electrochromatography (CEC). In this technique the capillary tubing is packed with 1.5-3-pm silica particles coated with a bonded, nonpolar stationary phase. Neutral species separate based on their ability to partition between the stationary phase and the buffer solution (which, due to electroosmotic flow, is the mobile phase). Separations are similar to the analogous HPLC separation, but without the need for high-pressure pumps, furthermore, efficiency in CEC is better than in HPLC, with shorter analysis times. 

FIG. 22-26 Types of arrangement for zone electrophoresis or electrochromatography. (a) Rihhon unit, with d > w cooling at side faces, (h) Block unit, with w > d cooling at electrodes. 

K.D. Bartle and P. Meyers, Capillary Electrochromatography, Royal Society of Chemistry Chromatography Monographs, London, 2000. ISBN 0854045309. 

Most of the criteria and features outlined above for liquid chromatography media also apply to the development of selectors for electrodriven separations such as electrophoresis and electrochromatography. 

Sander, L.C. et ah. Separation of carotenoid isomers by capillary electrochromatography with C30 stationary phases. Anal. Chem., 71, 3477, 1999. 

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... 

Pusecker K, J Schewitz, P Gfrorer, L-H Tseng, K Albert, E Bayer (1998) On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy. Anal Chem 70 3280-3285. 

Breadmore, M. C., Macka, M., and Haddad, P. R., Manipulation of separation selectivity for alkali metals and ammonium in ion-exchange capillary electrochromatography using a suspension of cation exchange particles in the electrolyte as a pseudo stationary phase, Electrophoresis, 20, 1987, 1999. 

Zhang, M., Yang, C., and Ziad, E. R., Capillary electrochromatography with novel stationary phases. 3. Retention behavior of small and large nucleic acids on octadecyl-sulfonated-silica, Anal. Chem., 71, 3277, 1999. 

Spikmans, V., Lane, S. J., Tjaden, U. R., and van der Greef, J., Automated capillary electrochromatography tandem mass spectrometry using mixed mode reversed phase ion-exchange chromatography columns, Rapid. Com-mun. Mass Spectrom., 13, 141, 1999. 

Monolithic columns, formed from the co-polymerization of divinylbenzene and vinylbenzyl chloride or styrene, were observed to be resistant to bubble formation.11 Application of pressure in electrochromatography, discussed below, also reduces bubble formation. A massively parallel detector capable of scanning up to 1000 capillaries using planar confocal fluorescence has been used for DNA sequencing.1213 Recovery of fluorescence following pho-tobleaching has been used to measure DNA mobility in agarose gel.14... 

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