Capillary Isoelectric Focusing CIEF

CIEF is the replication in the capillary of slab gel isoelectric focusing, a widely used separation mode, particularly suitable for protein separation. In isoelectric focusing, substances are separated by applying an electric field in a buffer system forming a pH gradient Analytes focus where the buffer local pH equals their isoelectric points. 

Isoelectric focusing is a steady-state technique, and its application in capillaries requires the partial or total supression of electroosmotic flow. Capillaries used for CIEF can be derivatized with methylcellulose or linear polyacrylamide, to block the surface silanol groups, whose ionization is responsible for electroosmotic 

A technique called salt mobilization may be used to elute the focused protein bands. The field is turned off, and one of the reservoir solutions is changed to a solution containing acid (or base) plus a different cation (or anion) than H+ (or OH ). When the electric field is turned on again, electroneutrality causes a deficiency of either H+ or OH to enter the capillary. The result is that the entire pH gradient moves toward the cathode if OH- is deficient, or toward the anode if H+ is deficient, and the focused pattern is eluted through a detector. 

This method has been tested with a variety of covalently modified silica capillaries. It has been found that the derivatized capillaries tend to be unstable if pH values 7.5 are used, and are especially unstable near the cathodic ends. 

An alternative method, recently proposed, involves the use of modifiers in the running buffers, where the modifiers interact with the capillary walls to reduce, but not eliminate, electroosmotic flow. A continuous slow migration toward the detector thus occurs throughout the run. Using 0.1% methylcellulose as a modifier, species having pi values of 0.01 pH unit can be separated. 

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Capillary isoelectric focusing (CIEF) is suitable for the separation of amphoteric analyses in a pH gradient. A continuous pH gradient is built up in the column by using ampholytes under a potential field. Amphoteric analyses migrate to the point where their net charge equal to zero and they form stationary and sharply focused zones. 

Currently, there are five major modes of operation of CE capillary zone electrophoresis (CZE), also referred to as free solution or free flow capillary electrophoresis micellar electrokinetic chromatography (MEKC) capillary gel electrophoresis (CGE) capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP). Of these, the most commonly utilized capillary techniques are CZE and MEKC (Rabel and Stobaugh 1993 Issaq 1999 Smyth and McClean 1998). 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

Schmitt, R, Garrison, A.W., Freitag, D., and Kettrup, A. (1997a). Capillary isoelectric focusing (CIEF) for the characterization of humic substances. Water Res. 31, 2037-2049. 

The second separation mechanism is found in capillary isoelectric focusing (cIEF), where analytes are separated on the basis of isoelectric points. The third mechanism is found in capillary isotachophoresis (cITP), where all of the solutes travel at the same velocity through the capillary but are... 

Capillary isoelectric focusing (cIEF) is used to separate zwitterionic compounds, such as peptides and proteins, on the basis of isoelectric point. The separation occurs as a result of a pH gradient that is established inside the capillary. It is important in cIEF to use coated capillaries to reduce or eliminate the EOF and to minimize solute adsorption if the EOF is too swift, it could sweep the analytes past the detector before the separation is complete. 

Capillary electrophoresis (CE) with ICP-MS detection is a technique that first started to receive attention in 1995 [100-102]. There are several modes of CE, including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary isoelectric focusing (CIEF), and capillary isotacophoresis. Capillary zone electrophoresis is the most common mode of CE to be used with ICP-MS detection to date, although coupling of other modes with ICP-MS may have significant potential. 

Amphoteric — a substance that can act both as an acid and a base - acid-base theories, subentry - base-antibase concept, -r ampholyte, - Bjerrum, Niels Jan-niksen, - capillary isoelectric focusing (CIEF), -+ metals. 

Capillary isoelectric focusing (CIEF) — An electrophoretic separation technique (- electrophoresis) for the separation of amphoteric analytes (-> ampholyte) according to their - isoelectric points by the application of an electric field along a pH gradient formed in a capillary. 

Capillary isoelectric focusing (CIEF) separates proteins based on differences in their isoelectric points. CIEF has been used to separate product-related impurities of recombinant proteins, mainly deamidated species, such as those of human growth hormone.71... 

Capillary isoelectric focusing (CIEF) is one of the separation techniques with the highest resolution power. Since the first experiments performed by Hjerten and co-workers in the mid-1980s [1-3], hundreds of papers have appeared about methodological aspects and utilization. 

The next four chapters discuss the basic principles underlying operation and method development of the most common electrodriven analytical techniques CE, capillary isoelectric focusing (cIEF), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE). Weinberger presents a comprehensive approach for method development in CE with an emphasis on small-molecule applications. This is followed by Kilar s chapter describing the principles of and method development in cIEF, as well as recent innovations... 

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