Electromigration Techniques

This group includes several related techniques such as classical gel electrophoresis, field-flow fractionation (FFF) and the capillary techniques capillary zone electrophoresis (CZE), capillary electrokinetic chromatography (CEKC) capillary isotachophoresis (CUP), capillary isoelectric focusing (CIEF) and capillary electrochromatography (CEC). 

Classical gel electrophoresis is well-established for the purification and preparation of charged compounds of biological importance such as peptides, nucleic acids, etc. However, it 

The challenges of capillary electromigration techniques in enantiomer analysis are obvious, and in opposition to their potential in enantiomer production [173]. 

The most important advantages of capillary electrophoresis (CE) are extremely high peak efficiency, small sample size, minute amounts of chiral selectors and buffers, low costs and less environmental problems. A capillary format leads exclusively to all these advantages. At the same time, the capillary format is a clear disadvantage from the preparative point of view. Thus, CE is an excellent technique for enantiomer analysis, but not for their preparative separation. Therefore, at this point the technique is not discussed further. 

Another capillary technique, also with significant potential but again just used for analytical enantioseparations, is CEC [173, 174]. This hybrid technique relies on electrophoretic migration and chromatographic separation principles. The enantioseparation of several chiral compounds in non-aqueous CEC using polymethacrylate-type (Chiralpak OP) packing material is shown in Fig. 10 [174]. 

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Scriba, G. K. E. (2006). Recent advances in peptide and peptidomimetic stereoisomer separation by capillary electromigration techniques. Electrophoresis 11, 222-230. 

Chankvetadze, B. (1999). Recent trends in enantioseparations using capillary electromigration techniques. Trends Anal. Chem. 18, 485—498. 

Electromigration techniques capillary electrophoresis capillary electrochromatography... 

Giibitz G, Schmid G, Chiral separation by choromatographic and electromigration techniques, A review, Biopharm. DrugDispos 22 291—336, 2001. 

B Chankvetadze, G Blaschke. Enantioseparations in capillary electromigration techniques recent developments and future trends. J Chromatogr A 906 309-363, 2001. 

A variety of microscale separation methods, performed in capillary format, employ a pool of techniqnes based on the differential migration velocities of analytes under the action of an electric field, which is referred to as capillary electromigration techniques. These separation techniques may depend on electrophoresis, the transport of charged species through a medium by an applied electric field, or may rely on electrically driven mobile phases to provide a true chromatographic separation system. Therefore, the electric field may either cause the separation mechanism or just promote the flow of a solution throughout the capillary tube, in which the separation takes place, or both. 

On-line conpling of capillary electromigration techniques with nuclear magnetic resonance spectroscopy [5] and matrix-assisted laser desorption/ionization (MALDl) time-of-flight (TOF) mass spectrometry [6] has also been reported. 

This chapter illustrates basic concepts, instrumental aspects, and modes of separation of electromigration techniques performed in capillary format. It shonld be noted that most of the fundamental and practical aspects of the electromigration techniqnes performed in capillary tubes also apply when the techniques are carried out in microchannels fabricated on plates of reduced dimensions, communally referred to as chips. 

INSTRUMENTATION FOR CAPILLARY ELECTROMIGRATION TECHNIQUES 6.3.1 Separation Unit... 

Typically, sample detection in electromigration techniques is performed by on-column detection, employing a small part of the capillary as the detection cell where a property of either the analyte, such as UV absorbance, or the solution, such as refractive index or conductivity, is monitored. This section briefly describes the major detection modalities employed in capillary electromigration techniques, which are accomplished using UV-visible absorbance, fluorescence spectroscopy, and electrochemical systems. The hyphenation of capillary electromigration techniques with spectroscopic techniques employed for identification and structural elucidation of the separated compounds is also described. 

Fluorescence detection is widely employed in electromigration techniques for samples that naturally fluoresce or are chemically modified to produce molecules containing a fluorescent tag. Indirect detection incorporating a fluorescent probe into the electrolyte solution is also employed. One of the most common fluorophores used for this purpose is fluorescein, which is a water soluble, stable, and relatively cheap compound. 

Other detection modes employed in capillary electromigration techniques include chemiluminescence [69-71], Raman spectroscopy [72,73], refractive index [74,75], photothermal absorbance [76,77], and radioisotope detection [78]. Some of these detection modes have found limited use due to their high specificity, which restricts the area of application and the analytes that can be detected, such as radioisotope and Raman-based detection that are specific for radionuclides and polarizable molecules, respectively. On the other hand, the limited use of more universal detection modes, such as refractive index, is either due to the complexity of coupling them to capillary electromigration techniques or to the possibility of detecting the analytes of interest with comparable sensitivity by one of the less problematic detection modes described above. 

The hyphenation of capillary electromigration techniques to spectroscopic techniques which, besides the identification, allow the elucidation of the chemical structure of the separated analytes, such as mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) has been widely pursued in recent years. Such approaches, combining the separation efficiency of capillary electromigration techniques and the information-rich detection capability of either MS or NMR, are emerging as essential diagnostic tools for the analysis of both low molecular weight and macromolecular compounds. 

Mass spectrometry provides detailed information regarding molecular weights and structures from extremely small quantities of materials. Several types of ionization sources can be employed for the on-line hyphenation of capillary electromigration techniques with MS, which include... 

The overall separation potential of an electromigration technique can be expressed by the peak capacity ( ), which is defined as the maximum number of peaks that can be separated within a given separation time, usually coincident with the time interval between the first and last detected peak in the electropherogram, while retaining unit resolution for all adjacent peak pairs ... 

The following subsections briefly describe the different separation modes of electromigration techniques. Most of the separation parameters utilized to evaluate performauce aud results of the differeut separation modes are common to the majority of them and have been discussed above, whereas terms and parameters related to a specific separation mode are introduced and discussed iu each specific subsectiou. 

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