Capillary electrophoresis operation

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

Guttman, A., Cooke, N., and Star, C. M., Capillary electrophoresis separation of oligosaccharides. I. Effect of operational variables, Electrophoresis, 15,1518, 1994. 

A number of developments have increased the importance of capillary electrophoretic methods relative to pumped column methods in analysis. Interactions of analytes with the capillary wall are better understood, inspiring the development of means to minimize wall effects. Capillary electrophoresis (CE) has been standardized to the point of being useful as a routine technique. Incremental improvements in column coating techniques, buffer preparation, and injection techniques, combined with substantive advances in miniaturization and detection have potentiated rugged operation and high capacity massive parallelism in analysis. 

Altria K.D., Capillary electrophoresis without method development—using generic operating methods, LC-GC, 18(1), 33, 2000. 

K.D. Altria (ed.), Capillary Electrophoresis Guidebook Principles, Operation and Applications, Humana Press, Totowa, NJ (1996). 

In the last decade, capillary electrophoresis (CE) has become one of the most powerful and conceptually simple separation techniques for the analysis of complex mixtures. The main reasons are its high resolution, relatively short analysis times, and low operational cost when compared to high-performance liquid chromatography (HPLC). The ability to analyze ultrasmall volume samples in the picoliter-to-nanoliter ranges makes it an ideal analytical method for extremely volume-limited biological microenvironments. 

Swedberg (1997), Fernandes and Flick (1998) and Frazier et al. (2000a) have described the principles and some applications of CE. A recent review by Kok (2000) describes capillary electrophoresis instrumentation and operation. The major formats of CE are ... 

Apart from the qualification dossiers provided by vendors there seems, at present, to be very little information published on the performance of an operational qualification for capillary electrophoresis (CE) instruments other than a chapter in Analytical Method Validation and Instrument Performance. The chapter, written by Nichole E. Baryla of Eli Lilly Canada, Inc, discusses the various functions (injection, separation, and detection) within the instrument and provides guidance on the type of tests, including suggested acceptance criteria, that may be performed to ensure the correct working of the instrument. These include injection reproducibility and linearity, temperature and voltage stability, detector accuracy, linearity, and noise. 

Mccomb, M. E., and Perreault, H. (2000). Design of a sheathless capillary electrophoresis-mass spectrometry probe for operation with a Z-Spray ionization source. Electrophoresis 21, 1354-1362. 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

The first work on pKa determination by zone electrophoresis using paper strips was described by Waldron-Edward in 1965 (15). Also, Kiso et al. in 1968 showed the relationship between pH, mobility, and p/C, using a hyperbolic tangent function (16). Unfortunately, these methods had not been widely accepted because of the manual operation and lower reproducibility of the paper electrophoresis format. The automated capillary electrophoresis (CE) instrument allows rapid and accurate pKa determination. Beckers et al. showed that thermodynamic pATt, (pATf) and absolute ionic mobility values of several monovalent weak acids were determined accurately using effective mobility and activity at two pH points (17). Cai et al. reported pKa values of two monovalent weak bases and p-aminobenzoic acid (18). Cleveland et al. established the thermodynamic pKa determination method using nonlinear regression analysis for monovalent compounds (19). We derived the general equation and applied it to multivalent compounds (20). Until then, there were many reports on pKa determination by CE for cephalosporins (21), sulfonated azo-dyes (22), ropinirole and its impurities (23), cyto-kinins (24), and so on. 

Operational procedure of capillary electrophoresis (CE) is similar to TEF. Proteins are separated in an electrical field, migrating until they reach the point where they carry zero charge. Analysis is carried out in a microcapillary tube which provides high resolution. CE can be coupled directly to a MS instrument. The superiority of CE to other analytical techniques is its high resolution. However, it is not widely used for proteomic analysis as there is no commercially available and reliable -MS instrument. 

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). 

For analytical equipment, qualification is broken down into four areas design qualification (DQ), installation qualification (IQ), operational qualification (OQ), and performance verification (PV) [2,3]. In this chapter we focus on the operational qualification of a capillary electrophoresis instrument. The tests used in the operational qualification are often used in the routine performance verification as... 

Agilent Capillary Electrophoresis System Operational Qualification/Performance Validation, Agilent Technologies, Waldbronn, Germany, 2000. 

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