Capillary electrophoresis separation parameters

Capillary electrophoresis (CE) has become a valuable technique in the analytical toolbox for pharmaceutical analysts. CE methods have been successfully applied for identification, assay, purity determination, and chiral separation. ICH guidelines should be followed in meeting regulatory approval if CE methods are used in a registration dossier. Here, the validation parameters required for different analytical procedures are described and a comprehensive overview of CE validation studies presented in literature is given. 

Capillary electrochromatography (CEC) is a miniaturized separation technique that combines aspects of both interactive chromatography and capillary electrophoresis. In this chapter, the theory of CEC and the factors affecting separation such as the stationary phase and mobile phase parameters have been discussed. The chapter focuses on the types and preparation of columns for CEC and describes the progress made in the development of open-tubular, particle-packed, and monolithic columns. The detection techniques in CEC such as the traditional UV detection and improvements made in coupling with more sensitive detectors such as mass spectrometry are also described. The chapter provides a summary of some applications of CEC in the analysis of pharmaceuticals and biotechnology products. 

Rathore, A. S., and Horvath, C. S. (1996). Separation parameters via virtual migration distances in high-performance liquid chromatography, capillary zone electrophoresis and electrokinetic chromatography /. Chromatogr. A 743, 231-246. 

JL Beckers, FM Everaerts, MT Ackermans. Determination of absolute mobilities, pK values, and separation numbers by capillary electrophoresis. Effective mobility as a parameter for screening. J. Chromatogr. 537 407-428 (1991). 

SG Penn, ET Bergstrom, DM Goodall, JS Loran. Capillary electrophoresis with chiral selectors. Optimization of separation and determination of thermodynamic parameters for binding of ticonazole enantiomers to cyclodextrins. Anal Chem 66 2866-2873, 1994. 

Capillary electrophoresis offers a set of important advantages that make it a premier technique for the investigation of enantioselective effects in the affinity interactions between chiral drugs and cyclodextrins. The most important advantage of CE is the inherently high separation efficiency offered by this technique. As already known, the most important contributors to peak resolution (R) are a separation selectivity (a) and an efficiency (N). A relationship between these parameters in CE is described by the following equation (2) ... 

Capillary electrophoresis (CE) is the most rapidly expanding separation technique in pharmaceutical analysis and is a rival to HPLC in its general applicability. The instrumentation is quite straightforward, apart from the high voltages required, but the parameters involved in optimising the technique to produce separation are more complex than those involved in HPLC. The technique is preferred to HPLC where highly. selective separation is required. 

K. Wang and X.-H. Xia, Microchannel-electrode alignment and separation parameters comparison in microchip capillary electrophoresis by scanning electrochemical microscopy, J. Chromatogr. A, 1110 (2006) 222-226. 

A) Design, integration, and alignment of an amperometric detector based on a metal-wire working electrode in a capillary electrophoresis microchip. (B) Evaluation of the amperometric detector and separation/ injection performance. (C) Determination of different parameters for the separation of L-ascorbic acid and hydrogen peroxide using PMMA and Topas CE-microchips. 

E. Rohde, A. J. Tomlinson, D. H. Johnson and S. Naylor, Protein analysis by membrane preconcentration-capillary electrophoresis systematic evaluation of parameters affecting preconcentration and separation , 7. Chromatogr. B 713 301-311 (1998). 

Jorgenson and Lukacs [8] have described a number of parameters, which may be helpful in characterizing CE separations. The linear velocity (v) of a given analyte in free-solution capillary electrophoresis may be represented by the following equation ... 

The key operational parameter in free-solution capillary electrophoresis is the pH of the running buffer, as the electroosmotic flow and ionization of the analyte can be regulated by this variable. The role of buffers in capillary electrophoresis has been discussed in detail, with emphasis on buffer concentration, buffer type, and pH effects [10]. The effect of organic solvents on separation and migration behavior has been studied for dipeptides [11] and somatostatin analog peptides [12]. The order of migration as well as the selectivity may be manipulated by organic modifiers in... 

The use of mass spectrometry (MS) as a detection system is inevitable in the evolution of any separation method, especially CE where the liquid flow rate ( 1 ml/min) is compatible with conventional mass spectrometers. The combination of a high-efficiency liquid-phase separation technique, such as capillary electrophoresis, with MS detection provides a powerful system for the analysis of complex mixtures. Analyte sensitivity and the mass spectrum obtained depend on the electrospray ionization (ESI) voltage, ion-focusing parameters, and buffer composition. In general, the greatest sensitivity is obtained by employing conditions that facilitate desolvation and minimize cluster formation.47 Three ways of interfacing for CE-MS... 

The same authors also applied capillary electrophoresis to the study of benazepril hydrochloride and several angiotensin-converting enzyme inhibitors [43]. Separation of the compounds was performed by means of two phosphate buffers (each 0.1 M) at pH 7 and 6.25, respectively [42], Due to the highest selectivity of the first mentioned running buffer, the same system has been applied for the quantification of benazepril and other compounds in their corresponding pharmaceutical formulations. It was found that the possibility of simultaneous identification and quantification of the active ingredient in the finished products was especially attractive, and that excipients do not adversely affect the results. This article deals with the validation of some parameters of the quantitative analysis, namely linearity, precision, accuracy, and robustness [43],... 

The solution contained within the capillary in which the separation occurs is known as the background electrolyte (BGE), carrier electrolyte, or, simply, the buffer. The BGE always contains a buffer because pH control is the most important parameter in electrophoresis. The pH may affect the charge and thus the mobility of an ionizable solute. The electro-osmotic flow (EOF) is also affected by the buffer pH. Table 1 contains a list of buffers that may prove useful in high-performance capillary electrophoresis (HPCE). As will be seen later, only a few of these buffers are necessary for most separations. 

Issaq, H.J. Atamna, I.Z. Muschik, G.M. Janini, G.M. The effect of electric field strength, buffer t)fpe and concentration on separation parameters in capillary zone electrophoresis. Chromatographia 1991, 32, 155-161. 

Capillary electrochromatography is a more complicated system than CE and HPLC due to the combination of both electrophoretic and chromatographic transport mechanisms. It is difficult to define an effective selectivity (separation factor) as in the case of general chromatography or general electrophoresis. To better illustrate the interactions that control selectivity, we defined a relative selectivity a,. =. tjt 2), and postulated a model that illustrates the effect of separation parameters on the enantioselectivity [10]. 

Although not a major separation parameter, as in gas chromatography, the operating temperature clearly affects migration behavior in capillary electrophoresis (CE). This alone should be reason to work under uniform, well-thermostated conditions. The temperature affects bulk viscosity, electro-osmotic flow, ionic mobilities, even pK values and buffer pH. 

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