Capillary electrophoresis optimization methods

This chapter discusses general considerations for improving capillary electrophoresis (CE) method performance from a robustness angle. Several method parameters are discussed and examples are offered of how CE procedures are handled to obtain optimal performance. The purpose of this chapter is to raise the awareness and help the development of more robust and sensitive methods. 

Persson Stubberud, K., and Astrom, O. (1998). Separation of ibuprofen, codeine phosphate, their degradation products and impurities by capillary electrophoresis I. Method development and optimization with fractional factorial design. ]. Chromatogr. A 798, 307—314. 

Madej et al. [76] used a non-aqueous capillary electrophoresis (NACE) method for the screening and quantification of seven phenothiazine derivatives in blood. The optimal medium for dissolving the examined blood extracts was tested. The linear dynamic ranges were between 0.25 and 4.00 xg/mL (correlation coefficients higher than 0.996), the RSD values going from 1.21 to 9.15%, according to the compound. The detection limits were 0.08 xg/mL for promazine and 0.15 pg/mL for the rest of the drugs under study. Finally, the proposed method was applied to two forensic blood samples, and concentrations of the examined phenothiazines determined by the HPLC and NACE methods were found to be comparable. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Analytical methods are ripe for attack using Al methods. Capillary electrophoresis is a routine separation technique, but like other separation techniques, its effectiveness is correlated strongly with experimental conditions. Hence it is important to optimize experimental conditions to achieve the maximum degree of separation. Zhang and co-workers41 studied the separation of mixtures in reserpine tablets, in which vitamin B1 and dibazolum may be incompletely separated, as may promethazine hydrochloride and chloroquine... 

Jimidar, M.I., Vennekens, T., Van Ael, W., Redlich, D., De Smef M. Optimization and validation of an enantioselective method for a chiral drug with eight stereo-isomers in capillary electrophoresis. Electrophoresis 2004, 25, 2876-2884. 

The origins, principles, methods, and modes of capillary electrophoresis (CE) are discussed. Massive application of electrophoresis methods started after Tiselius s moving boundary method that was optimized by the use of paper or a gel as a semiconducting medium. The applications of paper and gel electrophoresis were situated mostly in the biochemical environment for the analysis of proteins, amino... 

Salas-Solano, O., Tomlinson, B., Du, S. B., Parker, M., Strahan, A., and Ma, S. (2006). Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies. Anal. Chem. 78(18), 6583-6594. 

McClean, S., O Kane, E. J., and Smyth, W. E (2000). The identification and determination of selected 1,4-benzodiazepines by an optimized capillary electrophoresis-electrospray mass spectro-metric method. Electrophoresis 21, 1381 — 1389. 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

Musenga et al. [55] described a capillary electrophoresis method for determination of vigabatrin in human plasma after precolumn derivatization with 6-carbox yfluorescein-N-s ucc i n i m i d i d yl ester. Optimal separation and detection were obtained with 50 mM borate buffer (pH 9.0) containing 100 mM N-methylglucamine with laser-induced fluorescence detector (Aexc = 488 nm). The assay was rectilinear over the concentration... 

The apparent differences in identified photoproducts between marine and freshwater systems may be due to fundamental differences in DOM composition (see Chapters 3 and 5) or to differences in analytical approaches (e.g., capillary electrophoresis has often been the method of choice to identify DOM photoproducts in freshwater systems, but this method is not appropriate for high-salinity marine samples Table I). The fact that most of the labile photoproducts found only in freshwater environments have been identified by more than one analytical approach, however, suggests that methods alone cannot explain the 14 nonoverlapping photoproducts. On the other hand, studies conducted by the same researcher(s) tend to report the same suite of compounds, even across marine/freshwater boundaries (e.g., Kieber et al., 1990), suggesting that optimization of the analytical approach and/or researcher focus may be influencing the data. More studies identifying DOM photoproducts have been conducted in freshwater environments than in marine environments (12 vs. 6), a factor that is also likely to influence the number of reported photoproducts. At this point, evidence is insufficient to determine whether DOM photoproducts that are currently unique to either marine or freshwater environments can be attributed to inherent DOM compositional differences or to analytical approach. 

Mass spectrometry is a valuble tool with which an abundancy of structural information may be obtained from a minute amount of material. Capillary electrophoresis may be interfaced with mass spectrometry by electrospray ionization [124-126] or continuous-flow, fast-atom bombardment methods [127,128]. Several reviews discuss applications of the interfacing techniques, and address the attributes and disadvantages associated with these methods [129,130]. Critical parameters involved in the optimization of CE-electrospray ionization mass spectrometry analysis have been reviewed as well [131],... 

The use of CE methods for routine quality control of synthetic or recombinant peptides-proteins necessitates optimization strategies for rapid method development. Ideally, the methods should be simple, fast, and robust. Because capillary electrophoresis in the zone format is the most simplistic, initial efforts should be directed toward the use of a simple buffer system [61]. The high efficiency and reproducibility in protein-pep-tide separations demands that interactions between the analyte and capillary wall be neglible. The use of low-pH buffers generally results in enhanced reproduciblity, and hence ruggedness, as slight variations in the capillary surface will have little impact on the already suppressed EOF. 

Siouffi AM, Phan-Tan-Luu R. Optimization methods in chromatography and capillary electrophoresis. J Chromatogr A 2000 892 75. 

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