Separation conditions electrophoresis

Figure 17 SDS-capillary gel electrophoresis of protein standards. Separation conditions 27 cm x 50 pm i.d. uncoated capillary 888 V/cm gel eCAP 200. (From Bene-dek, K. and Guttman, A., ]. Chromatogr., 680, 375, 1994. With permission.)...

This instrumentation has exquisite sensitivity, which allows the analysis of single cancer cells (Hu et al., 2004). Our earlier work employed slow separation conditions and a rather primitive photodetection system. Our current system takes roughly 1 h to complete the two-dimensional capillary electrophoresis separation and employs state-of-the-art photodetectors. 

Chip-based enantioseparations using an electrophoresis principle were presented by Gao et al. [59]. They used mono-, two-, and four-channel chips to develop chiral separations of fluorescein isothiocyanate (FlTC)-labeled basic compounds. To obtain the chiral separations, seven neutral CD were screened (i.e., a-CD, fi-CD, y-CD, HP-a-CD, HP-y-CD, and DM-fi-CD). Using the monochannel chip, the seven selectors were screened sequentially. Using the two-channel chip, between-channel repeatability could be demonstrated using the same separation conditions. Using two different selectors in the channels, the analysis time for the screening of the seven CD can be reduced to half, compared to the time needed in the monochannel... 

Jimidar, M., Bourguignon, B., and Massart, D. L. (1996). Application of Derringer s desirability function for the selection of optimum separation conditions in capillary zone electrophoresis. J. Chromatogr. A 740(1), 109-117. 

Tomlinson, A. J., Benson, L. M., Gorrod, J. W., and Naylor, S. (1994). Investigation of the in vitro metabolism of the H2-antagonist mifentidine by online capillary electrophoresis-mass spectrometry using non-aqueous separation conditions. /. Chromatogr. B 657, 373-381. 

A determination of PQQ by capillary zone electrophoresis was also developed <2000JCH(739)101>. The optimal separation conditions were a 50mM /3-alanine HCl pH 3.0 buffer, an applied voltage of 25 kV (negative polarity), and a temperature of 25 °C. The linear detection range for concentration versus peak area is that this assay is from 5 to 500 mM with a detection limit of 0.1-0.2 mM. 

Transfer of proteins onto nitrocellulose or PVDF membranes is usually performed after separation of complex protein mixtures by polyacrylamide gel electrophoresis (PAGE). Proteins can be separated on the basis of their molecular weight or isoelectric point, under reducing or nonreducing conditions, and it is therefore for the investigator to establish the most appropriate separation conditions for particular samples. Full details of PAGE can be found in refs. 4 and 5, and the reader is encouraged to use these as sources of further details... 

TABLE 2. Experimental electrophoresis protein separation conditions using membranes prepared by reacting polyvinyl alcohol having a M of roughly 20,000 daltons 97.5% to 99.5% hydrolyzed and selected crosslinking agents. 

Chang, H.-T. Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions. Electrophoresis 2001,1 (11), 2281-2290. 13. 

The cationic analyte of Problem 33-14 was separated by capillary zone electrophoresis in a 50.0-cm capillary at 10.0 kV. Under the separation conditions, the electroosmotic flow rate was 0.85 mm s toward the cathode. If the detector were placed 40.0 cm from the injection end of the capillary, how long would it take in minutes for the analyte cation to reach the detector after the field is applied ... 

One of the most important variables, as with many techniques, is the state of the sample to be analysed. Questions which should be considered prior to CE analysis include What is the sample dissolved in What are the known or likely impurities/additives present How might these impurities affect electrophoresis results Are sample modifications possible under the separation conditions employed (e.g. pH or temperature-sensitive compounds) Could aggregation or precipitation be a problem What is the concentration of the sample - does it need to be diluted or concentrated Finally it is important to consider any known properties of the samples to be analysed, for example charge, spectral absorption and adsorption characteristics, as these will aid in the setting of some of the other variables for the analysis. 

FIGURE 4 Indirect laser-induced fluorescence detection of 19 amino acids by microchip electrophoresis. Separation buffer 1.0 mM sodium carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH at pH 10.3. Separation condition /eff 5.5 cm, 15 s injection at 417 V/cm (reversed polarity), 183 V/cm separation voltage, sample amino acid concentrations of 0.4 mM in 1.0 mM sodium carbonate and 0.2 mM CTAOH. Reprinted with permission from [66]. Copyright 2000, The American Chemical Society. 

Internal markers or standard proteins have most often been used by researchers to assure themselves that separations by electrophoresis are consistent and reproducible (21). In addition, charge markers In Isoelectric focusing are also used, although standard preparations of these materials are less reliable, especially under denaturing conditions. Other Internal standards that are often reported are materials for assuring radlolabel activity, enzyme activity, or other materials to monitor repeatability of measurements. In all cases, the stability of the standard material Is the key to long-term quality control. Large, batches of standards that are reproducible from lot to lot would also be useful. 

The following tables provide examples of buffer systems, some containing additives or using surface-modified capillaries, that have provided successful separation conditions for the select analytes. Culled from the capillary electrophoresis (CE) literature, this list is not comprehensive, but is... 

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