Differential migration processes in analytical separation: Capillary zone electrophoresis and electrochromatography

A novel concept of virtual migration distances is proposed to facilitate the recognition of the underlying similarities as well as the expression and comparison of the various separation parameters in high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). The treatment is extended further to the analysis of capillary electrochromatography (CEC) and micellar electrokinetic chromatography (MEKC) leading to a consistent and unified description of the characteristic parameters of these separation systems. The detrimental effect of the interference of on-column reaction with the separation by displacement chromatography is studied via computer simulations. The results serve not only as caveat for conditions to be avoided but also as guidance to the selection of those with which good yield and productivity can be achieved. The scope and potential of CZE as a major high performance analytical technique for separation of complex biological substances in the field of analytical biotechnology is explored and further extended by carrying out CZE at low temperatures. The equilibrium and kinetic parameters of cis-trans interconversion of L-phenylalanyl-L-proline are investigated using computer simulations and the results are found to agree with those reported in literature and obtained from various other experimental techniques including the ad hoc dissolution method with nuclear magnetic resonance (NMR). Further, the dimensionless Damkohler number (Da) is used as a quantitative measure of the interference of the isomerization reaction with the separation by CZE and a simple and rapid method is proposed for estimation of the above mentioned kinetic parameters. The effect of addition of the various beta-cyclodextrin derivatives including 6-monodeoxy-6-monoamino-beta-cyclodextrin, carboxymethylated-beta-cyclodextrin and heptakis (2,6-di-O-methyl)-beta-cyclodextrin on the selectivity in the CZE of peptides as well as a variety of acidic and basic proteins is investigated and the results support the use of cyclodextrins as selectivity enhancers in CZE of peptides and proteins. Theories dealing with electrosmotic flow (EOF) in CEC in porous media are reviewed with particular regard to the use of packed capillaries in CEC. In view of the discontinuities of the electric field strength and the flow velocity that exist at the interface of the packed and open segments of a CEC column, a framework is offered for measuring and interpreting the relevant electrochromatographic parameters such as the electric field strength, the potential drop and the flow velocity in the packed segment of the column in CEC. It is found that in most cases a "flow equalizing intersegmental pressure", which is different from the pressures at the two ends of the column, develops at the interface of the packed and open segments and is shown to have a significant effect on the magnitude as well as the radial distribution of the flow velocity in the open segment. The properties of "duplex" columns having a pre-detection open segment between the frit and the detector window are examined with particular regard to the effect of the relative lengths of the packed and open segments on the separation of mixtures containing neutral and charged components. The results are expected to facilitate CEC method development for optimum separation.