Electrophoresis retention mechanism

Capillary electrophoresis (CE) provided an orthogonal separation technique. The retention mechanism can be manipulated with buffers or addition of surfactants to form micelles for the analysis where size and charge differences... 

Enantioresolution in capillary electrophoresis (CE) is typically achieved with the help of chiral additives dissolved in the background electrolyte. A number of low as well as high molecular weight compounds such as proteins, antibiotics, crown ethers, and cyclodextrins have already been tested and optimized. Since the mechanism of retention and resolution remains ambiguous, the selection of an additive best suited for the specific separation relies on the one-at-a-time testing of each individual compound, a tedious process at best. Obviously, the use of a mixed library of chiral additives combined with an efficient deconvolution strategy has the potential to accelerate this selection. 

A constant observation when the MRP were separated by various methods was that antioxidative effect was found in many different fractions. Both the dialysates and the retentates from dialysis were antioxidative to some extent. All the electrophoresis fractions exhibited some antioxidative effect. Attempts to separate the MRP by column chromatography on Sephadex G-50 have resulted in several fractions with some antioxidative effect, and so on. This indicates that several antioxidative products are formed by the Maillard reaction, possibly differing in molecular size and chemical structure, but perhaps with one single antioxidative functional group in common, such as a free radical function. However, it can not be excluded that the MRP contain a few entirely different antioxidants with different modes of action. Various mechanisms have also been suggested. Eichner (6) claimed MRP to inactivate the hydroperoxides formed by the lipid oxidation. There are also reports on the complex binding of metals by MRP (18, 19). 

There are a few relatively simple ways to increase retention times. If they fail, consider another separation mechanism, such as ion exchange or ion exclusion chromatography or move to capillary electrophoresis or capillary electrochromatography. 

Isocitrate dehydrogenases have been studied in extreme thermophiles mainly from the point of view of elucidation of mechanisms of thermostability. This has included the studies of Hibino et al. [32] on the enzyme from a Bacillus stearothermophilus strain grown at 65 °C. In the presence of substrate the enzyme was markedly stabilized as judged by retention of tertiary conformation, reflected by the dramatically improved resistance to denaturation by urea. The thermostability of the partially purified isodtrate dehydrogenase from Thermus aquaticus was shown to be dependent upon the buffer used and on the addition of isodtrate [135]. This enzyme has also been purified from Thermus thermophilus HB8 [136]. The enzyme had a dimeric structure with identical 57.5 kDa subunits as judged by SDS-polyacrylamide gel electrophoresis, whilst gel filtration of the native enzyme on two different matrices gave values of 95 kDa and 120 kDa. The enzyme had a pH optimum of 7.8, required a divalent metal ion for activity (Mn " > Mg " ) and was NADP dependent, although NAD" was shown to replace NADP" with low efficiency. 

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