Organic substrate extraction, dissolved

It should be noted also that the redox couple may be solution-soluble or may be immobilized within an electrode coating and that, in the former case, the chemical step may be carried out (1) with the organic substrate dissolved within the electrolyte (2) at the interface between the electrolyte and the substrate present as a separate phase or dissolved in a solvent immiscible with the electrolyte (3) within a solvent immiscible with the electrolyte, the redox reagent in its active oxidation state being extracted into the other solvent (maybe by phase-transfer catalysis) and (4) at the interface between the electrolyte and the organic substrate present as a solid. 

Boy et al. [79] used lyotropic liquid crystals (LC) for the immobilization of HfeHNL. The solid LC phase is not used because of the high viscosity. Therefore, the LC is used in a biphasic system consisting of the LC and an organic solvent. Such biphasic liquid crystal systems consist of organic solvent, water, and surfactant, where poorly soluble substrates and products are dissolved in the organic solvent and the liquid crystal matrix, which contains the enzyme, has a protective effect on it. By optimization and by virtue of the immobilization, it is possible to establish an extractive continuous process [79]. 

The standard reaction mixture contained 0.1 mL of 10 mM substrate dissolved in acetonitrile, 200 fiM NADPH, 0.1 mL of cytosol, and 10 mg/mL bovine serum albumin in 0.1 M potassium phosphate buffer (pH 6.2). The final volume was 2.0 mL. The reaction was continued at 25°C for 10 minutes before being terminated by the addition of 0.2 mL of 2 M HC1. The product was extracted twice with 5 mL of ethyl acetate in the presence of 50 nmol of 3-acetyl-7-dimethylaminocoumarin as the internal standard. After centrifugation, the organic layer was evaporated in vacuo. The residue was taken up in 1.0 mL of the mobile phase and a 5 /xL aliquot was used for HPLC analysis. The formation of both enantiomers was linear with time for up to 30 minutes. The rate of formation of the S enantiomer was about ninefold higher than that of the R enantiomer. 

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