Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Abstract

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20α-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.