Liquid-chromatographic separation and on-line bioluminescence detection of creatine kinase isoenzymes

We separated isoenzymes of creatine kinase by anion-exchange chromatography, with use of an elution of gradient containing lithium acetate (0.1 to 0.6 mol/L). A stream splitter was used to divert a 5% side stream of column effluent, which was subsequently mixed with the reagents necessary for bioluminescence assay of the separated isoenzymes. The use of the stream splitter greatly decreased the rate of consumption of reagent and, when combined with a peristaltic pumping system, permitted independent control of the side-stream flow rate. Thus both the residence interval in a delay coil in which the ATP reaction product is formed and the bioluminescence response could be increased. Bioluminescence emission was monitored in a flow-through fluorometer without use of an external light source or filters. Separation and detection of the isoenzymes of creative kinase were rapid, sensitive, and highly selective. The incremental decrease of bioluminescence response owing to inhibition by the ions in the eluent was less than 31% across the entire gradient.