Dual-detector-post-column reactor system for the detection of isoenzymes separated by high-performance liquid chromatography. I. Description and theory.

We describe a dual-detector-post-column chromatographic reaction detector system that corrects for substances present in biological samples that interfere with the measurement of isoenzymes separated on a chromatographic column. The response observed at the detector in front of the reaction coil is mathematically dispersed, time transformed and subtracted from the detector behind the coil to produce a blank corrected chromatogram. The same computer program calculates peak areas and other chromatographic parameters such as height equivalent to a theoretical plate and retention time. In addition, we have evaluated the dispersion effects caused by various changes in our experimental system.

Dual-detector-post-column reactor system for the detection of isoenzymes separated by high-performance liquid chromatography. II. Evaluation and application to lactate dehydrogenase isoenzymes.

We describe the separation of lactate dehydrogenase isoenzymes by high-performance liquid chromatography-anion-exchange columns and their quantitation by a computer-controlled, dual-detector post-column reaction system. The recoveries from the separation column were ca. 90%. The dynamic range of the system was linear over about three orders of magnitude from 3 to 1500 U/l. The coefficient of variation for isoenzyme peak areas was ca. 2%. The method is compared to the classical electrophoresis measurement and shows increased speed, resolution, precision and accuracy.