Calibration of a detector for nonlinear chromatography

In many studies of nonlinear or preparative chromatography, chromatographic signals must be recorded for relatively concentrated solutions and the detectors, that are designed for analytical applications and are highly sensitive, must be used under such experimental conditions that their responses are often nonlinear. Then, a calibration curve is needed to derive the actual concentration profiles of the eluates from the measured detector response. It becomes necessary to derive a relationship between the concentration of the eluent and the detector signal at any given time. The simplest approach consists in preparing a series of solutions of known concentrations and in flushing them successively through the detector cell, recording the height of the plateau response obtained. However, this method requires relatively large amounts of the pure solutes being studied and these are not always available or they may be most costly, although these solutions can be recovered. We describe and validate an alternative procedure providing this calibration from a series of peaks recorded upon the injection of increasingly large pulses of the studied compound.