Computerized Interpretation of H P L C Chromatogramms by Means of Absorbance Ratio Method and Derivative Spectroscopy

Abstract

As in other chromatographic processes it is the aim of high pressure liquid chromatography (HPLC) to identify the components of a mixture, whereby there may be some information on the class of substances in question. The analytical procedure can shortly be desribed as follows. The mixture is injected in a mobile phase (solvent) and passes a column where the separation takes place. The separated components leave the column at different retention times. A following detection device generates a signal as a function of concentration (chromatographic peak). Usually the components are identified by relating retention times of external standards to the times of occurence of chromatographic peaks. A System for the computer aided evaluation of such chromatograms was developed by the authors (1). That system was designed in such a way that all components of the mixture to be analyzed are supposed to separate completely, e.g. only chromatograms with distinct peaks were allowed, while overlapping peaks are rejected by a checking algorithm. An inevitable shortcome of this algorithm is, that total overlapping of peaks — which result in a single peak (with undetectable shoulders) — cannot be recognized, so that it was the responsibility of the HPLC- operator to ensure the absence of such envents.