Automated instrumental method for on-line fraction analysis and peak deconvolution in gradient multicomponent overloaded high performance liquid chromatography

An automated method for deconvolution of overloaded band profiles in gradient elution is described. The instrumental set-up consists of a pseudo-bidimensional HPLC system, where overloaded band profiles generated in the first direction are sampled on the second one. The method, previously employed under isocratic conditions, has been now extended to gradient elution, where one has to face the problem of band compression (and possibly band interference) during gradient, which decreases the time-window available along the first direction for sampling the overloaded profiles. The effect of the gradient steepness on the problem of defining a minimum number of sampling points to reconstruct single component bands from overloaded profiles is investigated. This approach is particularly useful in the framework of using inverse method (IM) to determine adsorption isotherm parameters for preparative gradient elution. In fact, it allows for the gathering of the information necessary to run IM calculations with minimum efford, also in cases where the individual component forming the mixture have different UV spectra.