Hélène Gras

Alternate pooling for optimizing high-performance liquid chromatographic fractionation of complex peptide mixtures

Complete resolution of multi-component mixtures is achieved following a single RP-HPLC run. The resolution is generally limited both by peak broadening, which occurs due to an overloading of the column, and by the limited number of fractions which can be collected. In order to take full advantage of the separation capacity of the gradient, each fraction is often individually submitted to a second chromatographic run under the same conditions. Starting from a complex mixture from which a large number of compounds are expected, this conventional process implies multiple parallel chromatographic steps and rapidly generates a large number of samples resulting in considerable time-consuming manipulations and an increase of errors. To overcome these major drawbacks we propose an original strategy which relies upon the rational pooling of fractions issued from the initial chromatography in two groups, A and B. Since Group A and Group B contain the odd- and even-numbered fractions, respectively, each fraction is removed from its closest neighbors. Each group is then submitted to an additional fractionation under the same chromatographic conditions as used previously. The separation of toxins from Dendroaspis viridis snake venom was performed according to this strategy.