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

Complete resolution of multi-component mixtures is achieved following a sin gle RP-HPLC run. The resolution is generally limited both by peak broadenin g, which occurs due to an overloading of the column, and by the limited num ber of fractions which can be collected. In order to take full advantage of the separation capacity of the gradient, each fraction is often individual ly submitted to a second chromatographic run under the same conditions. Sta rting from a complex mixture from which a large number of compounds are exp ected, this conventional process implies multiple parallel chromatographic steps and rapidly generates a large number of samples resulting in consider able time-consuming manipulations and an increase of errors. To overcome th ese major drawbacks we propose an original strategy which relies upon the r ational 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-number ed fractions, respectively, each fraction is removed from its closest neigh bors. Each group is then submitted to an additional fractionation under the same chromatographic conditions as used previously. The separation of toxi ns from Dendroaspis viridis snake venom was performed according to this str ategy. (C) 1999 Elsevier Science B.V. All rights reserved.