Alkyl-silica columns (e.g., C8, C18) are presently the most popular by far for use in analyzing samples by means of high-performance liquid chromatography (HPLC). Unfortunately, column manufacturers are currently unable to guarantee that different production batches of nominally equivalent column packing will provide the same separation of different samples, while user surveys indicate that column irreproducibility is a serious problem that can result in major delays and added costs in routine HPLC analysis. The present proposal is intended to solve this problem in two ways. First, a review and extension of previously published studies suggests that it should be possible to characterize different columns in terms of the fundamental column properties that determine sample retention and separation, by the use of a suitable standard sample (mixture of test compounds) and test procedure. It may also be possible to modify these column properties by suitable adjustments in other separation conditions (mobile phase, temperature, flow rate), so as to reduce (and hopefully eliminate) minor differences in column retention for any sample. Second, it is proposed to develop computer software that will allow the user to conveniently predict separation as a function of small, simultaneous variations in any number of experimental conditions (e.g., mobile phase pH, column temperature, etc.) The software would also automatically correct for column-to-column differences in retention by recommending suitable adjustments in separation conditions. This would minimize the impact of column irreproducibility, as well as allow unattended correction for any change in the column during use. This capability is currently needed for high-throughput applications of HPLC, as are found in combinatorial synthesis. Only uses of this new software are described in the proposal.
The proposed software and accompanying protocols can be used by (a) column manufacturers to better control column manufacture and testing and (b) chromatographers to develop HPLC methods, to troubleshoot assay procedures and to adapt procedures for column irreproducibility.
