A trace-enrichment technique for the loading of gel-permeation high-performance liquid chromatography columns

Abstract

Gel-permeation high-performance liquid chromatography (GP-HPLC) columns provide rapid high-resolution separations but are frequently limited to analytical tasks because the injection volumes must be small. The reduction of volume required for the loading of solutes can often be impractical and lead to poor recoveries. We have developed a trace-enrichment technique to circumvent this problem. By placing a Waters Guard Pak within the loop of a Valco injector and connecting a pump to the injection port it is possible to concentrate proteins and peptides onto the guard column from relatively large volumes. Enrichment onto a reversed-phase guard column insert is achieved by loading solutes in an aqueous solution or one of low organic solvent concentration. Provided that the GP-HPLC is mean-while equilibrated with a solvent system of sufficiently high organic solvent concentration (i.e., 40% acetonitrile containing 0.1% trifluoroacetic acid) it is possible to elute material that has been loaded in this manner by simply placing the injection loop in line with the column. The solvent strength abruptly increases and the peptide or protein sample is loaded onto the column in a very small volume. We have applied this loading principle to both analytical and semipreparative problems. The amino-terminal fragment of pro-opiomelanocortin (POMC) has been extracted from a single human fetal pituitary (18 weeks gestation) and characterized in terms of its molecular weight. This study indicated that no proteolytic processing of the amino-terminal fragment of POMC takes place at this stage in development. In a larger scale application the amino-terminal fragment of POMC was purified from bovine anterior pituitaries. The trace-enrichment GP-HPLC loading technique was used as an initial fractionation procedure. The isolation of the polypeptide by subsequent reversed-phase HPLC was greatly facilitated by the initial removal of high- and low-molecular-weight contaminants.