The role of short microbore high-performance liquid chromatography columns for protein separation and trace enrichment

The use of short microbore columns (7.5 cm × 2.1 or 1.0 mm I.D.) for the separation of proteins is described. The loading capacity for protein standards of such columns has been investigated and shown to be compatible with the purification of sufficient amounts of protein in a form directly suitable for automated amino acid sequence analyses (pmole or nmole levels). The trace enrichment potential of these columns for proteins is demonstrated by the ability to load large sample volumes (greater than 2 ml) recovered from analytical (4.6 mm I.D.) high-performance liquid chromatography columns onto the microbore columns and recover the protein quantitatively in eluent volumes as small as 25 μl. In addition it is shown how the increased levels of detectability (ca. five-fold for the 2.1 mm I.D. column), arising from the increased solute concentration, may be used to quantitate ng quantities of high specific activity radioiodinated proteins.