The effect of on-column structural changes of proteins on their HPLC behavior

Whenever proteins are found in environments different from those provided by physiological conditions, structural alterations can occur which can dramatically affect their adsorption and chromatographic behavior. The resultant behavior is often kinetically controlled and thus dependent on such factors as contact time of the protein with the adsorbent surface. Examples are given of the appearance of multiple peaks from seemingly pure species, as a result of these structural changes. In one case (papain in reversed-phase LC), multiple peaks are shown to arise from different conformational states. In a second case (beta-lactoglobulin A in hydrophobic interaction chromatography), a series of three peaks is a result of self-association or aggregation. Finally, recent work on an examination of structural changes of proteins on chromatographic supports, by means of intrinsic fluorescence and HPLC, is presented. The value of these studies for the elucidation of the retention mechanism in HPLC and the assessment of purity of proteins is demonstrated.