Protein conformational changes (denaturation) under chromatographic conditions.
Some proteins have very sensitive structure which can be disturbed during purification or analysis. In most cases the reason for these conformational changes are the interactions of the protein with the chromatographic surface. The surface and mobile phase induced conformation changes frequently occuring, however one not always recognizes the change. The various conformations, from a separation point of view, should be considered as new species of the sample mixture and as such they are visualized as new peaks.
Proteins, which are apperently pure, often demostrate odd peak shapes or multiple peaks. The multiple peak phenomena of pure proteins originates from the kinetics of unforlding-refolding equilibrium. When the kinetics of conformational equilibrium is much slower or much faster than the time scale of chromatography, only one peak, either the native or the denatured can be observed. When the conformational equilibrium is on the time scale of chromatography, we can see multiple peaks or odd peak shapes.
Our experience is that this phenomena occurs with most proteins but it is rearly recognized. However in some cases it could create confusion. We are studying these types of surface induced conformational changes for over two decades and it is clear that HPLC conditions are excellent to study surface induced conformational events, which are frequently occuring during the formulation of protein and peptide based biopharmaceuticals.