Investigation of the parameters for reversed-phase high-performance liquid chromatography of collagen types I and III

A detailed study was carried out on the behaviour of collagen and its subcomponents on large pore size reversed-phase high-performance liquid chromatographic columns. Investigation of laboratory-prepared C18 and CN 50-nm pore size supports using a simple solvent system showed that heat-denatured collagen chains could be retained and resolved and that the C18 packing had a greater selective capacity. However, because of inconsistencies in the chromatography obtained using laboratory-prepared supports commercial 33-nm pore size packings were subsequently studied with both pyridine-based and trifluoroacetic acid-based solvent systems. An optimised solvent was defined and used to check the resolving capacity of both C18 and CN commercial columns. These studies led to the description and interpretation of the effects of pH, counter-ion concentration, solvent strength, type of support and molecular weight of protein solutes on the chromatographic behaviour of this large protein and its CNBr peptides. An explanation of the major forces acting to bring about retention and resolution is presented and suggestions are made for the application of this methodology to other proteins.