MULTIPLE PEAKS IN HPLC OF PROTEINS - BOVINE SERUM-ALBUMIN ELUTED IN AREVERSED-PHASE SYSTEM

Elution of a commercial sample of bovine serum albumin (BSA) in a reve rsed-phase (RP) HPLC system at room temperature gives a distorted peak . If a shallow gradient is used during elution a split peak is observe d. The nature of the several parts of this multiple peak is studied us ing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAG E), size-exclusion chromatography (SEC), amino acid analysis, re-eluti on in RP-HPLC of collected fractions, capillary electrophoresis (CE), and matrix assisted laser desorption ionization-mass spectrometry (MAL DI-MS). This study demonstrates that the split peak of BSA observed in these chromatographic conditions is due to the monomer, dimer and oth er aggregates existing in the commercial sample of the BSA used. Moreo ver, it is proved that typical RP-chromatographic conditions do not ca use aggregation of BSA.