Sample Preparation Affects Separation of Whey Proteins by Reversed-Phase High-Performance Liquid Chromatography

Measured concentrations of whey proteins in single milk samples often differ by more than 20% when analyzed by different separation methods. In the current study, we examined the effects of using guanidine hydrochloride or urea at zero and 8.5 h after sample preparation and the effects of using dithiothreitol or 2-mercaptoethanol on the separation of bovine milk proteins by reversed-phase high-performance liquid chromatography. Treatment with guanidine hydrochloride or urea resulted in similar separation of milk proteins when samples were injected immediately after preparation. Separation was repeatable over 8.5 h for samples prepared with guanidine hydrochloride, whereas the resolution of α-lactalbumin and β-lactoglobulin decreased for samples treated with urea. Treatment with dithiothreitol improved the resolution of α-lactalbumin and β-lactoglobulin in comparison with treatment with 2-mercaptoethanol. Quantitation of whey proteins is more reliable when milk samples are treated with dithiothreitol and guanidine hydrochloride than when treated with 2-mercaptoethanol or urea. Keywords: Casein; whey; milk protein; reducing agent; chaotropic agent; reversed-phase high-performance liquid chromatography