Multidimensional analysis of denatured milk proteins by hydrophobic interaction chromatography coupled to a dynamic...

Multidimensional analysis of denatured milk proteins is reported using high-performance liquid chromatography (HPLC) combined with dynamic surface tension detection (DSTD). A hydrophobic interaction chromatography (HIC) column (a TSK-Gel Phenyl-5PW column, TosoBiosep), in the presence of 3.0 M guanidine hydrochloride (GdmHCl) as denaturing agent is employed as the mobile phase. Dynamic surface tension is measured through the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 4 s) is achieved, for each eluting drop of 4 s length, providing insight into both the kinetic and thermodynamic behavior of molecular orientation processes at the liquid-air interface. An automated calibration procedure and data analysis method is applied with the DSTD system, which allows two unique solvents to be used, the HIC mobile phase for the sample and a second solvent (water for example) for the standard, permitting real-time dynamic surface tension data to be obtained. Three-dimensional data is obtained, with surface tension as a function of drop time first converted to surface pressure, which is plotted as a function of the chromatographic elution time axis. Experiments were initially performed using flow injection analysis (FIA) with the DSTD system for investigating commercial single standard milk proteins (alpha-lactalbumin, beta-lactoglobulin, alpha-, beta-, kappa-casein and a casein mixture) denatured by GdmHCl. These FIA-DSTD experiments allowed the separation and detection conditions to be optimized for the HIC-DSTD experiments. Thus, the HIC-DSTD system has been optimized and successfully applied to the selective analysis of surface-active casein fractions (alpha s1- and beta-casein) in a commercial casein mixture, raw milk samples (cow's, ewe's and goat's milk) and other diary products (yogurt, stracchino, mozzarella, parmesan cheese and chocolate cream). The different samples were readily distinguished based upon the selectivity provided by the HIC-DSTD method. The selectivity advantage of using DSTD relative to absorbance detection is also demonstrated.