Reversed-phase high-performance liquid chromatography on-line with the second and fourth derivative ultraviolet spectroscopy as a tool for identification of milk proteins

This paper reports the possibilities of high-performance liquid chromatography on-line with photodiode-array detection for identification of bovine milk proteins. The application of this method for qualitative analysis of milk proteins on the basis of UV spectra discriminants is described. These discriminants characterise the most frequently occurring extrema of derivatives of UV spectra of proteins separated using HPLC. The measurements have been carried out in the wavelength range of 270-300nm, corresponding to the absorbance of aromatic amino acid residues. The experiment has allowed to estimate the minimal value of absorbance, at which a given protein may be identified and to determine the method specificity, i.e. statistical significance of differences between discriminants characterising the spectra of individual proteins. The findings show the possibility that milk proteins, except alphas1-casein (alphas1-CN) and alphas2-casein (alphas2-CN), can be distinguished also proving the method robustness. This means that the method is independent of environmental interference, such as the temperature influence in 20-30°C range, a change of column, the content of acetonitrile and trifluoroacetic acid in the solvent and a sample concentration. The method presented is suitable for identification of individual proteins in such preparations as calcium caseinate, sodium caseinate, milk protein coprecipitate and whey protein concentrate.