New chromatographic method for separation and determination of denatured αs1-, αs2-, β- and κ-caseins by hydrophobic interaction chromatography

Separation and determination of denatured α-, β- and κ-caseins by hydrophobic interaction chromatography (HIC) was improved by using a TSK-Gel Ether-5PW column (Tosoh Biosep). The method, already proposed and performed by a TSK-Gel Phenyl-5PW column (Tosoh Biosep), is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate and HIC analysis in the presence of 8.0 M urea in the mobile phase. Employment of the less hydrophobic ether phase had the main advantage of separating casein fractions in less than 22 min and, additionally, of separating α-casein in αs1- and αs2-casein fractions. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certified for total nitrogen content. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5–40 μM. The detection limit for α-, β- and κ-caseins ranged between 0.33 and 0.65 μM. The precision of the method was evaluated, the RSDs for αs1-, αs2-, β- and κ-casein determination ranging between 2.3 and 5.5% for standard solutions and between 4.4 and 6.2% for real sample solutions. The mean value of casein content found in eight aliquots of BCR-063R calculated with respect to the total protein content (estimated on the basis of certified total nitrogen content) was 78.3±6.1%. Results of linear fitting of standard additions data of αs1-, αs2-, β- and κ-caseins to BCR-063R were compared with linear fitting of αs1-, αs2-, β- and κ-casein calibration data. The method was applied to commercial caseins and to 30 real, raw samples. A statistical comparison was performed between results on quantitation of α-, β- and κ-caseins obtained by TSK-Gel Ether-5PW and TSK-Gel Phenyl-5PW HIC columns, showing more accurate results for chromatographic analysis performed by the ether column.