Separation and determination of denatured a-, b- and k- caseins by hydrophobic interaction chromatography (HIC) was improved by using a TSK- GelÒ Ether-5PW column (Tosoh Biosep GmbH). The method, already proposed and performed by a TSK-GelÒ Phenyl-5PW column (Tosoh Biosep GmbH), is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate (GdmSCN) and HIC analysis in the presence of 8.0 M urea in the mobile phase. Employment of the less hydrophobic ether phase allowed the main advantages of separating casein fractions in less than 22 minutes and, additionally, of separating a-casein in as1- and as2- casein fractions. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated 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 to 40 mM. The detection limit for a-, b- and k-caseins ranged between 0.33 and 0.65 mM. The precision of the method was evaluated, the coefficient of variation for as1- , as2-, b- and k-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 8 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 as1- , as2-, b- and k-caseins to BCR-063R were compared with linear fitting of as1- , as2-, b- and k-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 a-, b- and k-caseins obtained by TSK-GelÒ Ether-5PW and TSK-GelÒ Phenyl- 5PW HIC columns, showing more accurate results for chromatographic analysis performed by Ether column.
New chromatographic method for separation and determination of denatured alfa S1, alfa S2, beta and kappa caseins by hydrophobic interaction chromatography
2002
Abstract
Separation and determination of denatured a-, b- and k- caseins by hydrophobic interaction chromatography (HIC) was improved by using a TSK- GelÒ Ether-5PW column (Tosoh Biosep GmbH). The method, already proposed and performed by a TSK-GelÒ Phenyl-5PW column (Tosoh Biosep GmbH), is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate (GdmSCN) and HIC analysis in the presence of 8.0 M urea in the mobile phase. Employment of the less hydrophobic ether phase allowed the main advantages of separating casein fractions in less than 22 minutes and, additionally, of separating a-casein in as1- and as2- casein fractions. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated 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 to 40 mM. The detection limit for a-, b- and k-caseins ranged between 0.33 and 0.65 mM. The precision of the method was evaluated, the coefficient of variation for as1- , as2-, b- and k-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 8 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 as1- , as2-, b- and k-caseins to BCR-063R were compared with linear fitting of as1- , as2-, b- and k-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 a-, b- and k-caseins obtained by TSK-GelÒ Ether-5PW and TSK-GelÒ Phenyl- 5PW HIC columns, showing more accurate results for chromatographic analysis performed by Ether column.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
