alfas1- , alfas2-, beta- and kappa- caseins from raw cow's, ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate (GdmSCN) followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimised by analysing commercial single bovine standard caseins and their mixture. In the optimised chromatographic conditions the four casein fractions were separated in less than 45 minutes. 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 microM. The detection limit for alfa-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alfa-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analysed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analysed and the ratio between casein peak areas (alfas1/kappa, alfas2/beta, beta/kappa and alfas2/alfas1) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.
Separation and Determination of Denatured alfas1-alfas2-, beta- and kappa-Caseins by Hydrophobic Interaction Chromatography in Cows', Ewes' and Goats' Milk, Milk Mixtures and Cheeses
Bramanti E;Onor M;
2003
Abstract
alfas1- , alfas2-, beta- and kappa- caseins from raw cow's, ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate (GdmSCN) followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimised by analysing commercial single bovine standard caseins and their mixture. In the optimised chromatographic conditions the four casein fractions were separated in less than 45 minutes. 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 microM. The detection limit for alfa-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alfa-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analysed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analysed and the ratio between casein peak areas (alfas1/kappa, alfas2/beta, beta/kappa and alfas2/alfas1) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
