Chromatographic Separation and Quantification of Major Human Milk Proteins

Abstract

High Performance liquid chromatography using a reverse phase column Chrompack P 300 RP, which contains a polystyrene‐divinylbenzene copolymer based packing (8 µm, 300 Å, 150×4.6 mm i.d.) was applied to separate human casein and α‐lactalbumin. The chromatographic separation was performed rapidly with this HPLC/UV method.

Calibration curves were constructed using human casein and α‐lactalbumin. Linearity between protein concentrations and UV absorbance at 280 nm was maintained over the concentration ranges of 0.09–1.5 and 0.45–3.6 g/L, respectively. Caseins were collected, after preparative HPLC separation, concentrated and lyophilised for further use as standards. The concentration of α‐lactalbumin was estimated by comparison with a commercial standard. The correlation coefficient for each standard curve invariably exceeded 0.99 for the two proteins.

The validity of the method was verified. Sample dilution with water proved to be a simple and adequate method for sample preparation. Repeatability was evaluated by ten consecutive injections and human milk. The RSD values for concentrations were all below 3.11%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 85 and 97%.

Keywords:

• Human milk
• Proteins
• Caseins
• α‐Lactalbumin
• HPLC