Reversed-phase liquid chromatography of egg white proteins. Optimization of ovalbumin elution

This paper is an experimental contribution to the optimization of ovalbumin elution in RP-HPLC with a C4 support. The effects of TFA concentration, gr adient duration, flow rate, and the nature of the solvent were studied usin g an experimental design. The results highlighted the major role of TFA con centration in the recovery yield of ovalbumin, with an optimum concentratio n range which is very narrow and much lower (0.025%) than the levels usuall y applied (0.1%). In this respect, ovalbumin seems to behave in a specific way. Among the four solvents tested, i.e. acetonitrile, isopropanol, ethano l, and a mixture of these three, the first one is definitely the most satis factory in the area of recovery yield and efficiency. By modifying the elut ion conditions alone, the recovery yield of ovalbumin may range from 50 to 85%. Finally, this work has enabled us to define the chromatographic conditions for quantitative egg white analysis of its major proteins in only six minut es and with minimal subsequent ghosting effects.