Separation of the apoprotein components of human very low density lipoproteins by ion-paired, reversed-phase high performance liquid chromatography

A number of crude apolipoprotein samples isolated from human very low density lipoproteins (VLDL) were analyzed by reversed phase high performance liquid chromatography. The mobile phase consisted of a 1% solution of the polar ion-pairing reagent triethylammonium phosphate. A slow, nonlinear gradient of acetonitrile (37-42%) was used to elute the apolipoproteins. The order of elution was as follows: apolipoprotein C_x, apolipoprotein C-I, apolipoprotein C-III₂, apolipoprotein C-III₁, apolipoprotein C-III₀ and apolipoprotein C-II. This order is consistent with the known polarity of the proteins, i.e., the most nonpolar, apolipoprotein C-II, was the last to be eluted, whereas apolipoprotein C-I, with the lowest nonpolar surface area eluted first. The recovery of the individual apolipoproteins was 80-95% and the individual peaks were characterized by amino acid analysis, UV absorption spectra and chromatography of pure protein standards.