Separation of human tear proteins with ceramic hydroxyapatite high-performance liquid chromatography

Human tear protein, which consists mainly of albumin, lysozyme, and lactoferrin, was assayed with high-performance liquid chromatography using a new ceramic hydroxyapatite column. Proteins were eluted at room temperature using a 20-min linear gradient from 95:5% A/B buffer to 0:100% A/B buffer (buffer A, distilled water; buffer B, 400 mM KH2PO4 containing 240 mM NaOH). The proteins eluted at 1.2 min for albumin, 8.5 min for lysozyme, and 20.4 and 21.7 min for lactoferrin, respectively. The assays may be performed in 30 min.