Separation of human tear proteins by high performance liquid chromatography

The optimal conditions for separating human tear proteins by high performance liquid chromatography (HPLC) using a Waters I-125 gel filtration column were investigated. Several elution buffers were tested including phosphate buffer alone and phosphate buffer to which varying amounts of NaCl or 0.1% Tween was added. The combination of phosphate buffer (pH 5.28), 0.5 M NaCl and 0.1% Tween gave the best resolution and a recovery of 90% of the proteins applied. Tear lactoferrin was shown to adhere to the column packing when the molarity of the elution buffer was not high enough. Using optimal conditions, the tear proteins IgA, lactoferrin and lysozyme were identified in distinct peaks after a preparative HPLC run. When used in combination with Schirmer strips as a tear sampling method, HPLC was shown to be a rapid, simple and reproducible way of investigating the composition of tear proteins.