INSULIN STACKING FOR CAPILLARY ELECTROPHORESIS

Stacking methods are very important in overcoming the poor detection l imits in capillary electrophoresis. Human insulin, a polypeptide, was concentrated on the capillary (stacked) based on three different and s imple treatment methods to the sample: dilute buffers, high salt conte nt, and acetonitrile (66%) were added to the sample to induce stacking . A dilute buffer in the sample caused a limited stacking, while aceto nitrile treatment and high salt content in the sample caused much grea ter (similar to 20-fold) stacking. High salt concentration in the samp le caused stacking presumably by a transient isotachophoretic method. In addition to stacking, the acetonitrile treatment removed the excess proteins in the sample. Insulin did not denature or precipitate in 66 % acetonitrile as confirmed by high-performance liquid chromatography (HPLC) and immunoassays. Acetonitrile treatment enabled one-third of t he capillary to be loaded with sample thus increasing the detection si gnal greatly. The insulin peak after acetonitrile treatment and separa tion by capillary electrophoresis (CE) was confirmed by HPLC and by CE fraction collection followed by immunoassay. Based on acetonitrile tr eatment, insulin detection in pancreatic tissue homogenates is shown t o be feasible. (C) 1998 Elsevier Science B.V.