Insulin stacking for capillary electrophoresis.

Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis. Human insulin, a polypeptide, was concentrated on the capillary (stacked) based on three different and simple treatment methods to the sample: dilute buffers, high salt content, and acetonitrile (66%) were added to the sample to induce stacking. A dilute buffer in the sample caused a limited stacking, while acetonitrile treatment and high salt content in the sample caused much greater (approximately 20-fold) stacking. High salt concentration in the sample 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 the capillary to be loaded with sample thus increasing the detection signal greatly. The insulin peak after acetonitrile treatment and separation by capillary electrophoresis (CE) was confirmed by HPLC and by CE fraction collection followed by immunoassay. Based on acetonitrile treatment, insulin detection in pancreatic tissue homogenates is shown to be feasible.