Optimization of electrophoretic determination of protamine and insulin

This work deals with optimization of a method for separation and detection of protamine and insulin using capillary zone electrophoresis. The composition of background electrolyte, the solution pH and the injection method were optimized. Citric acid in a concentration range of 80 to 240 mmol L-1 and chloroacetic acid ranging from 50 to 150 mmol L-1 were tested sequentially. The optimized method uses a fused silica capillary with inner diameter of 50 μm. The total length of capillary is 50.0 cm, effective length is 8.5 cm. The injection of the sample is performed on the short end of the capillary. The method uses chloroacetic acid of 100 mmol L-1 concentration as the background electrolyte. Driving voltage is 20 kV. Sample is injected hydrodynamically with a pressure of 5 kPa for 3 s. The analytes are detected spectrophotometrically at wavelength of 200 nm. The method allows for determination in case of protamines in concentration range between 4 μg mL-1 and 300 μg mL-1 and insulin from 5 μg mL-1 to 300 μg mL-1 . The limits of detection are 1 μg mL-1 for protamine and 2 μg mL-1 for insulin. Repeatability of migration times and peak areas tested at 30 μg mL-1 and 200 μg mL-1 concentration levels using hydrodynamic injection showed values of relative standard deviation lower than 6 % suggesting..