Peptide stacking by acetonitrile-salt mixtures for capillary zone electrophoresis

Many small natural and synthetic peptides can be stacked for capillary zone electrophoresis by dissolving the peptides in a mixture containing acetonitrile and high concentrations of inorganic salts. In many instances one third of the capillary can be loaded with peptides dissolved in a mixture of 2 volumes acetonitrile and 1 volume of 1% sodium chloride leading to about 20-fold enhanced detection. This stacking is dependent on the presence of both salts and acetonitrile. Natural peptides such as enkephalins, angiotensin and insulin chain B in addition to peptides released from the action of proteolytic enzymes on proteins were concentrated by this method. From a practical point of view, the stacking in acetonitrile is more useful since it removes proteins, counteracts the deleterious effects of high concentrations of inorganic ions present in the sample and stops the enzymatic reaction. Furthermore, it allows a larger volume of the sample to be loaded on the capillary increasing the sensitivity of the CE. This stacking produces a greater sample concentration and better resolution than the traditional stacking obtained in aqueous low ionic strength buffers. The mechanism is also different since it is improved by a high concentration of ions in the sample. Furthermore, since proteins are eliminated, the electropherograms are cleaner and the capillary does not require thorough washings between samples, speeding up the analysis and extending the capillary life.