Stacking and separation of coproporphyrin isomers by acetonitrile-salt mixtures in micellar electrokinetic chromatography

The effectiveness of the addition of salt and acetonitrile in the sample ma trix to induce narrowing of the analyte zones is demonstrated for the first time in micellar electrokinetic chromatography (MEKC). Using coproporphyri n (CP) I and III isomers as test compounds, the use of sodium cholate (SC) as the micelle in the separation buffer and a high concentration of sodium chloride in the aqueous sample solution (without the presence of an organic solvent) were found to provide enhancement in peak heights for both CP I a nd III, but yielded very poor resolution of these two positional isomers at sample size of 10% capillary volume or larger. With the addition of aceton itrile as the organic solvent in the aqueous sample solution (acetonitrile- salt mixtures), baseline/partial resolution of CP I and III was obtained ev en at large injection volumes, along with significant increase in peak heig hts for both isomers. Possible mechanisms responsible for the narrowing of analyte zones are briefly discussed. The effects of experimental parameters , such as concentrations of salt and acetonitrile, on peak heights and reso lution of the test compounds were studied. Importantly, the usefulness of t he present method was demonstrated for the MEKC determination of endogenous CP I and III present in normal urine samples with good separation and dete ction performances.