Analysis and separation of enkephalin and dalargin analogues and fragments by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been applied to qualitative and quantitative analysis and separation of synthetic analogues and fragments of enkephalins ([Leu5]enkephalin, H-Tyr-Gly-Gly-Phe-Leu-OH, [Met5]enkephalin, H-Tyr-Gly-Gly-Phe-Met-OH), and dalargin (H-Tyr-d-Ala-Gly-Phe-Leu-Arg-OH), biologically active peptides with morphin-like effects acting as ligands for the opiate receptors in the brain. These oligopeptides (dipeptides to hexapeptides) were analyzed as cations in two acidic background electrolytes (BGEs), BGE I (100 mM H3PO4, 50 mM Tris, pH 2.25), BGE II (100 mM iminodiacetic acid, pH 2.30), and both as cations and anions in alkaline BGE IV (40 mM Tris, 40 mM Tricine, pH 8.10). Purity degrees of peptides, expressed in three different ways (relative peak height, relative peak area and relative corrected peak area), were determined by their CZE analyses in the above BGEs, and their values were compared with respect to the peak shapes and migration times of the main synthetic products and their admixtures. Selected analogues and fragments of enkephalins and dalargin were successfully separated by CZE in acidic isoelectric buffers, 100 and 200 mM iminodiacetic acid, pH 2.30 and 2.32, respectively. The effective electrophoretic mobilities at standard temperature 25 °C, and effective and specific charges of all analyzed peptides in the above three BGEs were determined. Correlation between effective electrophoretic mobility of the analyzed peptides and their charge and size (relative molecular mass) was investigated, which revealed different molecular shape of analyzed peptides in acidic and alkaline BGEs. In addition, the selected characteristics of the UV-absorption detector (noise, signal to noise ratio, sensitivity, and limits of detection and quantification) were determined.