Separation of selected peptides by capillary electroendoosmotic chromatography using 3 mu m reversed-phase bonded silica and mixed-mode phases

The retention behaviour and selectivity of selected basic, neutral and acid ic peptides have been studied by capillary electroendoosmotic chromatograph y (CEC) with Hypersil C-8, C-18, Hypersil mixed-mode, and Spherisorb C-18/S CX columns, 250 (335) mmx100 mu m, packed with 3 mu m particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model (HPCE)-C-3D capillary electr ophoresis system. The selected peptides were desmopressin (D), two analogue s (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, dep ending on the concentration of acetonitrile used and the buffer ionic stren gth. The retention and selectivity of these peptides under CEC conditions w ere compared to their behaviour in free zone capillary electrophoresis (CZE ), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it ca n be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both e lectrophoretic migration processes and retention mechanisms involving hydro phobic as well as silanophilic interactions. This CEC method when operated with these 3 mu m reversed-phase and mixed-mode sorbents with peptides is t hus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides d iffers significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal anal ytical separation procedure that is complimentary to both of these alternat ive techniques. (C) 1999 Elsevier Science BN. All rights reserved.