ABSTRACT
A new experimental device for high-performance capillary electrophoresis (HPCE) with a double UV detection system and with thermostating of the whole separation compartment was developed. UV detection is doubled by producing two apertures placed symmetrically close to the axis of the optical path of the single-beam UV detector and by the adjustment of the capillary loop on these two apertures. The double-detection system allows exact measurements of electrophoretic and electroosmotic flow velocities. A procedure for the determination of effective mobilities from the data obtained by the double UV detection system was developed and applied to determine the effective mobilities of synthetic peptides (diglycine, triglycine, growth hormone releasing peptide and its derivatives and fragments). The measurements are performed at constant temperature (25 degrees C) and low input power at which temperature increase in the capillary can be neglected and temperature corrections of temperature-dependent magnitudes need not be included in the calculations.
Subject(s)
Electrophoresis/instrumentation , Peptides/isolation & purification , Spectrophotometry, Ultraviolet/methods , Amino Acid Sequence , Electrophoresis/methods , Molecular Sequence Data , Peptides/chemistry , TemperatureABSTRACT
Two carrier-free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free-flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purifity of peptide fractions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correlation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow-through cell, respectively, have to be taken into account and corresponding corrections have to be made.
