0000000000402060
AUTHOR
Karin Walhagen
Capillary electroendoosmotic chromatography of peptides
This review focuses on the current state of peptide separation by capillary electroendoosmotic chromatography (CEC). When carried out under optimised conditions, peptide separation by CEC methods represents an orthogonal and complementary technique to micro-HPLC (micro-HPLC) and high-performance capillary zone electrophoresis (HPCZE). The origin of the selectivity differences that can be achieved with these three separation techniques (CEC, micro-HPLC and HPCZE), respectively are discussed, and the current limits of performance with CEC methods documented. Peptide separations by CEC methods with n-alkyl bonded silicas or mixed-mode phases are also illustrated. The development of different v…
Capillary electrochromatography analysis of hormonal cyclic and linear peptides.
The retention behavior of linear and cyclic peptides has been studied by capillary electrochromatography (CEC) with a variety of different n-alkyl silica reversed-phase sorbents and also with mixed-mode phases containing both strong cation-exchange (sulfonic acid) and n-alkyl groups bonded onto the silica surface, using eluents ranging from pH 2.0 to pH 5.0. Depending upon the amino acid sequence, electrochromatographic retention of the peptides was strongly affected by the composition of the eluent, its pH value, and the choice of sorbent packed into the capillaries. The dominant separation processes operating with these charged analytes could be modulated inter alia by the content of orga…
A critical appraisal of capillary electrochromatography.
Although still in its infancy, CEC promises some advantages over competitive techniques for separating charged analytes including biomolecules.
The CEC behaviour of several synthetic peptides related to the activin ��A-��D subunits
The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-…
Separation of selected peptides by capillary electroendoosmotic chromatography using 3 μm reversed-phase bonded silica and mixed-mode phases
The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentrat…
Life-time studies with capillary electrochromatography columns operated under different conditions.
A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed …
On the nature of the forces controlling selectivity in the high performance capillary electrochromatographic separation of peptides
In this minireview, the nature of the forces controlling selectivity in the high performance capillary electrochromatographic (HP-CEC) separation of peptides has been examined. For uncharged and charged peptides, a synergistic interplay occurs in HP-CEC systems between adsorptive/partitioning events and electrokinetically driven motion. Moreover, at high field strengths, both bulk electrophoretic migration and surface electrodiffusion occur. Thus, the migration behavior of peptides in different HP-CEC systems can be rationalized in terms of the combined consequences of these various processes. Moreover, in HP-CEC, the buffer electrolyte interacts with both the peptide analytes and the sorbe…
Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV-1 gp120 epitope
In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic(CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular,10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1-9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form alpha-helical secondary structures in solution. Electrochromato…
Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography
The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature ov…