Characterization of Open Tubular Capillary Electrochromatography Columns for the Analysis of Synthetic Peptides Using Isocratic Conditions

Abstract

In this paper, we report on investigations related to the performance characteristics of two different types of etched chemically (n-octadecyl- and cholesterol-) modified capillaries in the open tubular format of capillary electrochromatography (CEC) for the analysis of synthetic peptides. The results confirm that the nature of the surface chemistry used to modify the capillary wall and type of chemically bonded group employed can affect the selectivity as well as the resolution of peptide samples. The results are consistent with the participation of selective peptide interactions with the bonded phase, although other factors, such as the morphology of the capillary wall surfaces, appear to be also involved. Moreover, several surprising observations related to peptide-specific multi-zoning effects have been observed. Additional experimental variables that can also be utilized to affect the retention of peptides in this approach to OTCEC include the type and percentage of organic solvent modifier employed in the eluent and the pH of the buffer system. To evaluate the reproducibility of different batches of the n-octadecyl- and cholesterol-modified capillaries and the stability of the chemically modified surface, the OTCEC selectivity and peak shape behavior of two small basic molecules (serotonin and tryptamine) and two proteins (turkey and chicken lysozyme) were also investigated. Finally, the use of the "bubble" cell technology for creating the detector window has been shown to provide significantly higher detection sensitivity with peptides, as compared with the conventional capillary format.

Polar stationary phases for capillary electrochromatography

“…However, open-tubular columns often suffer from low surface coverage, which leads to a low phase ratio, and hence low sample loadability. Pesek et al [49][50][51][52][53] developed a method to prepare the etched capillary to address this problem. Starting with a 20 mm ID capillary and etching with caustic reagent, a variety of surface structures ranging from ripples to coral-like rods can be produced.…”

The separation of biomolecules using capillary electrochromatography

“…Pesek and coworkers [2,3] modi®ed the wall of etched capillaries with n-octadecyl and cholesterol groups, so that the large n-octadecyl and cholesterol groups impeded the contact between the negatively charged silanols and the positively charged peptides. Batch-to-batch reproducibility for both capillaries was excellent, as characterized by the selectivity and peak shape behavior of two basic small molecules and two proteins.…”

“…The use of CEC is also hampered by its low detection sensitivity, which is inherent in the short optical path length, when on-line absorption detectors are used. Some methods such as sample stacking [28], on-line enrichment [45,46] and larger detection cell [2] have been adopted to increase the detection sensitivity. However, highsensitivity detectors coupled to CEC are needed to ful®ll the demand of biological analysis.…”

Capillary electrophoretic separation of biologically active amines and acids using nanoparticle‐coated capillaries

“…Although HPLC is still the most common method for the analysis of biogenic amines and their metabolites, CZE, MEKC, and CEC have recently become attractive [7][8][9][10][11]. CZE is easy but does not provide enough resolving power for the separation of the analytes.…”