Performance and selectivity of polymeric pseudostationary phases for the electrokinetic separation of amino acid derivatives and peptides

Abstract

Two polymeric pseudostationary phases, one an acrylamide polymer and the second a siloxane polymer, have been investigated for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized amino acids and small peptides. The dervatized amino acids were detected by UV absorbance and laser-induced fluorescence (LIF) detection. The polymers provided very high efficiency and good selectivity for the separation of the amino acids. The separation selectivity using the polymers was significantly different from that of SDS micelles, and there were subtle differences in selectivities between the polymers. Although very good detection limits were obtained with LIF detection, a significant background signal was observed when the polymers were not washed to remove fluorescent impurities. The polymers did not separate the peptides very well. It is postulated that the fixed covalent structure of the polymers prevents them from interacting strongly or efficiently with the peptides, which are large in relation to the analytes typically separated by electrokinetic chromatography using polymers.

MEKC: An update focusing on practical aspects

“…9.0. An interesting method providing better efficiency and good selectivity was recently reported by Palmer and coworkers [204] that uses polymeric PSPs for LIF detection for the first time. Two types of polymeric PSPs (viz acrylamide and siloxane polymers) were evaluated for the separation of NDA-labeled amino acids and small peptides.…”

“…Naphthalene-2,3-dicarboxaldehyde (NDA) is a another typical labeling reagent for amino compounds (especially amino acids). NDA derivatives have been detected by LIF after separation by MEKC, using different types of laser, including argon-ion [202], He-Cd (442 nm) [203], and krypton ion (413 nm) [204]. As a rule, the separation of the labeled analytes requires the addition of b-CD [204,205] or even methanol [202] to a BGE consisting of SDS and borate buffer at pH ca.…”

Recent developments in CE and CEC of peptides

“…The highly selective separation of tryptic BSA peptides was achieved in an optimized BGE, composed of 50 mM ammonium formate buffer, pH 2.0, and containing 32 mM PAPS and 0.6% m/v Brij 35. CEKC with two polymeric pseudostationary phases, acrylamide-and siloxane-based polymers, provided very high efficiency and good selectivity for separation of NDAderivatized amino acids with selectivities different from those observed by SDS micelles but these phases were found to be generally unsuitable for separation of NDAlabeled peptides [130].…”

Electrokinetic chromatography with polymeric pseudostationary phases

“…Peptides and derivatized amino acids were separated by EKC with both UV absorbance and LIF detection using a lauryl methacrylamide/AMPS copolymer and a siloxane polymer [65]. Efficient and selective separations of the derivatized amino acids were achieved with both polymers (Fig.…”