Separation of native amino acids at low pH by capillary electrophoresis

Abstract

SummaryAmino acids are cations at low pH and can be readily separated by capillary electrophoresis provided an alkanesulfonic acid is added to the electrolyte carrier. Formation of a positive net charge on the bare fused-silica surface at low pH was confirmed by measurement of an anodic electroosmotic flow. The addition of ethanesulfonic acid or octanesulfonic acid to the electrolyte carrier causes a reversal of the EOF. A mechanism is proposed in which the alkanesulfonic acid adsorbs to the positively-charged capillary wall through electrostatic attraction. Adsorption of a second molecule of alkanesulfonate by hydrophobic attraction to the carbon chain forms a negatively-charged coating on the capillary wall. The alkanesulfonate also imparts selectivity to the system by participation in ionpairing interactions with the native amino acids to improve resolution. The CE separation of a mixture of the twenty common amino acids at pH 2.8 with direct absorbance detection at 185 nm resulted in 17 amino acid peaks in 20 minutes with a 30 kV applied voltage. The effect of several variables was studied including electrolyte carriers containing different alkanesulfonic acids, the influence of pH, applied voltage, and concentration of electrolyte carrier.

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection

“…The direct UV detection at 185 nm of underivatized native amino acids was reported in the literature [19,20]. However, the instrument used in our work permitted the lowest detection wavelength of 195 nm, at which only some of the essential amino acids, namely Arg, His, Asn, Met, Trp, Phe, Tyr, and Cys, could be detected.…”

“…In the literature, CZE separations of underivatized amino acids in their protonated form using 1 M formic acid [24], 50 mM phosphate buffer at pH 2.5 [48], 50 mM sodium ethanesulfonate at pH 2.3 [20] or 10 mM NaH 2 PO 4 with 30 mM octanesulfonic acid at pH 2.4 were reported and a good separation selectivity for native amino acids was obtained in BGEs having pH 2.3 and 2.4. Nevertheless, all native amino acids were not totally separated and some comigrations were observed in these BGEs.…”

“…As most of the native amino acids absorb only slightly or not at all ultraviolet light (UV), different detection approaches were used in combination with the CZE and MEKC separation techniques. UV and fluorescence or laser-induced fluorescence (LIF) detection after a suitable precolumn, postcolumn and on-column derivatization with a UV chromophore [8][9][10][11][12][13][14] and fluorophore [7,[15][16][17][18], respectively, direct UV detection at a very low wavelength [19,20], indirect UV detection [21], electrochemical detection [22,23], mass spectrometry [24,25], and some other detection techniques [26,27] were applied to the analyses of amino acids.…”

Floating resistivity detector for microchip electrophoresis

“…Most underivatized native amino acids are only detectable at the UV wavelength of 185 nm [29,30]. This often poses a detection problem for amino acids as the minimum detection wavelength that most UV detectors provide is at 195 nm.…”

Plastic substrates based separation channels in electromigration techniques

“…The selectivity problem arises from the fact that capillary zone electrophoresis (CZE) separations are based on the mass-to-charge ratio which does not differ too much with most members of the classical set of 20 amino acids. This was attempted to be overcome by using buffer additives like alkylsulfonic acids at acid pH [16,17].…”