Separation of nucleosides and nucleotide‐3′‐monophosphates by micellar electrokinetic capillary chromatography

Abstract

Abstract. Simultaneous separation of ionic and neutral compounds in micellar electrokinetic capillary chromatography (MECC) is a consequence of a combination of charge/mass ratios, hydrophobicity and charge interactions at the surface of the micelles. The type of resolution can be chosen mainly by modification of the buffer composition and pH. Reproducibility depends on the capillary tubing characteristics, operating conditions, and temperature effects. We report here on the different buffer compositions, pH, temperature, operating conditions, and capillary tubing treatments for the optimal separation and best reproducibility of deoxynucleosides and deoxynucleotide-3'-monophosphates. The effects of different buffer compositions were tested when operating in a constant current mode at 38 pAmps. Lithium phosphate buffer improved the separation speed with little loss in resolution. The best concentration was found to be 20 mM. A concentration of 5 % acetonitrile substantially improved the efficiency of the nucleoside peaks and therefore their resolution. The optimum SDS concentration was found to be at 100 mM. The pH has a significant influence on retention time and resolution, and was adjusted for maximum resolution at pH 9.6. Capillary surface treatments were also investigated. Effective heat dissipation occurred below an operating voltage of 15 kV, and a temperature of 35°C was found to give a good compromise between efficiency, resolution, and time of analysis. Time of analysis can be decreased by using higher operating voltages without compromising the resolution. Efficiencies were 350,000 and 235,000 plates m-l for the nucleosides and nucleotides, respectively. The reproducibility of retention times was good with coefficients of variation below 1 %, which allows the possibility for automatic fraction collection.

Method development and validation for the analysis of didanosine using micellar electrokinetic capillary chromatography

“…This method was first introduced by Terabe et al [18] in 1984. MEKC can resolve both neutral and charged analytes by a combination of charge/mass ratio, hydrophobicity, and charge interactions at the surface of the micelles [19]. Although SDS is one of the most common surfactants used, lithium dodecyl sulfate (LiDS) was used in this work.…”

Separation ofD-lysergic acid diethylamide derivatives using micellar electrokinetic capillary chromatography

“…The basic objective of thermostating capillaries in MEKC is to remove Joule heat. The solute electrophoretic mobility [13] and the solute distribution [14] are temperature-dependent processes. Furthermore, the temperature variations will also induce important changes in mobile-phase viscosity [15], critical micellar concentration [16], solute diffusivity [15], the degree of ionization of the buffer [17], and its pH [18].…”

Capillary electrophoretic method for nucleotide analysis in cells: Application on inherited metabolic disorders

“…Separations in acidic electrolytes were also published [19][20][21][22], however generally with long analytical times. Some workers used MEKC to separate nucleotides [19,[23][24][25]. Other approaches derived benefit from the complexation of nucleotides with transient metals for better resolution [26][27][28][29][30].…”