Electrophoretic behaviour of oligonucleotides and mono-, di- and triphosphate nucleotides by capillary zone electrophoresis

Abstract

A systematic investigation has been made into the mechanisms of the capillary zone electrophoresis (CZE) separation of 12 common nucleotides (mono-, di- and triphosphorylated) and polydeoxythymidylic acid oligonucleotides (pd(T)5-18) using electrophoretic mobility values calculated from migration time data. Relationships between electrophoretic mobility and the physicochemical characteristics of the analytes (charge, dissociation constants, charge-to-mass ratio) and the background electrolyte conditions (buffer strength, percentage organic modifier and buffer pH) were characterised. Nucleotide migration was dominated by the negatively charged phosphate groups. Additionally, there were important contributions to migration behaviour from the ionised amide groups of the nucleobases guanine and uracil at higher buffer pH values or with the presence of methanol in the electrolyte. Calculated electrophoretic mobility values for the nucleotides showed a substantially improved (5-fold) inter-run repeatability compared with migration time data. These studies show the value of representing nucleotide migration data as electrophoretic mobility in CZE for obtaining a more thorough analysis of separation mechanisms and to compensate for variation in migration time data caused by small changes in electrosmotic flow. Oligonucleotides pd(T)5-11 could be adequately resolved from their nearest neighbour, but the limit of single-base separation was pd(T)10 from pd(T)11 under the conditions used. It was calculated that a difference in charge-to-mass ratio of 2.64 x 10(-5) was required for resolution under the CZE conditions used.

Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse

“…Capillary electrophoresis can be used to separate ionic analytes with a high resolution, which makes it very suitable for the analysis of nucleotides (Uhrova, Deyl, & Suchanek, 1996; McKeown, Shaw, & Barrett, 2001; Zinellu et al, 2008). A combination with MS makes the technique even more powerful, as shown by many applications for nucleotide analysis (Willems et al, 2005).…”

Determination of limiting mobilities and dissociation constants of 21 amino acids by capillary zone electrophoresis at very low pH

“…They utilized isotachophoresis which offers accurate results in the moderate pH regions. Recently, especially the pK a values were determined by CZE for pharmaceutically and nutritionally important compounds [23][24][25][26]. Pospíchal determined the mobilities of various analytes by CZE as well as reviewed of the data published in the literature [27].…”

Dynamically coated silica monolith with ionic liquids for capillary electrochromatography

“…In general, nucleoside monophosphates, which bear negative charges at pH .2, are often separated at pH above 10 or in co-EOF mode in the presence of modifiers [40,41]. In this study, [BMIM] [BF 4 ] DCSM column, which generated anodic EOF, could be a promising alternative for the separation of them.…”