Capillary electrophoresis of oligonucleotides using a replaceable sieving buffer with low viscosity-grade hydroxyethyl cellulose

Short model homo-oligomeric deoxynucleotides ranging in length from 12- to 24-mer were separated using a 20 mM Tris(hydroxymethyl)aminomethane-N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid buffer containing 4% hydroxyethy cellulose of low viscosity as the polymer additive (pH 7.0). The separation was performed using a DB-17-coated capillary. The influence of instrumental parameters such as field strength and temperature was evaluated. It required a separation voltage of 12 kV at reversed polarity and a temperature of 25 degrees C. Efficiencies of up to 2.5X10(6)/m were obtained. The most important parameters influencing the separation were the concentration and the viscosity of the polymer used. Different viscosity grades of hydroxyethy cellulose were evaluated for their loading time on the capillary and their ability to separate p(dA)(12-18) and p(dA)(19-24). The use of low viscosity-grade hydroxyethy cellulose at a relatively high concentration (4%) made it possible to replace the buffer after every run and permitted the use of hydrodynamic injection of oligonucleotide samples. The entangled polymer solution system was found to be applicable on automatic capillary electrophoresis (CE) equipment. For quantitation, the use of an internal standard has been shown to improve both migration time and peak area repeatability. This method using low viscosity-grade hydroxyethy cellulose has been demonstrated to have the repeatability, linearity and selectivity required for stability studies of oligonucleotides.