K. Khan et al., CAPILLARY ELECTROPHORESIS OF OLIGONUCLEOTIDES USING A REPLACEABLE SIEVING BUFFER WITH LOW VISCOSITY-GRADE HYDROXYETHYL CELLULOSE, Journal of chromatography, 742(1-2), 1996, pp. 267-274
Citations number
33
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
Short model homo-oligomeric deoxynucleotides ranging in length from 12
- to 24-mer were separated using a 20 mM 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 instrumenta
l parameters such as field strength and temperature was evaluated. It
required a separation voltage of 12 kV at reversed polarity and a temp
erature of 25 degrees C. Efficiencies of up to 2.5X10(6)/m were obtain
ed. The most important parameters influencing the separation were the
concentration and the viscosity of the polymer used. Different viscosi
ty grades of hydroxyethy cellulose were evaluated for their loading ti
me on the capillary and their ability to separate p(dA)(12-18) and p(d
A)(19-24). The use of low viscosity-grade hydroxyethy cellulose at a r
elatively high concentration (4%) made it possible to replace the buff
er after every run and permitted the use of hydrodynamic injection of
oligonucleotide samples. The entangled polymer solution system was fou
nd to be applicable on automatic capillary electrophoresis (CE) equipm
ent. For quantitation, the use of an internal standard has been shown
to improve both migration time and peak area repeatability. This metho
d using low viscosity-grade hydroxyethy cellulose has been demonstrate
d to have the repeatability, linearity and selectivity required for st
ability studies of oligonucleotides.