Analysis of Oligonucleotides by On-Column Transient Capillary Isotachophoresis and Capillary Electrophoresis in Poly(ethylene glycol)-Filled Columns

Abstract

Capillary electrophoresis in poly(ethylene glycol) (PEG) solution-filled uncoated silica capillaries was demonstrated to be suitable for separation of oligonucleotides in 100 mM ammonium formate (pH 4.5) buffers. Reversed polarity (−30 kV at the injector end) was used, because the electroosmotic flow (EOF) at 4.5 was considerably lower than the mobility of the anionic analytes. On-column transient isotachophoresis (ITP) was developed for preconcentration of oligonucleotide samples in PEG solution-filled capillaries. The ITP focusing step allows injection of 20% of column length without loss of peak resolution. The detection limit of the method was 100 ng/mL of 16-mer oligonucleotide by using UV detection at wavelength 254 nm (signal-to-noise ratio 10).