Preparation and evaluation of packed capillary columns for the separation of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Abstract

Oligonucleotides and double stranded DNA fragments were separated in 200 μm I.D. capillary columns packed with micropellicular, octadecylated, 2.1 μm poly(styrene–divinylbenzene) particles by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Both the length and the diameter of the connecting capillaries (150×0.020 mm I.D.) as well as the detection volume (3 nl) had to be kept to a minimum in order to maintain the high efficiency of this chromatographic separation system with peak widths at half height in the range of a few seconds. Three different types of frits, namely sintered silica particles, sintered octadecylsilica particles, and monolithic poly(styrene–divinylbenzene) (PS–DVB) frits were evaluated with respect to their influence on chromatographic performance. Best performance for the separation of oligonucleotides and long DNA fragments was observed with the PS–DVB frits, whereas the short DNA fragments were optimally resolved in columns terminated by octadecylsilica frits. The maximum loading capacity of 60×0.20 mm I.D. columns ranged from 20 fmol (7.7 ng) for a 587 base pair DNA fragment to 500 fmol (2.4 ng) for a 16-mer oligonucleotide. Lower mass- and concentration detection limits in the low femtomol and low nanomol per liter range, respectively, make capillary IP-RP-HPLC with UV absorbance detection highly attractive for the separation and characterization of minute amounts of synthetic oligonucleotides, DNA restriction fragments, and short tandem repeat sequences amplified by polymerase chain reaction.