High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers

Anal Biochem. 1993 Aug 1;212(2):351-8. doi: 10.1006/abio.1993.1340.


Phosphodiester-oligonucleotides were separated by ion-pair reversed-phase HPLC on nonporous polystyrene-based particles having a diameter of 2.1 +/- 0.12 microns. With unmodified poly(styrene-divinylbenzene) beads it was not possible to resolve oligonucleotides efficiently. However, upon addition of polyvinyl alcohol during polymerization baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases was obtained, with triethylammonium acetate serving as ion-pairing reagent. An even higher separation efficiency was achieved by Friedel-Crafts alkylation of poly(styrene-divinylbenzene) particles. At a column temperature of 50 degrees C, the number of theoretical plates exceeded 6 x 10(5) per meter. The maximum loading capacity for a 50 x 4.6-mm i.d. column still resulting in the highest resolution was 0.1 microgram. Calibration curves showed excellent linearity over a range of at least 4 magnitudes, with a lower mass detection limit of 0.16 ng. Recoveries ranged from 96.7 to 100.8%. The same stationary phase also allowed the separation of phosphorylated from dephosphorylated oligonucleotides, the former ones being eluted earlier.

Publication types

  • Comparative Study

MeSH terms

  • Alkylation
  • Base Composition
  • Chromatography, High Pressure Liquid / methods
  • Oligonucleotides / analysis
  • Oligonucleotides / isolation & purification*
  • Particle Size
  • Polymers
  • Polystyrenes*
  • Vinyl Compounds*


  • Oligonucleotides
  • Polymers
  • Polystyrenes
  • Vinyl Compounds
  • divinyl benzene