High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers.
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.
Huber
C G
CG
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
Oefner
P J
PJ
Bonn
G K
GK
D000478:Alkylation; D001482:Base Composition; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis / Q000302:isolation & purification*; D010316:Particle Size; D011108:Polymers; D011137:Polystyrenes; D014753:Vinyl Compounds
D009841:Oligonucleotides; D011108:Polymers; D011137:Polystyrenes; D014753:Vinyl Compounds; C004985:divinyl benzene