Analysis of Phosphorothioated Oligo-DNA (VN-50 2D)

Phosphorothioated oligonucleotides are often used as nucleic acid medicines, because of their stability compared to ordinary oligonucleic acids. During the development and quality control of nucleic acid drugs that are expected as next-generation pharmaceuticals, ion-pair reversed phase mode is often used to analyze the oligonucleotides. However, deposition of the ion-pair reagent on the column can be a problem. In this application, an unpurified product of synthetic phosphorothioated oligo-DNA of 20 mer was analyzed using HILICpak VN-50 2D, a polymer-based HILIC column.
A good separation of oligomers were achieved without the use of ion-pair reagent under the HILIC mode. The gradient mode developed used 50 mM ammonium formate (pH9.5) and acetonitrile.


Sample : 1 µL
Synthesized phosphothioated oligo-DNA 20 mer (crude)
(A*T*A*C*C*G*A*T*T*A*A*G*C*G*A*A*G*T*T*T ; * means phosphorothioated position)
1.0 mg/mL (in H₂O)

Column       : Shodex HILICpak VN-50 2D (2.0 mm I.D. x 150 mm)
Eluent       : (A); 50 mM HCOONH4 aq. (pH9.5)/(B); CH3CN
               Linear gradient ;
               (B %) 64 to 58 % (0 to 10 min), 58 % (10 to 15 min), 
                    58 to 64 % (15 to 15.01 min), 64 % (15.01 to 20 min)
Flow rate    : 0.25 mL/min
Detector     : UV (260 nm) (small cell volume)
Column temp. : 40 °C