While doing anion exchange chromatography of a short oligonucleotide, I have noticed that at $\rm pH=12$, the oligo retains longer on the column than the same oligo- at $\rm pH=8$. I thought the reason for this could be the change in the net charge of nucleobases.
Could anyone suggest what is the net charge of RNA nucleobases (adenine, guanine, cytosine, uracil) at $\rm pH=12$ (denaturing conditions)?
The conjugate acids of the adenine and guanine have pKa's between 9-10. Guanine also has a nitrogen with a pKa of 12.3, which would be mostly protonated at pH 12. The pKa for the phosphoric acid proton of a nucleotide is significantly lower than 7.
Therefore, in a pH 8 buffer, the basic nitrogens of each adenine and guanine are protonated, while the phosphate is deprotonated. This gives an overall net charge closer to zero than you would see at pH 12.
In a pH 12 buffer, where the pH is above the pKa of both the phosphoric acid protons and the nitrogen conjugate acid protons (except the last one on each guanine), all of these positions would be deprotonated. Therefore, the formal charge on the nitrogens would be zero (again, except the most basic nitrogen on guanine), but the phosphates would still have a charge of -1 each. This would lead to a net charge of significantly higher charge magnitude because there would be very few positive charges to balance out the negative charges.
At pH 12, the net charge should be approximately: $$(n_N +n_u) - (n_c + n_g).$$ Approximate net charge at pH 8: $$n_N - (n_a + n_c + 2n_g).$$
nN= number of nucleotides
nu= number of uracils/thymines
nc= number of cytosines
ng= number of guanosines
na= number of adenosines
