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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)?

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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

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  • $\begingroup$ Thank you for your explanation! Do you think do Uracil and Cytosine influence the net charge of the oligo? I have found that Uracil and Cytosine may get a negative charge on oxygen: "Uracil is a weak acid. The first site of ionization of uracil is not known. The negative charge is placed on the oxygen anion and produces a pKa of less than or equal to 12. The basic pKa = -3.4, while the acidic pKa = 9.389." (from wikipedia) $\endgroup$ – alec_djinn Feb 3 '16 at 17:23
  • $\begingroup$ What do you think, in case I run the anion exchange chromatography at ph12.3, will Uracil have 1 or two negative charges and cytosine 1 negative charge on oxygen? $\endgroup$ – alec_djinn Feb 3 '16 at 17:23
  • $\begingroup$ Actually, I hadn't thought about the acidity of uracil/thymine. I had only thought about them in terms of basicity. Also, looking again, cytosine does have a basic nitrogen (pKa = 12.2). Uracil's imide nitrogen has a pKa of 9.39, so it would actually be deprotonated in the process. Thymine's imide has a pKa of 9.7. I don't have a chance at the moment, but I will update those equations to show those changes over lunch. In short, cytosines will have a positive charge in both cases (though it will be partially deprotonated at 12), uracil/thymine will go from neutral to negative charge. $\endgroup$ – SendersReagent Feb 3 '16 at 17:45
  • $\begingroup$ @alec_djinn Okay, so it's updated. Realize these are approximate because you're kind of near the pKa for some of them. The higher the pH, the more negative it will be, and I have no idea as to what conditions cause things start to react (e.g. deamination of cytosine to uracil, for instance). At pH 12.3, the imide on uracil is going to be deprotonated, but the amide won't, so it would have be -1. Cytosine, however, would be approximately half deprotonated. The protonated species would be +1, while the deprotonated would be neutral. Neutral cytosine doesn't have any especially acidic protons. $\endgroup$ – SendersReagent Feb 3 '16 at 22:38

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