Thank you for the reply. Do you mean triethylamine for TEA? If so, I didnot use any TEA in the previous wash. The ion exchange (IX) solid phase (SP) contains a linker-piperazine with pKa of 6, so I load the orotic acid (ORA, pKa1=2.8) with a pH 4.4 (half of 2.8+ 6) solution to make sure both SP and ORA are ionized. Then I use 2%Formic Acid/water (pH=2) to wash (should not elute any ORA out), and then use NH4OH in MeOH/ACN to elute ORA. But the result shows the acidic wash step elute a lot ORA. So I am wondering that the 2%fomic acid may ionize ORA with its pKa2 or pKa3. But I don't know how.
【在 s****2 的大作中提到】 : if it forms TEA salt, say orotic acid.2(3)TEA salts, it readily soluble in : DMF/ACN, even DCM.
【在 c******m 的大作中提到】 : Thank you for the reply. Do you mean triethylamine for TEA? If so, I didnot : use any TEA in the previous wash. : The ion exchange (IX) solid phase (SP) contains a linker-piperazine with pKa : of 6, so I load the orotic acid (ORA, pKa1=2.8) with a pH 4.4 (half of 2.8+ : 6) solution to make sure both SP and ORA are ionized. Then I use 2%Formic : Acid/water (pH=2) to wash (should not elute any ORA out), and then use NH4OH : in MeOH/ACN to elute ORA. But the result shows the acidic wash step elute a : lot ORA. So I am wondering that the 2%fomic acid may ionize ORA with its : pKa2 or pKa3. But I don't know how.
Thanks. I tested the post-loading solution, there is no ORA left in the solution. so I think it is not because of overloading. I tried 0.1%formic acid/water (pH 2.7) instead of 2%formic acid/water (pH 2) to do the wash. This time no ORA is washed out. So I guess the 2%formic acid may have very high ion concentration that could shorten the ORA retention. But I am not sure about this hypothesis.