I made a buffer as follow:
275 ul TEA
550 ul phosphoric acid
then adjusted the pH to 3.30 with KOH (10%).
My peaks came out allmost perfect. I had tailing on one of the 4 components, which is a weak base and thus is protonated.
So when I changed the amount of TEA to 325 ul it resulted in one big tailing peak.
what am I doing wrong?
Dear Michel,
Not everything is clear for me from you information, however I think the problem is in solubility of TEA in water. You do not provide volume so, it is difficult to advice exact solubility. TEA is a tertiaryamine and it is weakly soluble in water. When it became protonated (in acidic pH) it turns to soluble product. However, this reaction requires enough strong acid. When you increase volume of TEA, I guess you get mixture of protonated TEA and unprotonated which is insoluble. I need to add more acid or use hydrochloride of TEA to increase solubility. Note that 1 mol of TEA requires 1 mol of available protons. Phosphoric acid has pKa1 ~2, but pKa2 is 7. It can provides only limited amount of protons required for TEA protonation. Add more phosphoric acid or other acid or oppositely, decrease amount of TEA. I hope, it helps little.
best, Artur
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