Chromatography Forum

Dear all,

I have developed a reversed phase HPLC method for the separation of two compounds with phenyl column. One of the compounds (A) is very polar and have very short retention time according to the other one (B). Mobile phase: ACN: Phosphate buffer (pH 5.5). But with sodium acetate buffer at the same pH value, B comes first. How can I explain this situation?


Thanks in advance for all answers

I'm going to guess that one or both compounds are acidic. Phosphate is a poor buffer at pH 5.5, but acetate is good. I suspect that the effective pH in the presence of organic solvent is different for the two mobile phases. You can use a pH meter to check the pH of the two different mobile phases. (The neutral point is composition dependent, and no longer 7.0, but that doesn't matter for the purposes of comparison.)

Have seen this phenomenon only a handfull of times in the 10 yrs been working with HPLC. My question is why is it something that has to be explained? Pick the method that gives you the best chromatography for your analytes and perform the analysis. The data should be perfectly sound.

The effective pH of the ACN/Phosphate and ACN/Acetate are different with respect to the pH-dependant separation of both compounds. There is a good chance that compound B is more or alsmost completely ionized in the presence of ther acetate buffer which is stronger under these conditions than is the phospate buffer. Uisng the phosphate buffer, the effective pH could be higher and if B is an acid, less ionized and better retained on the phenyl bonded phase.

The thing that I have been surprised was the polar molecule (A) retained less when using acetate buffer. Because I have tried almost every method (including ion pairing) for less retention of A and have failed.

pH of the mobile phase containing ACN:Phosphate buffer is 6.7

pH of the mobile phase containing ACN:Sodium acetate is pH:7.2.

A is a basic molecule, the other one is acidic.

Thanks for your replies,

I would try to adjust pH of mobile phase after mixing of organic solvent and buffer at pH 5.5 (certainly acetate buffer that has higher buffer capacity than phosphate buffer at pH 5.5). Acetonitril is not very suitable for phenyl stationary phase as organic modifier (try methanol or mixure of methanol/tetrahydrofurane ?).
As well pH > 7 is not very good for phenyl stationary phase.

I still have not found a satisfying explanation.
pKa value of A:12, pKa value of B: 6.8.
I understand that small change in the pH of mobile phase (6.7 to 7.2) affects the retention of B, because its pKa is close to the pH. But A’s pKa is much higher than the pH of the mobile phase, why does its retention be affected so greatly?

A is a base. Therefore it may interact with residual silanols on the packing via ion-exchange. If this is the case, its retention would be sensitive to the concentration of counterions, i.e the sodium concentration in your buffers. So what is the sodium concentration in both buffers, and is this an explanation?

Also, is there a possibility that acetate is acting as a weak ion-pair reagent?

Retention of A is decreasing with increasing buffer concentration (acetate or phosphate, in both cases)
Elution order of A and B is changing only in acetate buffer, I think acetate acting as a ion pair reagent is major possibility.

Thank you very much for your replies,