Chromatography Forum

My compound has 2 pKa: 3 and 8. Is the buffer pH at 5-6, like ammonium acetate, the best choice for RP HPLC mobile phase A? Can I use 0.1%TFA in H2O?

There are four major issues with pH selection:
- column lifetime
- method robustness with respect to minor pH variations
- retention
- selectivity.

Taking them in reverse order:

1 selectivity is always a gamble. The only way to find out is to do the experiment.

2. Retention is reversed-phase will depend on the degree of ionization, with the neutral form of a molecule being more strongly retained than the ionized form. If the rest of the molecule is sufficiently hydrophobic, you can get reasonable retention even with an ionized functional group.

3. Robustness with respect to pH is generally the worst at or near the pKa (that's where retention changes most rapidly as a function of pH) and generally the best 2 or more units away from the pKa. The exact meaning of "worst" and "best" depend on the details of the specific molecule and system. Note that "lack of robustness" is essentially synonymous with "extremely fine control of selectivity" .

4. Column lifetime is generally best between pH 3 and pH 7, and falls off outside that range, unless you are using columns designed (and promoted!) for high or low pH operation.

I'm assuming your compound has two acidic groups. In that case, running near pH 6 will have the low-pKa group ionized and the high-pKa group neutral. If the rest of the molecule is sufficiently hydrophobic to give reasonable retention, you should have a reasonably robust method. Running at pH 2.1 or thereabouts (0.1% TFA) will have the low-pKa group slightly ionized (if you are 1 pH unit below the pKa, you will have approximately 10% ionization). That should give you greater retention, and may give you some control over selectivity, but may also result in less robustness with respect to pH.

It's hard to tell what is the "better" choice without trying both!

If your compound has one acidic and one basic group, then you will get best retention where the compound has a net neutral charge (halfway between the pKa's or 5.5). If it has two acidic groups, then your best retention will be at low pH where both groups are neutral. Check your column specs, but most columns don't like mobile phase with pH < 2.5. If your compound has two basic groups, then you will get best results with a column used for basic drugs. Are you using HPLC/UV? If you are using mass spec detection, then compound ionization is also a factor in detection.

You can use a column with hybrid particules like XBridges (from Waters)
You can easily work at pH 2.0 or 2.5 with these columns.

Have fun

If your analyte has one carboxylic acid group and one aliphatic amino group, it is doubly charged at intermediate pH and will have the lowest retention around pH 5. With other words, you will get the best retention at pH 2 or pH 10.

Thanks everyone for the tips! The compound has 2 basic groups. I tried both 0.1%TFA and ammonium acetate pH5, they both gave good separation except with 0.1%TFA, one impurty peak was slightly asymmeric.

The compound has only about 0.02mg/mL solubility at pH 5, do I need to worry about compound precipitation on column when I use ammonium acetate as buffer?

Overall, which buffer should I choose?

Analyte solubility is only an issue in preparative chromatography. In analytical chromatography, dilution with mobile phase is so fast that I would not worry about it. In addition, if you would have a problem, you could get a distorted peak, which would tell you that you need to improve the method...
Since everything is rather equal, I would go for ammonium acetate, pH 5. It is milder on the column, and thus your column life may be longer.