Chromatography Forum

Hi, I would like to know if anyone can explain the advantages and disadvantages when choosing among phosphates or acids like acetic or formic as modifiers. I am trying to separate in a C18 small acidic metabolites, which are very soluble in water and polar solvents. I understand that I need them as unionized as possible for RP separation. I know that phosphates are messy (headache for cromatographs) because you would have to clean a lot, but there is a lot of references with phosphate methods...why?

For your application, you may need low wavelength UV detection. Phosphate has very low UV absorption <230 nm so you get a clean baseline. Phosphate also buffers around pH 2--3, which is where you need to be for organic acid analysis. Acetic or formic are much weaker acids, and you would need so much that the UV signal would be useless. Phosphoric acid is available in excellent purity at a moderate price.

For your application you will want high ionic strength which you can get with 40--100 mM phosphate. As an alternative, you can use sodium sulfate acidified with sulfuric or methansulfonic acid; we recommend that for our Acclaim OA column.

If you want to use MS or ELSD detection, then you must avoid non-volatile acids and salts. Then UV does not matter, and you just load up the mobile phase with formic acid and ammonium formate.

Thank you Mark! So, the main difference lyes in pH stability and UV absorption... I asked this because my column providers don´t really liked very much phosphates and this gave me some doubts about their use.

Phosphates are only a problem with high organic concentrations in the mobile phase, particulrly acetonitrile. In your case you will be using fairly low organic concentrations so it shouldn't be a problem.

you might also want to consider that phosphate tends to attack silicates with prolonged exposure.

mdfenko, how does phosphate attack silicates?

I wouldn't say that "phosphates attack silicates" but according to this reference:

J.J. Kirkland and al., J. Chromatogr. A, 762 (1997) 97-112

phosphate buffers at higher concentrations and higher temperatures can cause significant hydrolysis of the bonded phase comparing to other buffers...

Best regards

I agree with what has been said on this subject thus far. But, I would like to suggest TFA (trifluoroacetic acid). I used to prefer acetic acid and formic acid, but it takes alot less TFA to bring the pH down. Its pH range is about 1.5 - 2.5. There are also ammonium formate and ammonium acetate with useful low pH ranges . . . . and heptafluorobutyric acid, pentafluoropriopionic acid, etc.

mdfenko, how does phosphate attack silicates?

phosphoric acid etches glass when in contact for periods of time.

While concentrated H3PO4 can etch glass, at the dilutions used in HPLC (<20mM), there is not a serious problem.

The observation that phosphate buffers shorten the life of columns is only true at alkaline pH, and is largely accounted for because of the shift of pKa in water/organic mixtures. So if you make a TRIS buffer and a phosphate buffer in water to pH 8, then dilute to 50% in acetonitrile, the pH will shift higher for phosphate and lower for TRIS. Not surprisingly, the HPLC column will have a shorter life with the phosphate mobile phase prepared in this way. Other anionic buffer salts (carbonate, borate...) behave similarly.

Thanks Mark, another myth deflated.
Incidentally, life thinks phophates are great (at the right concentration and pH).

Completely agree with this last comment. It's been an illuminating discussion. Thank you all!