HPLC buffer pH 4.0 reading at 206 nm

[WillyOne]

Hi

I'm looking for a buffer able to work at 206 nm.
The only one I have found is Phosphate Buffer but its buffer capacity ranges to pH 3.1.

Thank you in advance

[tom jupille]

If the concentration is low enough, you might be able to get away with formate.

[WillyOne]

Thank you.
I'm going to check 5 mM potassium formate...
I need to separate Citric and Gibberellic acids.
Typical Buffer for Gibberellic is pH 3.
It works well when analyzing Soluble Liquid formulations.
But in soluble tablets there is something coeluting, perhaps Citric acid.

Regards

[mattmullaney]

Hi Guillermo,

Just a question...if you need to separate gibberelic and citric acids, could you not use, say, 0.5% or 1% (v/v) TFA in water as the weaker eluent and work closer to pH = 1 or so? TFA is fairly transparent at low UV, or maybe perchloric acid? May not want these in a MS detector, but either should be okay with UV, I'd think.

This way, you'll be working around two pH units less than the pKa values for gibberelic acid (ca. 4) and citric acid (lowest pKa ca. 3.15).

For a column, you could then use a Type A silica like Waters Spherisorb ODS, Phenomenex Superspher ODS (LiChrosphere in U.S.) or Agilent Zorbax ODS, all non-endcapped, or maybe something newer such as Waters Atlantis T3 or Agilent Bonus-RP or Polaris. You may not need endcapping at all if you only must separate acids, just an acidic eluent and a low ligand coverage silica, or limited coverage on newer phases.

In any case, best luck and best wishes!

Matt

[WillyOne]

Thank you for your helping.
The case is that I analyze Gibberellic formulations as soluble liquids without problems.
Typical conditions are pH 3 Phosphate buffer 100 mM/ Acetonitrile 60:40; flow 1 ml/min. UV detector reading at 206 nm.
Sample is a Methanol solution of 500 mg/l and injection volume 10 µl.
The difficulties begin when I tray to analyze autodispersible (fizzy) tablets. I know that formulators use Citric acid supposedly to react with NaCO3H to disperse the tablets.
The Upper method doesn't work. So I suppose that I have a coelution of citric and Gibberellic acids.
I have tried to reduce the pH but the problem persist. (pH 2.1 Phosphate Buffer).
Next idea has been to increase the pH in order to keep citric as anion water soluble and Gibberellic unprotonated.
Citric pKa 3, Gibberellic 4; this is not a big difference....
So I was looking for a pH 4.0 buffer to work at 206 nm.
I've conducted some experiments having bad results.
Really I don't have any idea how can I get over it.

Sorry by tears

[mattmullaney]

Understood...is it an option to try the second pKa of phosphate, try ammonium phosphate, around pH 7.2 or so...should still be okay with UV at 206 nm, again using a phase like Atlantis T3 or Bonus RP, mid-range in coverage, or an older Type A silica with ion-pairing such as a tetrabutylammonium phosphate? In this case, everything would be in base form and paired with the TBA cation...

[Mattias]

An unorthodox solution could be to use (non-pH adjusted) dihydrogen phosphate. That will give a pH of about 4.

I know that the buffer capacity is very low and that it is against all HPLC theory. But it could work... And it is UV transparent.

[mattmullaney]

Matias...pretty cool idea, that May Work! Wish I could've thought of that myself...certainly worth a try!

[WillyOne]

Thanks a lot everybody.

I will work on your suggestions next week.
I'll keep you informed of the results

Thanks again