Chromatography Forum

I would like to detect Lactate, Acetate and sulfate using PRP-X 100 column with indirect UV method. The problem is, I am not able to get separation of lactate and acetate. (mobilephase 4mM p-hydroxy benzoaic acid pH 10) I need some help in this regard.

thanks in advance

Try decreasing the pH.

There is a pretty old paper:

The determination of lactate, acetate, chloride and phosphate in an intravenous solution by non-suppressed ion chromatography; Haddad,P.R. Croft,M.Y.; Chromatographia, 1986 / 21 / 11, P. 648 -650.

Eluent: 0.3 mM phthalate (pH=6.0), 30% ACN

Acetate and lactate are well separated within 9 min. But no information about sulfate. Iassume that ic comes pretty late.
The paper shows as well that acetate/lactate separation strongly improves from co-elution at 0% to approx. 4 min. RT difference at 50 % ACN.

You might try ACN with your eluent as well, may be with pH change as well.

Use a lower concentration of phthalate(1 mM) and add some MeCN (5-15%).

Bear in mind that conditions that facilitate separation of these early eluting organic acids will likely lead to excessively long retention times for sulfate. You may consider a step gradient to 10 or so mM phthalate once acetate and lactate elute in order to get sulfate off in a reasonable time.

Thank you very much for nice suggestion, will try and get back to you.

Thank again

Separation of lactate and acetate by HPLC :
- mobile phase : 1000 ml distilled water containing 1.5 ml phosphoric acid .
- column : 150 × 4.6 mm , C18
- detector : UV 210 nm
- injection volume : 20 micro litre
- column temp : 25
- flow rate : 1 ml/min
I am applying this simple method usually and it gives very good results

ph/Amr Tarek
Instrumental Unit Head at Otsuka pharmaceuticals

Thank you very much for nice suggestion. will try one by one and get back to forum if get some success.

Hiren