Chromatography Forum

Hello,

We are working on a method to determine organic acids in food samples with HPLC-DAD. We are using a strong anion exchange SPE cartridge (Strata-X-A 33u Polymeric Strong Anion) from Phenomenex for the purification of organic acids (Formic acid, malic acid, lactic acid, acetic acid, propionic acid and citric acid).

The SPE-cartridge has a capacity of 0.3 meq/g. Because we’re using cartridges of 200 mg, our capacity is 0.06 meq. I’ve calculated that the six organic acids in a phosphate-buffer have a capacity of 0.056 meq in 0.5 ml.
The procedure was:

1) Conditioning: 6 ml MeOH, followed by 6 ml H2O
2) Sample loading: 0.5 ml of organic acid standards (500 ppm) in a phosphate-buffer (0.1 M)
3) Drying for 5-10 seconds
4) Eluating with 4 ml of 0.5M H2SO4

We became high recoveries (80-90 %) with this procedure, but when we use a washing step (4 ml MeOH or 4 ml Phosphate-buffer), the recovery decreases to 20-40 %.

Can anyone help and explain why this is happening? Or what solution we can use to keep the high recoveries? We will need a washing step when we use this purification on food samples, because they contain a lot of interferences.

Thanks for your input!

I'm actually surprised that you got as good a recovery as you did without the wash. I've always used the rule of thumb that you should load no more than about 1/3 of your total capacity to avoid the risk of breakthrough. What you're seeing is your wash solution eluting a substantial portion of your acids. If this were my problem, I would use a smaller sample and/or a larger cartridge. If that's not feasible, using a lower ionic strength buffer for the wash might help.

The pH of the sample solution is important. The more acidic, the less organic acids are kept. Also the phosphate from the buffer starts eluting. Therefore you get lower recoveries after washing with phosphate buffer. MeOH may also elute as long the organic acids are not totally dissociated.

pH of the sample should be 2 pH units higher than the highest pKa of the organic acids of interest. To make sure that all acids are deprotonated.
Also all strong acid anions will be retained on the SPE and at least partially will be eluted by the sulfurc acid elution.