Chromatography Forum

Hello everyone,

My HPLC mobile phase has a pH of 8.5 (ACN: Phosphate buffer) while my sample for injection has a pH of 10.0. The column tolerates high pH. I was wondering if this difference in pH may cause problems in my analysis. Is it usually recommended to keep the pH of mobile phase and the sample the same?

Thank you,
Jeegar

It depends upon the property of the analyte. It is advisable to have the identical pH but u may have different pH.

Thank you Pawan for your reply. The analyte is acid sensitive therefore I thought of raising the pH of the sample to avoid its decomposition. But I am still wondering if it would work in the HPLC.

You may want to take a look at the contributions to syx´s "Story of 2 active substances in a dosage form".

jeegar,

There is no a priori reason why you can't use a sample pH different than your eluent pH (any more than you always need to match the solvent content of your mobile phase in your sample) but several factors need to be considered:

1. Will the sample pH cause column damage? This isn't too likely in your case as the exposure to sample pH should be mitigated by the buffering effect of the mobile phase buffer system.

2. Will the sample at the selected pH overload your buffer system? Again, this is also not too likely but should be tested chromatographically (look for retention time shifts and peak shape abnormalities as you vary sample pH)

3. Is the pH difference between sample and mobile phase likely to result in focusing or defocusing? If the analyte will be more retained at the sample pH than at mobile phase pH, the system is focusing and the pH difference will have no effect on chromatographic performance (or might actually improve it). If the inverse is true, you might see peak distortion if the mobile phase buffer system is overloaded.

As HW mentioned, pH differences can cause a problem, but if the pH of your analyte solution and mobile phase are both not too close to the pKa of the analyte, you may be able to get away with it - assuming that there are no pH dependent solubility issues, that your injection volume isn't too large, and that the buffering capacity of the diluent is not significant when compared to that of the mobile phase...

Thank you all for your helpful messages. The analyte has pH-dependent solubility. As pH goes higher it's more soluble. It is a weak acid with a pKa of about 9. So, at pH of 10 (the sample pH) it is ionized and more soluble.

Thanks again,
Jeegar

jeegar,

Based on the properties you describe, I would expect that a sample pH of 10 will be defocusing since in reversed phase highly ionized solutes are invariably less retained (assuming they are monobasic or monoprotic) and as such, injecting a sample with a pH of 10 has a significant potential for distorting the analyte peak shape unless the buffer capacity of your eluent system is sufficient to eliminate this problem. If you have a strong practical motivation to use pH 10 for the sample pH, you might want to consider boosting the buffer strength of your mobile phase if peak shape or retention time stability suffers with your current buffer system when the sample pH is 10.

Although many column manufacturers are claiming the stability of column a higher pH but the column life is comparatively low. With time the change in the retention time and resolution may occur. I will suggest to consider this factor before finalizing ur method at high pH like 8.5.