Problem with Sample's pH

[cheebeng]

Hi all,
I am new to this forum.

I have a problem with my HPLC analysis.

I need to analyse a sample contain Shikimic acid (pKa 4.48) and Caffeic acid (pKa 4.62). HPLC solvent system that I am currently using is gradient elution of 97parts 1%H3PO4 solution :3parts MeOH, holding for 3.5 minutes then slowly increase the portion of MeOH to 99parts at 7minute and hold for 5 minutes before back to original status.

My problem is:
During normal analysis, my sample is prepared in the HPLC solvents, the separation is fine.

But, when I prepare my sample in different solvent system (water at pH 4.6 buffered with 25mM Potassium hydrogen phthalate (due to experimental need), I cant get the separation anymore. I am using 150X4.6mm C18 Column.

I know it is due to the pH change in the sample, but I have no idea how to solve this problem, maybe someone in this forum can help, thanks.

I have tried to change the HPLC solvent from 1%H3PO4 to the same buffered solvent, but doesn't seem to work.

[Alexandre]

If you have enough of sensitivity just dilute your sample to needed pH with acid or acid/MeOH. If needed infect more of this.

[cheebeng]

If you have enough of sensitivity just dilute your sample to needed pH with acid or acid/MeOH. If needed infect more of this.

Thanks. But my instrument sensitivity does not allow me to make such dilution as suggested.

Anyway, thanks for reply

[prepcolumn]

Your diluent pH is so close to pKa so acidification is important..if you cannot dilute your sample so much for sensitivity issues you may try this for ex:

Take 8 ml of sample add 1 ml conc acid (you need to optimize the acid amount and conc. to get your target pH) and dilute to 10 ml. In this case you may get the required pH and do not loose so much sensitivity.

[cheebeng]

hi guys,
the problem solved using the suggested solution. thanks to all of you.