Chromatography Forum

I have two synergi Hydro RP columns 4.6x250, 4um and 10 um.
Isocratic 100% 0.1% H3PO4, 1 mL/min, room temperature, 210nm,

4-hydroxy butyric acid gave a nice peak at ~8min; but the cyclic gamma butyric lactone gave a tailing peak (relative broader too) under both columns.

I tried two diluents 1) 90% ACN + 10% H2O, and 2) 0.1% H3PO4. Both gave the same tailing peak for both columns.

The concentration was about 2mg/mL, injection 10ul. signal at 0.08AU.
Was the column overloaded? If not, what could be the reason causing the tailing?

Hello YM3142,

I am not sure if this post might help, but i thought i'd post it anyways. I think you should try reducing the concentrations and see what happens with the peak shape. Long time back, almost 10 years, i worked with the hydroxycitirc acid lactone and i remember using a Potassium hydrogen pthalate/phosphoric acid buffer at pH 2.5-3.0 with Methonol/Water mobile phase (i don't quite remember the proportions) and we had very good separations.

I do remember me and my colleagues quizzing on the lactone breaking up to form the free acid and vice versa, but i believe that the lactone should be quite stable.

Suresh.

Can you tell us the retention times, peak widths and asymmetric factor of the two peaks?

Suresh.

I guess these column may be old.

Suresh, Thanks for your input.

Here: ref to my 1st post for other info

column 10um
lactone RT 15.1, tf 3.0, plates 1345
OH BA Rt 7.7, tf 1.4, plates 4745,

column 4um
lactone RT 9.2, tf 3.8, plates 4299
OH BA Rt 5.1, tf 1.2, plates 15348