Chromatography Forum

Hello,
I am new to this forum and hello to everyone. I recently had a problem with my LC which has split peak of API. I did reverse the column and back flush to clean the inlet frit. It seems to be my peak has improved alot but has FRONTING. can any one plz help me to reduce this?

Thanks,
Kish

Kish,

If you provide more chromatogram details and/or a picture it would be helpful to determine the problem.

One possibility is that the fronting and peak splitting is occurring because of overload, i.e. you are injecting too much analyte on the column. Try reducing either the analyte concentration or the injection volume.

Again, if you give more details, then the forum members can be better able to assist in the problem solving.

Regards,
Dan

Other possibilities include limited solubility in the mobile phase (which, I suppose, would be included under "overloading"), or a sample diluent which is stronger than your mobile phase.

As Dan said, more information would be helpful.

I am using 4.6X150 mm Waters Spherisorb 5um CN RP and for API in Cough suspension. I am injecting 20uL and The maximum analyte should be 0.12 mg/mL. I didnt have this probem earlier. I found after inhjecting almost nearly 1000 samples. I thought after reading valuable infomation in the forum due to clogged frit inlet and did reverse the column, peak shape is good but fronting.

Mobile phase is : 75%Acetonitrile, 10% Methanol and 15% 5mM Dibasic Phosphate Buffer adjusted the pH ~5.3


thanks,
Kish

KISH,

Offering my best hunch here---you are over-loading your column by injecting too much sample and there are not enough theoretical plates to enable selective binding of your target analyte.

Please reduce your maximum injection volume to 100 uL, then set up the x2 as "dilution factor" in your HPChemStation software sample table to correct your quant on the back-end.

By injecting less volume the peak fronting should dissipate to a level that is visually acceptable to you.

Hi Kish,

Your so called buffer has absolutely no buffer capacity at pH 5.3 and that could cause both peak splitting and fronting. The reason you didn’t have the problem before could be changes in the cough suspension’s pH. If it had pH 5 before and now changed to 6 or 7, it would be a perfect explanation.
If you want to avoid situations like this in the future, maybe you should try another buffer e.g. acetate.
Or play around with the pH – maybe a low one e.g. 2.5 phosphate buffer or something like that.

Good luck