Chromatography Forum

We are facing a problem in analysis of Stavudine. The mobile phase is as follows,
MP-A:0.77g of Ammonium acetate in 500mL of water pH adjusted to 8.0 with ammonia.
MP-B: Acetonitrile: Methanol (250:250) and 0.5mL of Ammonia.
Diluent: Mobile phase-A: Mobile phase-B (50:50)
Sample concentration is 0.5mg/mL and injection volume is 20 µL.
For 20 µL injections we observe peak split for stavudine. Again we injected a 10 µL injection the peak shape is good. We think because higher response it’s happened.
So we prepared a 0.2 mg/mL solution and injected 20 µL & 10 µL injections. Again Peak split observed for 20 µL injection and good peak shape for 10 µL injections.
At lower level i.e. (0.1% & 1.0%) also peak split observed for Stavudine for 20 µL injection and good peak shape for 10 µL injections.
Remaining peak shapes are good in both 20 µL & 10 µL injections.
What will be the reason, can anyone?
Thanks in advance.

It sounds like a concentration issue. When kind of detector are you using? UV detectors when overloaded can give peak shapes that make it appear as if the peak is splitting into 2 peaks. What is actually happening is the center of the peak is over saturating the detector so the response goes down.

Determine upper limit of detection by making a standard curve. Inject the same volume of sample but make seperate dilutions. Don't just inject different volumes to make standard curve because then there is no way to tell whats causing the problem.

Also are you dissolving the sample in your mobile phase or some other solvent? Its possible the high volume of solvent being injected (if the solvent is different them mobile phase) is causing the chromatography to change.

we observed this problem on two different detectors waters-2996 pda detector and water-2489 uv detector,and the diluent is mobile phase only.

Yea then try to figure out the upper limit of detection and just never inject more then that. Its most likely an overloading problem.

From the symptoms and troubleshooting I do not see how this can be anything to do with the detector.

Check the pH of your mobile phase B and of your diluent - I suspect that they are sufficiently different from mobile phase A to disrupt the chromatography as a 20 ul injection plug hits the column, but not with a 10 ul injecton plug. More or less ammonia in phase B, to get the pH of A and B similar might be a way forward.

Peter