I am doing reverse phase HPLC using C18. Keeping the flow rate and composition of solvent constant, I should get my target peak at 14 min, but I am getting the peak sometime at 14 minutes sometime at 4 min and sometime at 6 min. I am keeping all the parameters constant at every run. Can anyone help me with my problem?
Dear Manojukumar
There are a few things that I can think off hand that may be happening during your chromatography:
1. your compound that you are separating may be unstable and are degraded or oxidised over time giving you the change in profile
2. your compound may be aggregating with time or at higher concentrations and you are seeing the aggregates at the earlier retention times
3. the column is not properly reconditioned (washed and equilibrated) before the next run. I normally use at least 3x the Vo (or injection peak volume) for washing with 90% organic solvent and 8 times the Vo for equilibration before injecting the next sample.
4.your HPLC program ito reconditioning is changing from one run to the next - make sure that if run is 30 min with reconditioning included, you inject every 30 min and not delay between runs - some separations are quite sensitive to such delays.
5. If you are working with methanol (or an alcohol) as solvent and you modify it with acid such as trifluoroacetic acid or formic acid as modifier, remember that with time the carboxylic acid and alcohol with forms esters and ageing changes the solvent system.
6. If your HPLC pumps are not working properly - both pumps must deliver the same volume regardless of back pressure the program, albeit isocratic or with gradient, will change because the flow through the system changed. Check for leaks, bubbles and volume delivery. Problems with latter is mostly due to faulty check valves.
Good luck with your HPLC runs. Hope my advice helps to to success.
Regards
Marina
Dear Manojukumar
There are a few things that I can think off hand that may be happening during your chromatography:
1. your compound that you are separating may be unstable and are degraded or oxidised over time giving you the change in profile
2. your compound may be aggregating with time or at higher concentrations and you are seeing the aggregates at the earlier retention times
3. the column is not properly reconditioned (washed and equilibrated) before the next run. I normally use at least 3x the Vo (or injection peak volume) for washing with 90% organic solvent and 8 times the Vo for equilibration before injecting the next sample.
4.your HPLC program ito reconditioning is changing from one run to the next - make sure that if run is 30 min with reconditioning included, you inject every 30 min and not delay between runs - some separations are quite sensitive to such delays.
5. If you are working with methanol (or an alcohol) as solvent and you modify it with acid such as trifluoroacetic acid or formic acid as modifier, remember that with time the carboxylic acid and alcohol with forms esters and ageing changes the solvent system.
6. If your HPLC pumps are not working properly - both pumps must deliver the same volume regardless of back pressure the program, albeit isocratic or with gradient, will change because the flow through the system changed. Check for leaks, bubbles and volume delivery. Problems with latter is mostly due to faulty check valves.
Good luck with your HPLC runs. Hope my advice helps to to success.
Regards
Marina
The most probably explanation for such large differences are leaks in one of the gradient components before mixing (if you know the correct retention time, you can figure out which one) or a huge leak after mixing.
thank you Marina Rautenbach and Niel Jensen. I will recheck the things u told me. Thanks a lot for your guidance.
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