Hello, I've been running Budesonide in my HPLC system and I was getting my spectrum with the separation of the two epimers as two separate peaks. But since a few days back the two peaks have poor resolution with a peak fronting which is very hard to differentiate. I have not changed any parameters. Can anyone tell me what could have caused this? My conditions are,
C18-A 150 x 4.6 mm column
Flow rate - 1.5 ml/min
Column Temp - 50 C
Mobile phase - ACN:Buffer:EtOH=34:66:2 (Buffer consists of 0.005 M disodium hydrogen orthophosphate pH adjusted to 3.2)
thanks in advance!
Assuming nothing has changed in the method used, the real reason could be related to column performance. Most fo the column manufacturers won't recommend phosphate buffer and high column temperature combination as this has effect on performance.
You may have to refer the column care section of your column manufacturer's website for things to avoid for better column performance.
If you are using wrong wash-off solvent, that could also cause precipitation of phosphate buffer salts eventually.
Hello Shehan Kaleel, there could be many reasons for your problem. Have you checked your HPLC system, whether all components such as pumps etc. are still working properly? It can often happen that a part of your HPLC system has to be repaired or replaced.
If everything is working correct, it could be the column. You may contact the manufacturer of the column. Most they find a solution for your problem.
Miss/Mrs Kaleel,
This fact does not represent significant problem for your analysis, including in quantitative terms, because of the most important step is the mass spectrometric based determination of the analytes.
Please, consider the following discussion, in this context, and the corresponding reference section, therein:
https://www.researchgate.net/post/How_to_use_Sephadex_Column_to_isolate_aminoglycoside_compounds
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