Hello! I am having a mobile phase problem in the HPLC-UV. Where are measuring peptidoglycan composition of S. aureus MRSA. We run the samples and the buffer used for the samples as a blank.
Solvent A: phosphate buffer with 0.00025% sodium azide
solvent B: meOH
When we substrate the bank to the samples we lost definition of the peaks (thing that did not happen in the past).
We also observed that the intensity of the new blank increases the double. We change columns filters prepare new solvent and try several injection.
Somebody could give an advice?. What could be the problem?
Just check the system with dummy run, if still problem persists then wash the column with minimal flow rate (0.5mL/min) about 2 hrs each by series of solvent IPA, MDC, IPA & water. Earlier I have faced this same issue for 2-Fluoro adenosine.
In addition to the previous answer:
I take that "intensity of the new blank increases the double" means an increase of the baseline?
Be sure that the water used for preparing solvent A is OK. Try water from a different source in case of doubt.
Always use HPLC solvents. Ultrafilter (0.22 um) both your mobile phases and sample prior to injection. Wash your detector cell.
Thank you Bruce and Balaji . We do all of that. Actually the runs where well and one day and start to go wrong. Thats why we change everything. we clean everything We stabilize again with the new column, etc etc. But still is still going wrong. Could be the detector??
Can you please provide column details viz chemistry, equipment info so that I can help yo0u better
Hi, It would help if you could further explain what you mean by 'When we substrate the bank to the samples we lost definition of the peaks ' and 'We also observed that the intensity of the new blank increases the double.' If your problem is with peak shape and/or separation of peaks then you might want to test the performance of the column using the manufacturer's test mix/mobile phase to check that the retention and peak shape/efficiency of your columns is good. What pH are you using and how are you checking this?
Are you using a reference wavelength on your UV/VIS detector (you should not be)? If so, that could cause the problem you describe. Otherwise, we really need more information to assist you (full method details).
You should only re-zero (balance) the detector one time. That time is at the very start of the analysis (Time zero). Is that what you are doing?
You did not respond with any information about what bandwidth you are using or if you are using a Reference Wavelength. Ask you teacher for help.
sorry I dint answer! Actually we called a technique and was the UV lamp. they change it and there is everything ok!. Thank you very much for all your advise and help!
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