Hello fellow researchers, i started to run several HPLC methods to quantify an enzyme activity. I incubate my Enzyme (different mutants) with my substrate (futalosine, quite hydrophobic). To seperate the molecules i run a RP-HPLC with a LC-18 column (analytical) and use a Water-Ethanol gradient to elute my molecules. Few months ago everything seemed fine, i could always identify the peak corresponding to my substrate and a product peak as well. Since we only have one HPLC a coworker was using the device as well but for his experiments he used acetonitrile instead of ethanol. After i started to use the device again i washed the column with 15 CL water and ethanol and started my experiments again. But since then my peaks have horrible shapes and sometimes there is literally no signal at the expected RT. Now there are also very high intesities at the end of the run (~2000 mAu) which was never the case. Therefore i used a standard to see if the effect is due to my sample preps. I attached 2 files of my standard (HPX) in which one it is visible that the same molecule and prep shows a different signal intensity. Setup and method for this runs are identical. HPX1 is my standard i used shortly after i started with the HPLC again and HPX2 is the same standard run few days ago, after i notice the very high intensities at the end of my runs. Does anyone had similiar problems in the past or could help me out with tipps, why my runs got wrose day by day?
I am looking forward to your response
Hello, the only thing that came to my mind, after reading your question, is that acetonitrile (ACN) is causing the problem. Do you know if your college purge de HPLC system with ACN and leaves it in this solvent for a few days??
Because, ACN is the worst solvent to purge the HPLC system due to this solvent polymerize and can cause saturation in the system. I mean “purge” the action to get the entire HPLC system in organic solvent to prevent the growth of biological “stuff”.
So, if the system is damage because ACN, maybe you can try to clean with hot water and inject again you standard. Another thing is that maybe your column is damaged because ACN polymerization has entered into the column. So, you have to replace the column.
Hope anything of that helps you.
Laura Trabalón Escoda
Dear Laura, thanks a lot for the answer. Since we are pretty new into the HPLC and only know the basics (we happen to have a HPLC device from previous coworkers). We followed the manual and everytime we put in a new solvent we "purge" the system. I dont know if u refer to that...but in our manual its meant to get rid of air bubbles in the cable connecting the lube in the solvent with the system. Since he used the hplc for around 2 weeks the whole system (and column) was stored in ACN itself. I hope i could answer your questions...but if its really ACN that polymerized in the column then do we need to purge the system with water/methanol after every run with ACN?
Best regards and thank you for this helpful information!
For a better picture i will attache 2 files with previous peaks (first picture) and the peaks of the same compound (same methode, setup and preparation) of the recent runs (second picture).
Hi Duc,
Yes, I refered to that when I said "purge". But is better to leave the system in methanol when you finish your analysis. And for the column, every type of column has a manual that explain in which solvent is better to leave to save it. So, I recommend you that use methanol instead of ACN to flush the system. Thanks
It appears that the analyses are incompatible with one another. Thus, it is more likely that the same column is being used for both analyses. Get a 'new' column and dedicate it to your analysis alone. Remember, other 'crap' is being injected on the column than just your molecule of interest.
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