Hello,
Due to high number of genotypes to be analyzed, I prefer to prepare 2 liters of mobile phase for daily use(acetonitrile:water 80:20) and I prepare it by weighing acetonitrile and water seperately because the volume of equipments is not reliable. However beginning from very top of the solution near to end, peak appearances have delayed about 3-4 min. I sonicate the mobile phase for 5 min (and actually I will degass it from now on) and the water we are using is not commercial HPLC grade water it is just ultra-pure water. I am trying to establish a new calibration curve every time that I renew mobile phase however even in the same bottle it makes differences in retention time. I am grateful for your help in advance.
There are many possible reasons for the changes in retention time that you are observing. It does sound like your mobile phase preparation may be the cause (Just use Volumetric flasks or Graduated cylinders and use the same procedure each time. We really need more details about your lab condition, instrumentation and method to provide better comments.
In the meantime, I would suggest that you review the following article I wrote on this topic, which I use in my training classes, on the many reasons for retention time drift and ways to solve it. "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it. " (attached)
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Dear Caner,
How accurately do you weigh the water and acetonitrile.
Do you physically shake the mixture of water and acetonitrile, in order to mix them.
Do you use a column heater/chiller, to maintain a stable and reproducible column temperature.
Kind regards,
Ade Kujore
Marketing
Cecil Instruments Limited
Milton Technical Centre, Cambridge CB24 6AZ
United Kingdom
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As Ade mentioned above, temperature gradients can influence your retention times. If you e.g. have your samples running over night, lab temperature might drop and hence, retention time changes if the temperature of the column is not controlled.
Other reasons for shifts in retention times can be different charges on your compounds. I usually prefer to not use water but try control pH of the mobile phase by e.g. preparing a buffer with a similar pH to your ultra-pure water. Ammonium acetate is good for this as it has a pkA of 4.8, i.e. can buffer from about 3.8 to 5.8. In addition, ammonium acetate is volatile and hence could be moved to the HPLC-MS system. You don't need a very high concentration of buffer and as little as a 10 mM solution will keep your pH in check.
Finally, is the bottle containing your mobile phase relatively tightly close? I it is left open, your ACN will evaporate which changes your mobile phase composition and hence may affect your retention time (I don't know how robustly you have validated the method, i.e. tested the affect of small changes in mobile phase composition).
I hope you find the root cause of your retention time shifts.
There are many possible reasons for the changes in retention time that you are observing. It does sound like your mobile phase preparation may be the cause (Just use Volumetric flasks or Graduated cylinders and use the same procedure each time. We really need more details about your lab condition, instrumentation and method to provide better comments.
In the meantime, I would suggest that you review the following article I wrote on this topic, which I use in my training classes, on the many reasons for retention time drift and ways to solve it. "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it. " (attached)
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Thank you, Willium Letter and Caner Yavuz,
Your answer regarding HPLC retention time shift fantastic and satisfactory.
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Best regards,
Ravi
Ravi Kumar Sonwani Did you found the reason why retention time shifts? I am also facing same problem..could you share with us?
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