Respected Sir,
I am doing method development.pKa of drug is 10.24. Column I am using YMC Pack Pro C18 250 mm in length and 3 micrometer in size. on one run of 100 ml of mobile phase pressure is 190kgf, second run of 100 ml of measuring cylinder of mobile phase pressure is 150 kgf, third run 120 kgf. This cylces appears as such. Same is the case with retention time sometimes 5, sometimes 5.5 sometimes 4.3. Mobile phase is 0.1 %formic acid and methanol 35 : 65.
These trials I tried in schimazdu HPLC PDA as well as schimazdu HPLC UV.
Hi, as shown, pressure is decreasing by time not fluctuating, and in this case it seems that your system require more time for equilibrium. If the problem still exist even with a new column and with another mobile phase, the pump system require a full calibration.
You have already asked this same question TWO TIMES, within the last week and received responses before which you did not reply to or respond.Why are you posting it again? Please respond to questions and participate in a discussion to solve the problem.
- https://www.researchgate.net/post/Why_there_is_pressure_fluctuation_in_hplc_how_to_overcome_this
- https://www.researchgate.net/post/What_is_the_reason_of_fluctuation_in_retention_time_in_hplc
You still have not provided the details of your chromatography conditions or provided any information about the column dimensions, system used or troubleshooting done so far. Isocratic methods should have the same backpressure throughout the run time. Significant variation as you have indicates you have not addressed the basics.
In the meantime, if you would like our help, please provide answers to the questions which were asked previously (and again) and review these linked articles to troubleshoot the problem.
- "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."
- "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)"
http://hplctips.blogspot.com/2015/1...lc-retention-time-drift-change.html
http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html
pka of drug is 10.24 you may change the mobile phase and adjust pH OF YOUR MOBILE PHASE
Thank You Sir for your valuable suggestions. I will try the suggestions given by you
First of all, you have to equilibrate for 15 min for every run.
Second, do you use gradient or isocratic system. In gradient system fluctuation of pressure is normal.
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