Hello,
I'm having problem with an intense dip between my runs of a HPLC method. It happens right after the first run.
When the first run finishes, the absorbance of the blank run is too high and it gets flatten at that high absorbance for two minutes while DAD is balancing. When the detector gets balanced, absorbance decreases to zero and in the next 2-3 minutes before the start of the next run absorbance goes down to -100, -120, -140mAU and more negative values till the next run starts.
This intense dip is not repeatable since starting negative values are different and because of this, subtracting sample chromatogram from the blank doesn't give a repeatable result in most cases.
This problem wasn't seen before and is happening for a month. Nothing in the method has change which is as below:
Runtime: 25min
Flow: 1mL/min
Gradient
Mobile A: Buffer of sodium dihydrgen phosphate with pH=3.00
Mobile B: 30/70 Mobile A:ACN
I'm attaching three images for the first blank run, the second blank run and the sample run.
According to your mobile phase condition, it is in isocratic mode.
The intense dip would be equilibrium may not be reached by running time.
You can try to test a run with extention to longer time but let see less than 60 min.
plese check whether the solvent used for sample is same as that of mobile phase.
Thanks Santi,
The method isn’t on the isocratic mode and as I mentioned it is on the gradient mode as below;
time: 0, A:80%, B:20%
time: 20min, A:32%, B:68%
time: 21min, A: 32%, B:68%
time: 21.01, A: 80%, B:20%
time: 25 min, A: 80%, B: 20%
Sample solvent is buffer similar to mobile phase A. Standard solvent is Water.
This problem of the positive shift from the minute 7 and the negative dip at the first of the run couldn’t be seen for the first 50 runs.
could the negative run be due to the system peaks?
Santi Kongmany
Dear Shirkhani,
Thank you very much for providing more detail on your problem.
If you think the problem is on the system, you can check the pressure of LC pump. UV/DAD signal is usually fluctuated by pump's pressure change. So, please make sure this point.
If LC system is not the problem, please make sure there is no contamination after running many samples. If your sample is dirty, contaminant accummulation may occur. If this a case, try to reverse column and back-flush with strong organic solvent, i.e. methanol, for 10-20 min at a flow rate of 0.1-0.5 mL/min. Then reinstall column in normal flow direction, purge mobile phase you use for separation, set flow from 0.1-1.0 mL/min slowly by making sure that the pump pressure is stable before increasing flow rate. note the flow vs. pressure. Keep the system run for 20 min. Finaly, try to run just DI water and mobile phase B as samples and compare the signal.
Good luck.
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