In HPLC analysis I am getting a negative dip in chromatogram before peak of compound. Mobile phase is Methanol:water 70:30 +0.01% acetic acid. After washing of column with hot water, the dip disappears and reappears again after few runs. Please suggest what should be done.
The negative dip always appear in HPLC chromatogram at retention time between 1-2 min due to the dramatic change of pump pressure when it switch from injection mode to running mode (flushing sample to column). The shifting of the negative dip is also dependent on the mobile-phase flow-rate.
Back to you problem, the disappearance of negative dip after washing with hot hot water could be due to some compounds from injected sample still remaining in column. Important point to check is to observe the change of pump pressure. Is your pump pressure increased after a few run of your sample?
Another reasons may be possible.
However, you can try to use higher detection wavelength. If you use 254 nm, you can try to increase to higher about 10-15 nm (250 nm to 260 nm). This is the correction of absorption by solvent and so on.
In the new HPLC system, the baseline correction function is available.
The negative dip always appear in HPLC chromatogram at retention time between 1-2 min due to the dramatic change of pump pressure when it switch from injection mode to running mode (flushing sample to column). The shifting of the negative dip is also dependent on the mobile-phase flow-rate.
Back to you problem, the disappearance of negative dip after washing with hot hot water could be due to some compounds from injected sample still remaining in column. Important point to check is to observe the change of pump pressure. Is your pump pressure increased after a few run of your sample?
Another reasons may be possible.
However, you can try to use higher detection wavelength. If you use 254 nm, you can try to increase to higher about 10-15 nm (250 nm to 260 nm). This is the correction of absorption by solvent and so on.
In the new HPLC system, the baseline correction function is available.
Substract the chromatogram of mobile phase from chromatogram of sample.
From your problem description, it seems you are running an isocratic method. You could change your elution to a pseudo-gradient analysis by leaving your method as is but adding a "Wash" step at the end of your present method.
If a rinse with water helps, you could change the flow to 100% water for several minutes before changing it back to your actual mobile phase composition (Methanol:water 70:30 +0.01% acetic acid). Depending on the length of your column and flow rate, I would recommend re-equilibration for about 5 min. before analysing your next sample.
I know this will lengthen your analysis and turnaround time but it may just be worth it.
If the dip is the system peak (t0) and it doesn't interfere with your measurements, just leave it.
You shouldn't remove it. It contains a lot of information about your system see work by Douglas Westerlund; Uppsala University, Sweden.
I also experienced this problem with HPLC-RID. I noticed that different solvents give different dip profiles and there were solvents that would give a peak between 1-2 minutes, however dips do not compromise your results. Just optimize the solvent and instrument parameters so that your desired component peak(s) will appear farther from the dip.
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