Hello.
I`ve been experiencing a negative baseline in my HPLC chromatograms. I don`t know if this can be considered a baseline drift and I also don`t know what is causing this problem. The first blank was good, the problem started on the subsequent chromatograms - second and third blank, two samples and standard. The substance we are trying to quantify is catechin. Can you please suggest the reason?
Mobile phase A: water/phosphoric acid 85% (99:1)
Mobile phase B: methanol/phosphoric acid 85% (99:1)
Column: C18 250x 4.6 (5 μm)
Please look at the following below links which may help you in your analysis:
https://www.chromacademy.com/chromatography-HPLC-Gradient-Elution-Baseline-Drift.html
http://www.ucd.ie/t4cms/VWR%20HPLC%20Troubleshooting.pdf
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Supelco/Bulletin/4497.pdf
Thanks
Hello
As I saw your files, I don't think it is a negative baseline. I think the initial change in the chromatagrams may refer to an impurity whether in the sample or in mobile phase or it may be referred to the gradient program you already used.
To fix the problem first, I think you should do the elution carefully before the initial injections. then you need to give enough time in the middle of injects for re-equilibrium the column with the initial fraction of the mobile phase. if you are using gradient program it is better to change it in a way that the percentage of A or B changes more slightly.
If the problem won't be fixed, you can think about changing the column to a new one or check the detector.
Hi Raissa,
I guess your system isn't properly equilibrated. Start a blank run and observe the system pressure. Important is the re-equilibration segment. Only when you observe a constant system pressure (horizontal curve) you can start the next run. Maybe you have to prolonge the equilibration time.
Regards
Markus
Hi. The negative baseline doesn't cause any problem in chromatography analysis because you are using the peak area as analytical signal in your calculation. Due to the use of standard solution in analysis of the real sample , peak area of the analyte
is not affect by negative baseline. In order to remove this problem, you can select *zero* in software of the instrument before analysis.
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