Hello,
I noticed a problem with my HPLC analysis and wanted to ask for help.
During my analysis I've noticed repetitive problems with the baseline: it is dropping and then (usually) going back up. I am adding some pictures so you know exactly what I have in mind. Sometimes this problem is regular like here, but most of the times it is random and do not occure everytime.
This problem occurred in two different C18 columns during two analyses using different sets of solvents (Acetonitrile with Water or phosphate buffer).
We tried to clean the pump and flow path filters as well as a long column wash. The problem occurred again during the preview run and was very regular.
Have any of you got a similar problem? Any advice?
Best regards!
The issue shown has nothing to do with the column so "washing" will not solve it.
In general, the chromatograms provided show several different problems occurring. Issues such as: Pump cavitation, loss of prime (air), sticking valve(s), lack of degassing, mixing or precipitation issues, inappropriate compression settings (?), lack of equilibration and so on. Before running any samples, you must first establish proper flow of the HPLC pump (under pressure) resulting in a smooth stable baseline. To do so requires that the method used (programmed) follow good chromatography fundamentals, the HPLC system is well maintained and operating properly and finally, run by an experienced user.
Here is an article from one of my many training classes that address some of the problems observed. Review it with an experienced chromatographer, on-site, as these issues should be very simple to resolve.
- Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline); https://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html
BTW: You did not include any HPLC method information at all so we have no context to associate with the chromatograpms shown (Please always include FULL method details with questions, including FLOW rate, mobile phase composition detector settings (wavelength & bandwith etc). We need this basic info to understand what we are seeing.
William Letter thank you for your response. As I mentioned the problem occurred during two separate types of analysis that we ran for a long time (several years) without this kind of problem. Before running the sample we are waiting until baseline and pressure are stable.
Two pictures (Przechwytywanie/Przechwytywanie2) present one type of analysis (analysis time about 30 minutes):
Column: Purospher® STAR RP-18 endcapped
Mobile phase: Acetonitrile: Water (80:20)
Flow rate: 1,4 ml/min
Detection at 220 nm.
Before I ran a sample there was nothing wrong during the Preview Run - the pressure was stable and the baseline was flat, the problems started about 1 hour into a sequence.
The other two pictures (SCHODY and 2) are from the later incident (about a week later, we had a week of analysis without problems between)
Column: Kinetex Evo C18, 2,6µm 100Å 150x 4,6mm
Mobile phase: Acetonitrile: KH2PO4 buffer pH 8 (50:50)
Flow rate: 1 ml/min
Detection at 220 nm.
At first, those steps were regular, then it changed into spike down, and now we are back to steps but they occur randomly. The pictures are from preview runs.
At first, I thought it could be a column problem therefore column wash but when it happened on a different column I started to look for another explanation.
As noted in the linked article provided, you will find troubleshooting info to solve the issues observed. Your "pump" and the related valves used are not performing properly. The reasons are many so please get some local expert help on-site to solve as web forums are not very useful for these types of things.
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