I was trying to analyze protein sample using HPLC, but unfortunately I am unable to get a good baseline of my blank run; it is sloppy with zigzag kind of pattern. I tried different things to fix this but not effective.
I would appreciate any expert advises on this.
P.S. I am using mobile phase of 0.1%TFA in Acetonitrile, and 0.1% TFA in water.
Hi, Mekdes -
It's difficult to answer your question without more information!
There are dozens (if not more) possible reasons for an unsteady baseline.
First of all, are you experienced in HPLC in general?
How bad IS your blank run (can you upload an example)?
What kinds of things have you tried, to fix the problem?
What us your detector - UV? DAD? MS?
Are you planning to analyze intact proteins? Digest?
What exactly is in your blank run?
Do you get a steady baseline with just an isocratic solvent stream (or maybe a problem in one of your pumps)?
Hi, Mekdes -
It's difficult to answer your question without more information!
There are dozens (if not more) possible reasons for an unsteady baseline.
First of all, are you experienced in HPLC in general?
How bad IS your blank run (can you upload an example)?
What kinds of things have you tried, to fix the problem?
What us your detector - UV? DAD? MS?
Are you planning to analyze intact proteins? Digest?
What exactly is in your blank run?
Do you get a steady baseline with just an isocratic solvent stream (or maybe a problem in one of your pumps)?
Mekdes: We would need far more detail (a chromatogram with X and Y scales shown and all of your running conditions) to provide you with good quality suggestions. No one can solve a broad problem of this type without more detailed information and a chromatogram to look at.
Note: Many new users experience problems in setting up and operating the HPLC system to obtain a nice flat baseline. A "zig-zag" pattern indicates that the system is not stable and can not be used yet. There is a problem which must be fixed BEFORE starting any sample analysis. .
Unstable baselines: Often, the cause is due to the pump having air, not liquid, stuck inside it. The air could be from a loose fitting, leak, lack of mobile phase degassing, buffer buildup, contamination, clogged filters or sticking inlet or outlet check valve(s). None of these things can be solved over the web. You should get help from the person at your school who is experienced using the HPLC system. They should always be your first contact and source of information while you learn this complex technique.
I have linked a few general troubleshooting articles below which may be of interest to you; "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)" & "Common Causes of Baseline Noise in HPLC, UHPLC".
https://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html
https://hplctips.blogspot.com/2014/09/common-causes-of-baseline-noise.html
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