What is (are) the reason(s) for changing of peak shape in RP-HPLC-UV in a sequence with same mobile phase (Na Acetate buffer-pH:5: ACN-10:90 isocrat.), column temperature (40C), flow rate (1 ml/min)?
Because you are running in isocratic mode only, the number one reason for changes in peak shape would be due to column fouling over time. This can be prevented by washing the column after each analysis using a solution which is stronger than your mobile phase to elute off any strongly retained material. A gradient can be used or just an isocratic solution with a higher organic content and better solubility of the samples. For example: Try a solution of 98% MeOH and Water (methanol is much better at dissolving buffers than ACN is), or 95% ACN and Water. You could also try a similar solution which is more acidic (pH 2.5 instead). The idea is that you need to find a better solution to wash the column down with AND run a separate wash method after each or a specified number of analysis runs.
There are many other reasons for a change in peak shape, but fouling is the most common (esp when it "worked" before).
You may also find the attached article links (below) useful too:
- "Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)";
- "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."
http://hplctips.blogspot.com/2013/12/two-common-hplc-problems-and-their.html
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Hi, the issue was well addressed by Giorgi and Letter!
There are many reasons and the column fouling is a great probability! I've solved a similar problem in UPLC system by replacing the pre-column!
Another common reason leading the peak shape alterations is the solvent used to solubilize the sample. In common RP system, high organic contents in the sample solvent can cause aberrant peaks on the chromatogram. Try to proceed the column cleanup and reduce the organic in your sample diluent.
Best wishes
Thank you Mario, Bill and Lauro for your suggestions and advice. I washed column with MeOH, ACN, water and their mixture many times. Moreover, I changed the pre-column. The problem was solved and the peaks look fine!
Thank you again for your support.
Kind regards
CG
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