I have a problem with my HPLC analyses. I keep getting split peaks and I am not sure if I can quantify those compounds. I do have reference standards and I should do a calibration curve to be able to quantify that but I am not sure if I should really quantify the peak especially when some peaks merge and causes split peaks. I have tried other methods (changing solvents and parameters, also did sample prep clean up) in order to probably isolate that peak but unfortunately i cannot really isolate a single peak which corresponds to the reference standard.
Can you give journals/articles which states how to deal with the quantification of such cases (or if should not even dare to quantify)..
thank you!
Not really. This split peak may be due to stereoisomers (D and L) which are chemically and structurally similar, though I suspect you have 'poor' separation due to alpha (improper solvents, detector, unbuffered mobile phase...).
No. Duri Eun, based on your comments, your method is invalid and not selective for the sample under analysis. For the HPLC method to be valid, it must follow good chromatography practices and fundamentals. Peaks need to be fully resolved and separated by HPLC. Please consult the literature to screen for possible examples of methods used for your sample type(s). If found, be sure to screen the proposed method to insure it also follows good chromatography fundamentals (as many published methods do NOT, and may be invalid). *Proper method development is required if the data is to have value.
BTW: Peak "splitting" is different from peak "co-elution". Please obtain some local help from an experienced chromatographer to determine which issue you have as the solutions are different for each.
You need experience to do method development effectively, or you can ask for free help here
https://sielc.com/Services_MethodDevelopment
- "HPLC Peak Splitting. Common Reasons For It "; https://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html
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