I'm using a PDA detector HPLC unit, I was wondering whether it is possible that the retention time can change for a particular compound when the detector is set at a different wavelength, all other parameters like mobile phase an flow rate remaining constant.
As long as you are running your method with the same flow cell in place and have no leaks, then the retention time should not change. Detection wavelength and bandwidth are a separate parameters and have nothing to do with it. How much variation did you observe relative to a standard (or the Tzero time)? Is it reproducible? Retention times will rarely be 100% perfectly reproducible on their own. Some variation is normal. This is one of the reasons why we often repeat an injection/analysis multiple times to document what the normal variation is with the specific method.
As Allan mentioned above, if you have sample co-elution (and/or just a poorly developed method), then you may indeed observe what appears to be a shift in retention time (but in fact, it is not).
Lastly, please make sure you have the "Reference Wavelength" feature in your DAD/PDA turned 'OFF'. If it has been accidentally turned 'ON' (then data is being destroyed/modified), then there is the possibility that it can alter the peak shape seen which can in fact result in small changes to the observed retention time. Always set it 'OFF. *More info can be found at this link: https://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html
Dear Satadru
The retention time is a chromatographic parameter that is dependent on the equilibrium of the solubility of the analyte between the mobile and stationary phases and should not change for a given analyte run under identical conditions. Some problems can result in shifting retention times such as flow rate changes, leaks in the system after the injector, changes in mobile phase composition, accumulation of contaminants on the column, changes in the sample solvent and injection volume etc but these are not related to the collection of the signal at the detector.
The shift in retention time cannot be related to the detector parameters.
You do not mention the actual sample origin so if comparing a pure standard made up in a solvent to the same analyte in a complex mixture or biological matrix there could be retention time shifts due to interfering compounds or matrix related co-eleution that is not seen at the wavelength monitored for the analyte.
No. Retention time is the result of chromatographic separation which happens in column (stationary phase). Wavelength is the parameter to be adjusted for detection, after separation in column.
Only one possibility I see that, if your sample is mixture of 2-3 compounds or impurities and the wavelength max of these compounds are reasonably different to detect only at specific wavelength, you may get false view of change in retention time at different wavelength.
These compounds could be already separated, but appear at only specific wavelength in the same chromatogram.
Based on my previous answer, you have to ensure that:
1. pH is the same
2. Buffer is the same and does not react with your molecule.
3. Solvent mixture is the same.
4. Sample matrix is similar.
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