I'm withdrawing samples after a 15 minute interval for 90 minutes. Hence, the 90th minute sample is to show the highest amount of degradation or the least peak area, but it shows the highest, this trend holds for the other time intervals as well. At first I thought it was due to evaporation that the reaction mixture is somehow getting concentrated, but I've conducted the experiments in an ice bath as well with the same pattern recurring. I'm unable to overcome or explain this, can anyone shed some light?
It might be possible that not all of your sample comes down in a single run so you accumulate the compound over several runs.
You could try running a blank after the first injection to see if you actually get a blank spectrum.
Could be that the original method is not a high quality one. IOW: It is allowing co-elution to take place and/or reveal other components that were not seen with the original method. This may happen if the original method shows poor capacity factors for the key peaks or it not comprehensive or selective enough for the samples. *The original method and all related techniques used to process the samples and the set up the instrument should be reviewed by a professional experienced in chromatography.
I agree with the co-elution possibility. That is especially likely if your product(s) co-elute(s) with the starting material.
It's not quite clear if your using a HPLC plot or looking at the spectrum or a max plot. If it's a HPLC plot try collecting at a 2nd wavelength say at a minimum in the UV spectrum if available. Look at the absorbance ratio of the two wavelength it may give some extra information regarding a co eluting peak. The ratio should be the same for a pure peak. It sounds like you have a co eluting peak with the same RT. This peak has a stronger chromephore or there is more than one peak. The method soulds like it needs further development. Try collecting the fractions coming off the system and run on a mass spec
There are two likely possiblities for this discrepancy. One is that the compound was not fully dissolved at the start of the degradation experiment. Thus, as it dissolves during the degradation experiment the parent peak would go up. Another possibility is that you are making a degradate that does not resolve from parent and that has a larger extinction coefficient so it looks like the parent peak is increasing. I had this occur in a photolysis study where the parent rearranged to an isomer that had a 10-fold higher extinction coefficient. MS was not helpful since it had exactly the same mass. We noticed a very slight shift in RT for "parent" during the runs. By changing the conditions slightly we were able to baseline resolve this isomeric compound.
there could be a possibility that the impurities elute at the same RRT.Kindly change the mobile phase and diluent and observe
Increasing area could be due to Diluent which may evaporate if it is an organic solvent and that leads to increased concentration and increased area..
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