I am working with the UV-degradation of bezafiberate in water. Using HPLC- UV detector Bezafiberate has abosrption wavelength of 232 nm and gives peak at 1.7min.
I am expecting that during the UV- degradation process 4-hydroxybenzoic acid is formed, so I changed the wavelength for 4-hydroxybezoic acid to 254 nm. Then again a sharp peak appears at 1.7 min. So, weather this peak is of 4-hydroxybenzoic acid or bezafibrate.
Yes Cristina becoz the pressure of the pump is 10MPa...If we decrease the pressure of the pump to 5MPa then the retention time of peak goes to 3.5 mins.
First inject the pure standards of both the compounds 4-hydroxybezoic acid as well as Bezafiberate. If their retention times are same, then you need to modify your mobile phase. What is the phase that you are currently using and the HPLC column? Have you checked the area under curve or height of the Bezafiberate peak before and after UV degradation? Is there an increase in the peak height/area or decrease? Bezafiberate may not be completely degrading into 4-hydroxybezoic acid and therefore you are still getting a peak at 1.74 min.
1 - I guess the wavelengths of 232nm and 254nm you are using to detect respectively bezafiberate and 4-hydroxybenzoic acid are the maximum of absorption of each of them. Were you expecting not detecting bezafiberate when using 254nm? Did you check the absorption spectra of bezafiberate and 4-hydroxybenzoic acid. The point is that in principle both absorb at both wavelengths but having the maximum absorption at different nm. If after your UV degradation you still have some bezafiberate, you will always detect both either with 232nm or 254nm. I suggest you choose the wavelength at which they have the same absorption units and not the maximum of one or each of them.
2 - Separation principle of chromatography stands exactly in the different interaction that the different compounds establish with the solid phase in the column. To have different RT you have to play with the mobile phase such that one compound will have more affinity for the solid phase, being released latter that the other, or vice-versa.
3 - it is always a good practice to test and optimize the separation and analysis of your compounds of interest using standards of your compounds, first separately and then mixed, so to mimetic your "true" sample.
In HPLC usually it is always a best practice not to consider peaks up to 2 minutes.We have to adjust the conditions in such a way that your peaks will come atleast after 2 minutes.
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