In my research, to check purity of my synthesized compound I use HPLC method with detector UV-Vis. So, first I did spectroscopy UV my compound and appear two peaks maximum (237 & 367 nm) . This peaks I use in HPLC, and the result chromatogram in 367 nm didn't appear peak but I use valley wavelangth in 350 nm this chromatogram appear peaks. Why this happen?
Please help me. Thank you.
which solvent you have used to dilute your samples to carry out UV-Vis analysis?? and what solvent system has been used in HPLC?? because the absorption can changed with solvent.
This indicates a hypsochromic shift in the wavelength of maximum absorption of the analyte in presence of solvent system. This may happen only if the analyst has used a ionizing solvent, like any buffer system. It can be concluded that the analyte may be a weak acid/base.
Hi, I agree with comments above from Avtar and Vivekanand. In addition, do you have proved the identity and purity of your target peak(s)? Identical Rts does not mean identical compounds; one peak does not mean it belongs to 1 compound only, it could be contaminated with other compound(s); To check the identity and purity of target peak, it is nice if you can do it with DAD or/and MS detector. If you have only UV-VIS detector, you can prove the purity and identity of the target peaks by running using at least 3 different HPLC conditions. Appearing peaks with different wavelength as described (367 or 350 nm) showed, that your peak might be not pure (or your HPLC solvent system can influence the absorption). Best regards
to dilute my compound I used acetonitril and hplc used gradient elution with solvent acetonitril and water.
thanks sir.
Hi, have you checked the identity and purity of peak the of target compound? Have you tried measuring the UV spectrum of the compound by using mixture of ACN and Water (by using spectrophotometer)? in what solvent did you measure the UV spectrum (you got 237 and 367 nm as maximum )
hello Sir Gunawan, I used acetonitril as solvent in spectroscopy UV-Vis.
But I did TLC and melting point to check purity of compound and the result one spot for TLC.
Hi, Have you checked the purity of your spot by 2DTLC or TLC with at least 3 different mobile phase compositions? Best regards
I was tried to check TLC with 3 system mobile phase and result 1 spot but i don't try 2DTLC.
thank you Sir Gunawan for answering my question.
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