Dear Aftab Ali,

Unfortunately, you do not write anything about the chromatogaraphic conditions you are working with.

Conceivably:

Your molecule has two charges, one for each oxygen.

With an acidic eluent or buffer, the 1st peak could be the molecular form, the 2nd peak the single charged form, the 3rd peak the double charged form. The order of the peaks would be determined by the separation column (ion exchang, RP phase, or other mechanism) and the eluent used.

A mixture of molecular , partially dissociated or completely dissociated form would at least explain why three peaks occur.

Alternatively, positional isomers of the functional groups may be responsible (1,2 ; 1,3, 1,4 position), which are parallel present in your sample. Here the purity of the substance(s) / standard would have to be considered.

I hope this answer helps you.

Good luck with your work and stay healthy

with kind regards

Joachim Horst

RP column, methanol is used as eluent, 220nm,

Ok,

can you give me more details on your conditions?

RP phase could mean C18 to amide phase (light ion exchange mechanism).

Isocratic or gradient elution?

Ion exchange conditions would speak for different charge states, standard RP conditions more for positional isomers.

Methanol as the organic Eluent is quite ok, but alcohols have acidic protons, do have tried Actonitril? I observed that aromatic compound mostly have a better peakshape using ACN.

Yes C-18 RP column

Gradient elution

Yes acetonitrile is also best and reported