Chromatography Forum

Hello,
Iam developing a hplc method for monitoring reaction . The starting material (7-Phenyl acetamido-3-Hydroxy-3-Cephem-4-Carboxylic acid diphenylmethyl ester i was told that keto -enol tautomerism is possible) gives better peak shape only at pH below 7 ( at pH above 7 the peak tails badly [it drags] , but the intermediates formed degrades at pH below 7. how to go about.

Thank you
Santosh Gandhi

How exactly are you monitoring the reaction?

Are you trying to quantify all items Including the intermediates?

or

Just measuring decease in starting material/increase in product?

If it is the last option does the degradation of the intermediates affect chromatography?

Also how long does it take the Keto - Enol tautomerism to reach equilibrium?

(I am asking as I once had a an assay where the analyte took 2 hours to reach a tautomeric equilibrium. Attempting analysis before this point gave very poor calibrations and irregular peak responses).