Peak Splitting

[Santosh Gandhi]

Hello,
Iam in the process of developing a HPLC method for monitoring the reaction in the synthesis of a cephalosporin (Intermediate stage). when the product is isolated and injected into the hplc system I get a single peak (say RT@7mins), the solid dissolves in buffer. But when I add a little amount of solvent and then makeup with buffer i observe two peaks well seperated (one @RT 7.min and other @RT 2.0min). the uv spectra of two peaks are same.I want to know what effect will solvent have on the structure of molecule to be seperated.
Thanking you,
Santosh Gandhi

[unmgvar]

Santosh Gandhi

in general in reversed phase chromatography compounds separation can be affected by several chemical ways.
different functional group of your compounds interact differently with your mobile phase and stationnay phase.generally different chemical structures will interact differntly with the mobile phase and stationnary phase.
a change in the pH of the mobile phase , a change in the proportion of the different solvents in your mobile phase.
especially if you mix a buffer with an organic solvent then you can be sure that the final pH will be different, this can effect the state of your compounds in solution and so there chemical interaction in the column.

for your example at hand, the change that you made in your method caused one of the compounds to be very much unretained by the column. close to no interaction with the stationnary phase and so it came out very quickly. the other compound was not affected at all.
the exact nature of the behavior will dependend on the change made and the chemical structure of your compounds.

a good book on chromatography will probably be of great help as wellif you really want to better understand all the chemistry involved.

[Uwe Neue]

Santosh Gandhi: insufficient information to solve the problem...

It is possible that the early peak is the same as your retained peak when your sample solvent represents a stronger eluent than your mobile phase. To judge well, one would need the details of the sample solvent make-up and the mobile phase make-up.