05-01-2002

Greetings,

I have a typical problem. Injection of the blank is giving drug peaks. I am giving below the background:

I am working on indirect chiral analysis of Ketoprofen and related drugs.The molecule contains one carboxylic acid. This is converted into its amide diastereomers. Ethylchloroforamte and L-Leucinamide HCl is employed as the chiral derivatizing agent.The derivatisation of ketoprofen occurs at room temperature within 3 minutes. The amide derivatives are separated on an regular C18-ODS column in the RP-mode. I have been following this procedure successfully without any problem.

But today when I first injected the blank, consisiting of ethylchloroformate and L-leucinamide, to my suprise I noticed the typical derivatized peak of R- & S-ketoprofen. In my earlier experiments I never found this sort of behavior.

This time the only difference was before carrying out this experiment (two days back) I determined the ketoprofen content using achiral HPLC method in the same column using ACN:MEOH-HOH (35:13:52; pH:3.2). After completing the analysis I washed the column with non-buffered mobile phase.

I have made sure there is no problem of carryover. I flushed the injector with mobile phase. What could be the reason for this observation? Is it possible that ketoprofen adsorbed on to the column, during the earlier experiment, might have got derivatized in the column with the blank (comprising of the derivatizing reagents) injected and generated
the diastereomeric peaks?

Looking forward for you comments to solve the problem. Thanks for your time.

Nice, keep these ghosting problems coming, to show this is a great problem, which is often conveniently neglected.
Now to your suggested solution: It could be a reaction at the start of the column, but one would expect the peaks to get broader with amt (especially volume) of reagent injected.
Itīs more likely that your injection valve is the culprit. Washing it with something may reduce ghosting but VERY seldomly removes it. See the earlier chains on this. Now this ghosting (one can call it mechanical ghosting) seems to get worse with age of the injector (scratching of the rotor.....) and lowering of the pressure between rotor and stator. (Mechanical ghosting is possible whenever you have some moving parts in the system)

Here it would also be very instructive to be informed on your findings.

The material of the rotor seal has sometimes caused significant carryover, by adsorption of the sample. Replacing the seal with an alternative material has solved the problem.

This topic may be related to the line of comments seen in the ghosting posts. Readers of this might want to comment on both situations.