Chromatography Forum

Dear all

I am trying to derivatise a drug, the response is very good the problem is that when i prepare a blank (everything except the drug) I get a small interference. Every time I change the chromatographic conditions the interference moves always with the peak, I can not get rid of it

I really appreciate your suggestions

Regards
Sarah

Are you certain it is not actually in your solvents or reagent? I would try some different batches or brands of the solvent to confirm before troubleshooting the hplc system.

Yes, I injected every reagent alone and they were ok.

If the interference *exactly* matches your analyte, there is a chance that it actually *is* your analyte contaminating your lab. You can confirm/deny that hypothesis by preparing a blank in a different lab with new glassware.

Have you run a blank after having run other clear injections on the system before running the analyte injection? This would eliminate carryover being the cause of this.

If this is carryover from the analyte, try a needlewash with higher composition of solvent or set the needle wash to double time of extended time needle wash.

Yes it is not carryover.

it this peak present when you run the method without an injection?
can you give more details of the method
like mobile phase, column and other things?

When I inject mobile phase there is no interferance, the method is very simple, isocretic elution 40% water 60%Acetonitrile, injection volume 25mcl, fluoresence detection 325 exitation and 390 emission. I tried different combinations of mobile phases, isocretic and gradiant elution and also changed the wavelength but still the result is the same.
Many thanks
Regards
Sarah

what is you blank exactly made of?

Have you tried preparing your blank in a different lab using new glassware. (If not, contamination is still a possibility).

Dear unmgvar

My blank is composed from the derivatising agent the catalyst without the drug.

Dear Tom
I tried using a different HPLC with the same result, unfortunatlly i can not use a different lab.

Dear Sarah
if it is only 2 products, then have you injected each one alone?
do you know if the peak is due to a combination of them?

I remember seeing something like this many years ago, so I forgot what reagents and analyte were. The analysis was running at maximum sensitivity so the derivatizing reaction showed one peak next to the other, including one at the rt of analyte. Concentrating the sample gave a mess, interest of the medical people for whom I did this waned, so I gave up to invest further time.

I afraid your problem is mainly come from interaction between components in your blank. Fluorescence detection is much more sensitive compared to UV detection. Do you have any idea is there any reaction between your derivatizing agent and catalyst? Would you mind to give some information about your catalyst? Fluoresecence detection would detect even a very small interference in your sample.
If i suggest you to run your sample using UV detection, can you check the persistent of your problem?

Dear gabriel85

Thanks for your reply, The catalyst is 18-crown-6 I also use few grains of sodium carbonate as the reaction needs basic conditions. I noticed today that when I pepare the blank without 18-crown-6 the peak gets smaller but doesn't dissapear, I am planning to repeat this tomorrow just to make sure.

Before using fluoresence I tried derivatisation with another reagent and I still had this problem, but I will try also to use the UV with this method.