Chromatography Forum

Hi guys,
I have a big problem with the determination of ethambutol hydrochloride and Meso HCl,
I take 0,2g of ethambutol in separating funnel and add 40 ml of NaOH solution and mix. Add 5ml of 0,1% 2,4 dinitro fluoro benzene in chloroform.
Extract with four quantities, each of 20 ml chloroform. Evaporating and dissolve the residue in 100 ml H2O/ACN 50:50.

HPLC system

column : Hypersil Silica 5µm
Mobile Phase : 20 ppm of CuSO4 5H20 in distilled Water :ACN 50:50
pH :7,0 adjust by very diluite Ammonia solution
Flow : 1,5 ml/min
Wavelenght : 254 nm

calculation :

Response factor of Ethambutol HCl and Meso HCl is almost same

ethambutol HCl : 1,00 Meso HCl : 0,97

Meso Hcl in Ethambutol HCl is calculated by area % method.

The problem is that in the chromatogram i get a single peak and have intensity very low to be a solution of 2000 ppm ethambutol.
I think that 2,4 dinitrofluorobenzene is a chiral derivating agents and extract only meso HCl......I don't know I'm not convinced by

Help me please
Thanks,
Antonio

First of all, DNFB is *not* a chiral derivatizing reagent. There is nothing chiral about it, it simply tags amino groups with a UV chromophore. That's why you get the same response factor for the two forms. Any enantioselectivity in this method comes from complexation with the Cu++ in the mobile phase.

The classical Sanger reagent method for amines indicates an absorbance maximum at 420 nm. 254 nm sounds suspiciously like a very, very, old LC method developed back in the days when fixed-wavelength 254 nm detectors were the norm. Unless you are running a validated method where you are forced to run it as-is, you might look at a different wavelength.

All of that said, I suspect that your derivatization reaction is not working properly. If this were my problem I would start with the ethambutal and establish that you are getting reproducible derivatization (look for a reasonably linear calibration plot). Once you have that, work on the separation.

Hi!
Thank you very much for your answer,
I managed to improve the extraction with your invaluable advice...I'm very happy to have found this forum because is an important help for young analist!

Many thanks
Antonio