I am developing a method for simultaneous determination of aniline and azo dyes in the effluent of a bioreactor. However, I am obtaining weird results for aniline. It seems the signal keeps getting lower with time. For example, if I prepare a fresh standard stock solution and prepare dilutions to inject it right away, I have a strong signal; if I prepare new samples with the same concentrations after a few hours, signal will be much lower. After one day, it seems aniline was nearly totally degraded. It does not make sense, since aniline is a recalcitrant compound!
I am preparing all my standard stock solutions in MeOH/water 50/50% and keeping it refrigerated at 4 ºC. I have also tried to prepare solutions in MeOH (100%) and to keep it refrigerated at -20 ºC, but it did not work. I have also tried to remove the other analytes (azo dyes) from the mixed stock solution suspecting that aniline could be reacting with something, but that was not the problem either.
P.S: I am currently injecting samples in milliQ water, not in the matrix yet.
It may be degraded due to atmospheric oxygen or photodegradation. It thinks if you acidify your diluent then the degradation may decrease or stop. Due to formation of anilinium. (Note your retention time may get change )
do you have any new peaks appearing over time? what concentration of aniline are you using?
Dear Tabrez and David, thanks for your responses. I am monitoring few mass transitions, so did not notice any new peaks emerging (if there is a new compound being formed, I cannot see in the chromatogram). I noticed the RT shifts a little bit (from 6.50 to 6.42 min). I think that degradation by atmospheric oxygen is more likely than photobiodegradation because I am using an amber flask for the stock solution. Do you guys think acidifying is a better option than flushing nitrogen gas or something? Once I read about auto-oxidation of aromatic amines on exposure to air, so the authors would add salts (phosphate buffer) to stabilize the compounds before qualitative analysis by UV spectrophotometry.
P.S.: my concentration range is from 10 - 400 ug/L. The concentration of my stock solution is 500 mg/L.
From the internet on Aniline stability
Darkens on exposure to air and light and polymerizes to a resinous mass.
IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: http://monographs.iarc.fr/ENG/Classification/index.php, p. V4 27 (1974)
Forms explosive mixture with air. Unless inhibited (usually methanol), aniline is readily able to polymerize.
Pohanish, R.P. (ed). Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens 6th Edition Volume 1: A-K,Volume 2: L-Z. William Andrew, Waltham, MA 2012, p. 227
Thanks, all! It turns out aniline is really reacting with MeOH (and possibly with atmospheric air, too!). I've read some papers in which aniline stock solution is prepared in MeOH, so don't know how they managed it!
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