[Distinctlyminty]

First of all forgive me, I'm not a chemist by training but a biologist having a failing little go....

I'm studying a drug metabolite (Mw around ~300) and have used LC-MS to identify the structure, however this structure corresponds to either a nitrile form, or a primary amine form. I'm trying to use FT-IR with the metabolite disolved in methanol to identify the presence/absence of a nitrile peak, however this doesn't appear to be working. Is there another way to specifically identify a nitrile (that's not NMR)?

[Dan]

I'm trying to use FT-IR ... however this doesn't appear to be working.

What do you mean by "working"? If IR shows no nitrile, chances are there isn't one.

[Nebelhom]

well, first of all, you should not dissolve it in methanol when checking for an amine. both OH and NH absorption has roughly the same wavenumber, therefore you would swamp out the region that you want to have a look at.

how do you prepare your sample? I never needed a solvent to run an IR spectrum, it usually makes your spectrum unreadable.

[Distinctlyminty]

I'm not sure if the drug metabolite is evaportating away with the solvent and I am just getting a spectrum of a contaminent (protein, or co-factor), which is why I'm uncertain of the results. I'm also now trying this with a metabolite that should produce a nitrile, not an amine (given the literature, and it's mass corresponds to only a predicted nitrile based breakdown product, not an amine). I'm not seeing the nitrile peak. Absorbance scans suggest we have metabolite present at around 5 mM.

The reason for the solvents is that we prepare the drug metabolite with an enzyme and drug reaction in a water based buffer, then try to purify the metabolite away from the protein using a hydrophobicity column, which we have final eluted with the methanol. Acetonitrile seems to work better as a solvent, but obviously contains a nitrile itself so is less than perfect. I've no idea if its possible to somehow precipitate out the metabolite from the enymatic reaction instead, or use a better solvent.

[Nebelhom]

since I don't know the structure of your compound all I can say is that it is doubtful that a compound with a mass of around 300 is just evaporating off.
If you look at the mass of common solvents, they all tend to be below 100, so I would not worry too much about that.

is it possible that your compound is not stable?

from what you told me, if the column was successful, then you should have a mixture of compounds (or just your compound) without any protein impurities in a methanol fraction, right? Unless the metabolite's stability relies on it being in solution, you could just concentrate the mixture in vacuo (using a rotary evaporator, which should remove most or all MeOH or MeCN) to obtain an oil or solid and then do analytical testing. This is how it is done in organic chemistry labs. By these means you would be able to do a full analysis (IR, Mass spec, NMR) which should tell you more. If you have to rely on IR, make sure it is completely devoid of all solvent before running it, otherwise you will get false positives either for amines (as I mentioned amines and alcohols are pretty similar in IR) or nitriles depending on the solvents.

I don't know how much of a chemical background you have or which tools you have at your disposal, but this is how I would do it. For a chemist, this is pretty basic, so if you have one floating around just grab him and tell him to show you how to do it.

[Distinctlyminty]

is it possible that your compound is not stable?

Good call. It seems that our compound is stable in water but once in a different solvent it dissappears fairly quickly. Cheers for all the help on this. Often it's just the simplist of things..