Fuckity fuck fuck fuck

So I'm doing this experiment where I'm trying to get a bioengineered polyketide pathway to incorporate weird starter units. I did a small scale feeding experiment and got a promising looking result in terms of retention time, MS-2, and UV spectra. So then I did a 20L fermentation with hopes of eventually isolating it and getting an NMR.

First I did a huge ass silica column to get a crude seperation of the ethyl acetate extract. Then I did a huge ass sephedex column to get a better seperation. Then I did preparative HPLC to get 2.6 mg of something. I took a proton NMR. It was garbage. There were just too many compounds with similar retention times in there to seperate it, and the proton NMR was entirely too dirty to be at all informative. Basically, I couldn't see a lot of the diagnostic peaks at all, and a bit disheartening. BUT! It could have been that it was just too dirty. So on to further purification!

So then I purified my 2.6 mg on the analytical scale HPLC that has a really intensely good column with really great seperation. I fussed with the method with my supervisor for several runs and eventually got 2 peaks that had UV spectras consistent with my compound of interest (with this really fancy-pants analytical column, there were about nine peaks that showed up as one peak on the LC-MS spectra and maybe 4-5 peaks on the prep HPLC column, it was intense). I collected both peaks and started to evaporate them down on the rotovap using a vial adaptor that you can attach to the bump trap. One of my fractions was fine--I can put it on the vaccuum pump overnight, see if there's enough compound for an NMR (for the 500 MHz, you can get away with a little under a mg and still get spectra, so maybe if I isolate 0.7-0.8 mg of it I have a hope of doing the whole proton, 13C, COSY, HMBC, HMQC, etc. thing to really confirm the structure) and if not at least LC-MS it to see if it is there. There are more fractions from the spephedex column that one could purify--ones that actually had better seperation than the one I did attempt to purify (long story involving leaving in less than a week and the time it takes to get LC-MS samples and access to the prep HPLC in a large lab where a lot of people need the equipment)--but I simply don't have time to do it.

But the other fraction...well, what happened was a disaster. The vial adaptor LEAKED AND THE WHOLE VIAL AND BUMP TRAP FILLED WITH WATER FROM THE ROTOVAP BATH! FML!

So now I rinsed the vial and bump trap with MeOH, and am re-evaporating it and putting it on the freeze dryer overnight, hoping to god that a) something comes out, goddamn it, and b) it's not my compound of interest anyway (it was the less promising of the two peaks..but who really knows).

Seriously, though. FML.