gormetkhef - 20-3-2016 at 12:55
sorry for the rush and wrong thread place and incorrect terms etcetera. Im not sure how stable this is. I am pretty new to this and am running an
experiment out of my backyard with 2 beakers and 2 flasks. I'm not following any recipe, just going off my own theories.
I have a lysine-bound substituted phenethylamine (which i am in legal possesion of) which is about half lysine by weight. I want to cleave at the
lysine to get the freebase substituted phenethylamine, and then precipitate it as a salt (which i am also legally entitled to posess). In theory,
trypsin should cleave them for me.
I got enzyme supplements with trypsin at health store. I mixed ~2g of the powdered pills (proported to contain 225mg trypsin total) into 60ml water
and heated it to 70° C and set it to stir for an hour or so, then filtered it through a buchner funnel 3 times to get rid of the useless shit in the
pills like stearic acid. I got a yellow-tan solution. I added baking soda to the solution until it tested about 8 pH.
I mixed 180mg of the lysine-phenethylamine powder into the solution and heated to about 80° C. In 15 minutes of stirring an oily layer had appeared
on the surface, and a bit of whiteish precipitate had collected in the bottom of the beaker. Hopefully that was the freebase and the unbound lysine
respectively.
I poured the solution into a flask with 80ml solvent (not tolulene but close enough) and agitated it. As expected, the aqueous solution and white
precipitate seperated to the bottom and the "tolulene" floated above, with a thin line of azeotrope between.
However, there is now another mystery layer seperated above both of them. It's the same muddy yellow as the aqueous solution but more transparent. I
have genuinely no idea what it is.
My plan was to seperate the "tolulene" off and bubble HCl gas through it to crystalize the freebase into hydrochloride salt. Now though I'm worried
that the upper layer might be my freebase, or might be something that will ruin my salt.
Please advise.
Again, sorry if this is the wrong section or I'm breaking rules or something.
alking - 20-3-2016 at 13:16
Do you have any pictures? I find it hard to believe that there is either an 'azeotropic' layer or a separate top layer unless your NP layer was fully
saturated, which doesn't seem likely with the amounts listed, though I don't see where you mention how much NP you used either. As to the 'azeotropic
layer' do you mean an emulsion? Two liquids will not form 3 distinct layers, they are either soluble or not and may form an emulsion or some may be
dissolved in the other, but I've never heard of a distinct third layer in between or on top. Was the typsin a dry powder, a pressed pill, or what? I'm
wondering if there was some type of oil in there that may have contributed to saturating the NP layer and thus formed a third layer on top. Also how
long did you wait, are you sure the layers have fully separated?
gormetkhef - 20-3-2016 at 13:32
I guess Iam using the term "azeotrope" wrong. I mean a milky emulsion f the solvent and water which slowly seperates, which it has since done.
It was a 750mg powdery pill which contains pancreatin 300mg, papian 180mg, anise fruit 150mg, fennel seed 150mg, bromelain 135mg, trypsin 75mg,
L-chymotrypsin 3mg, and unknown ammounts of dicalcium phosphatr, stearic acid, modified cellulose gum, and magnesium stearate.
(EDIT: just realized that adds up to more than 750mg, but the pill weighs 750mg and that's what the label reads for one pill, so i dunno.)
What do you mean by NP? The solvent I am using is naptha.
As of now I have waited around an hour.
Here is a picture of the flask:
[Edited on 20-3-2016 by gormetkhef]
aga - 20-3-2016 at 15:03
Trypsin is good stuff.
Anyone even vaguely interested in Cancer should look up Dr. John Beard, Edinburgh around 1908. Some Clinical trial he did.
Seems he got it right over a hundred years ago.
Doubt that he knew the precise details, yet the basis is certainly there.
gormetkhef - 20-3-2016 at 15:11
Update: even with 100ml more naptha the layer still isn't going anywhere.
I,m going to wait a bit longer then bubble the "tolulene" with HCl and pray.
alking - 20-3-2016 at 15:14
NP = non polar, a non polar solvent (such as naptha). Did you make sure your naptha evaporates clean btw? I tried a local hw store naptha awhile back
and lots of black tar would crash out and be left in the pot upon distillation, I would def not use that for any sort of product I tend to ingest. You
can clean it out with a recrystallization or wash later, but still, better not to start with it at all.
Anyway that is definitely a third layer on top. I'm wondering if maybe they are of similar density and you just have a layer trapped up there by
surface tension or something. I have no idea what all that other stuff in the pills is, but I would try adding some salt to increase the density of
the aqueous layer. That's normally a trick you can use to break an emulsion, but I would expect it to work the same here, assuming that is the problem
anyway. Add some amount of salt, your guess is as good as mine, somewhere between a little bit and full saturation, shake, allow to separate.
[Edited on 20-3-2016 by alking]
gormetkhef - 20-3-2016 at 19:57
Yep, I'm using coleman camp fuel and it distills clean. Also I never said I was going to ingest it, this is for 101% scientific purposes.
I'll try adding salt. This batch is a proof of concept anyways so I am not too worried if I can't get whatever it is dissolved. When i start using
grams of the lysine-phenethylamine, I'll just get pure trypsin from a chem supplier or something.
My HCl gas apparatus is not working properly. Is there a way to get the salt using aqueous HCl?
[Edited on 21-3-2016 by gormetkhef]
alking - 20-3-2016 at 20:59
You should be able to just add it into your naptha directly and agitate or stir it, (after separating it from the aq of course). I don't see why you'd
have to bubble it in as a gas, If it works you'll see it precipitate out if it's concentrated enough. If it doesn't then make an acidic solution and
it will extract into that as it's converted to a salt, separate it from the NP and then evaporate the aqueous off to get the amine, and possibly other
things from that pill *shrug*. That's a basic acid/base extraction.
[Edited on 21-3-2016 by alking]
gormetkhef - 21-3-2016 at 10:51
I added another 100ml naptha and 100mg salt and let it sit 24 hours and the layer reduced about 50%. My only guess is that it could be something non
water soluble and slightly NP soluble that was produced from the breakdown of a mystery pill ingredient when the solution was turned basic with baking
soda and more basic with the eventual addition of substituted phenethylamine. I'm going to leave it on when I add the HCl.
The enzyme pills look pretty sketchy so I guess there's a possibility that the lysine was never cleaved in the first place, and the white precipitate
is just the powdered lysine-bound phenethylamine.
Ozone - 21-3-2016 at 11:02
My guess is that you denatured the enzyme during isolation. 70°C is too hot for this. Also, even if your enzyme is good, you will need to operate in
a buffered solution a pH 7.5 or so (optimum range is 7-9) and at, maybe 37 °C.
O3
gormetkhef - 21-3-2016 at 19:18
I read that trypsin denatures reliably only at 100°C for a few minutes. Either way, I bubbled it with HCl and got fine white product (as of yet
unmeasured, but it appears to be about half the volume of the lysine-bound phnethylamine) so clearly the enzyme worked at SOME capacity.
The enzyme is really cheap compared to the other reactant too.