I think we are ready to send the following sample out,



Isolated by a 4% NaOH extract of rue seeds in a 75um nylon bag at room temp, acidifying the extract to pH 5 with HCl (discarding a precipitate that formed at this step), bringing pH up to 8.5 with NaOH, adding 45g of (NH4)2SO4 per 100ml, and collecting/drying precipitate.

Precipitate dissolves in both basic and acidic solutions. Green fluorescence appears in the acidic solution only:



Arrangements are being made for lab testing (thanks to endlessness). Will update the team with that result.

Beautiful
If these turn out to be harmalol it means we are losing it when decanting the base solution, so we should do 2 base steps from now on
What was the yield?

Yes, very good point, the 1st strong NaOH basing step after a standard acid extract should have our harmalol candidate in solution. To check for it post decanting, green fluorescent clouds under UV should form as this first base is acidified. I have not done this as I have been starting with NaOH extracts while working on this.

Regarding yield, about 1/3 of the first 100g rue seed liquid NaOH pull received this treatment. Yield from that was 315mg, but there will be salts in it (let's say 50% as a worst case). Rest of the NaOH pull was lost to (failed) side experiments with other salts and solvents. So, should be about 0.5% yield from the entire first pull. Maybe we could see 1-2% after more pulls and isolation improvements, I don't know.

Also, here is a plain scope image of the isolate from post #61 (focused on the smaller chunks at the bottom),

I tested this by doing an acid extract of 90g of rue (in a bag made with the outer liner of a 0.5um wunderbag which was pleased in a mason jar with vinegar and squeezed between five 20 minute PC cycles - warning that I'm not sure if the wunderbag is inert), cleaning that up with everything I got (ending with a centrifuge cleanup to force out the last bits of what looked like a fine dust), and basing with NaOH to pH 12. This first base turned milky brown:



After settling over night the top layer looks like a deep red. It looks a lot like the straight NaOH extract from post #45 (just as Sakkadelic implies), but now it has harmine/harmaline precipitated at the bottom.



So as Sakkadelic said this first base solution should have the harmalol candidate we have been working on. I'll try to get it out with the methods used in this thread which were applied to a straight to base extraction.

Surely this first deep red base must have been seen by many folks before when basing hard (weaker bases may not dissolve the harmalol candidate as well). Is there a consensus on what makes the first strong base step red from prior work? If anyone has access to chloroform they should be able to lower the pH close to the isoelectric point where water solubility is minimal (see theoretical approximation plot below) and simply pull the harmalol candidate out like FISHER reported over 100 years ago as described in this book (page 469):

Again i never got or seen this deep red color with harmalas, it almost looks like acacia/mimosa base soup, for me the (first)base solution after precipitation almost look like the acidic tea.. maybe your solution is very concentrated? or maybe your seeds have a different spectrum?... when i squeeze seeds The fabric gets stained either brown or yellow and sometimes it's a nice red color

From the work and research that you did so far i think we can conclude that the mystery substance is not harmalol since it does precipitate at high PH and also we are probably losing the harmalol before reaching the manske stage. another important thing you showed is that we can recover the harmalol with a simple method.. so thank you for the great work loveall

Also did we get a final conclusion on whether Harmalol does or does not precipitate with manske?

Sorry Sakkadelic, I don't understand your comment I bolded above. I don't think that the harmalol candidate precipitated when basing (maybe I posted a typo earlier). I think it is still in solution at high pH and only harmine/harmaline precipitated in the picture with pH12. The only way I can get the harmalol candidate to precipitate is loading it with salt when at a slightly basic pH (roughly 7.5 to 8.5), and so far ammonium sulfate is the best salt tested (with sodium sulfate ready to be tested next).

I cannot get the candidate to precipitate during manske (slightly basic and 20% NaCl) conditions, and those manske tests where done without harmine/harmaline present in solution. I want to try manske on the candidate again after adding harmine/harmaline too, maybe in that case some candidate will precipitate out together with the harmine/harmaline needles? The precipitating harmine/harmaline may have an affinity for the candidate harmalol structure and pull it down during crystalization (pure speculation at this point). My intuition says that this could result in red crystals, but I'll need to run the test to see what actually happens.

And yes, the solution is pretty concentrated (300ml for 5 PC cycles of 90g of rue after boiling down and centrifugating - surprisingly 100ml evaporated during centrifugation). However, liquid is all ways a reddish brown even when coming out of the PC unconcentrated. You may be on to something with the seed variability.

oh i meant that the mystery substance does precipitate at high PH unlike harmalol which doesn't.. so that's how we know it is not harmalol. maybe the way i wrote it was a bit misleading
my tea looks like here it doesn't get considerably darker when i base it.. at first when you based the seeds ii thought maybe there's something in the seeds causing the deep red color that is not extracted in the tea, but now you got the same result with the tea. what color is your tea before basing?

But there is no mystery substance precipitating at high pH. My current thinking is that harmine/harmaline precipitated after basing the acid extract, and the deep red in the basic solution is the red harmalol candidate. This red will not show up after a second base step I think (and plan to test).

I think my acid extracts are redder than yours. I'll post more pictures next time. Do you have any pictures of what going from acid to base for the first time looks like (no manske in between)?

Oh, and I use a lot of vinegar (or even phosphoric acid). I use at least half water half vinegar: want the solution to really be acidic. Acidity does not seem to matter when extracting harmine/harmaline, but maybe it does not matter when a 3rd item is considered (or maybe not), but that is one difference between us - I'll do another test with less vinegar to see if that makes my tea look like yours.

precipitating from the manske solution...
no but i'll take pics next time of the first base
i think it's most probably just the concentration

But what I was trying to explain is that the harmalol candidate only precipitates in a salt solution at a slightly basic pH (7.5 to 8.5). At an acidic pH it does not precipitate when salt is added, and at a very basic pH (12) it does not precipitate either.

Yes we are agreeing on this, so the substance that precipitated in the first pics of the thread(the blobs) cannot be harmalol that's all i'm saying

Ok, got you know. Sorry it took me so long

Yeah, the initial investigation into the basified manske blobs has taken us down a new path in this thread. To avoid future confusion, if the GC/MS analysis comes back as there being harmalol in the red solution candidate we can isolate I think we should start a new thread with a new subject.

Sample from post #61 was delivered to the lab a little over a week ago, and we are waiting for results thanks to endlesness.

Sample results are in. There is a clear harmalol peak (5.4 minutes)

However, there are other larger peaks of harmine and harmaline. There is also a background (could be some proteins that precipitate out during the salting out technique as pitubo pointed out).

Not a lot of harmalol was found overall, but it is clearly present. I think there could be various candidate reasons for this:

- There is little harmalol to begin with
- Harmalol is oxidized, during the basing step (<- which may also be consistent with previous results from burnt were Harmalol was observed but subsequently disappeared), or it may have self-oxidized while waiting for analysis (in queue for several weeks).
- The salting out method to precipitate the zwitterion at the isoelectric point is not very effective.

Since harmine and harmaline are present they are being carried out during the basing step (at least in the way I'm doing it with NaOH). That means I'm having some losses of these two alkaloids when I base.

This also means that some of us have harmalol in our first acid extract. A lot of it will be discarded at the first basing step when the solution is dumped. Since it does not seem to manske, more will be lost during manske. It may be important to remove all of the harmalol to get the golden color (instead of red).

This also suggests that if one is stuck with a lot of red needles and wants the yellow ones they can try to wash the base precipitate repeatedly with more base several times before going to manske. This should pull the harmalol out of the wet free base precipitate. If anyone observes this it would be very interesting. On the other hand, oxidation of harmalol's -OH group could make oxidized harmalol precipitate out of the base, so a long base time could backfire. One strategy could be to have vitamin C or fumaric salts present during the base washes to keep harmalol from oxidizing so it stays in the solution and can be removed (I have not idea is this would work, but may be worth a try).

Setting future work on this on the side for now. Will be working on a more interesting zwitterion next: psilocin

Thanks for everyone who helped in this thread with their feedback and ideas. Special thanks to Sakkadelic, Jess, Mindlusion, downwardsfromzero, Pitubo, Endlessness, and Mindlusion. Apologies that because of how the cookie crumbled we went off a tangent different from the original subject.

Thank you for all that tinkering.
I've been sitting fence a bit on this one, because I wasn't seriously interested in harmalol, maybe unfairly because it's such an unknown substance.

Is there a special reason why you consider the harmalol being the reason of red-ish needles colorization? I always presumed it's just plant gunk of sorts, I kept washing as you said to get golden.

Hey Jess (rue sensei). There is some evidence that makes me say that, but it is by no means conclusive.

1) Burnt tested the red color and he found it contained phenolic compounds. Harmalol is a phenolic compound. In the same thread endlessness got a red compound starting from relatively pure harmaline after hearing it in IPA. geeg30 suggested the red appeared because harmaline was converted to harmalol in the hot alcohol.
2) It is known that rue can produce both red and yellow dies. To get yellow, extract with water. To get red, extract with alcohol.
3) When we extracted with basic water in this thread (basic water would pull harmalol and not harmine/harmaline) we got a deep red on the first pull that diminished in subsequent basic pulls.
4) At this time it is reported that rue contains 0.6% harmalol. The way we do extracts, it will be present as a residue in the wet base precipitate. So the first manske should have some harmalol traces too. And it gives red needles. The weight of the red needles and yellow needles suggests we are only removing a trace of something that has a strong color. Harmalol simply fits the bill to be that something. There could also be other things besides harmalol I'm the red needles, but I'm pretty confident that at a minimum harmalol is present.

Also the harmalol content can change for several reasons because of the extract procedure itself. If warm alcohol is used, some harmaline may be converted to harmalol as suggested by geeg30. Also the seeds themselves could biologically convert harmaline to harmalol (hypothetically) during the extract process depending on how it is done (e.g. long water soak or any condition that may activate demethylating enzymes).

A striking example of this kind of situation is P. Cubensis. Initially it can contain psilocybin, but after a hot acid extraction enzymatic action can turn most/all of that into psilocin. That adds an extra complication when interpreting extraction results.

Finally, there is some counter evidence for what I'm saying. Pure harmalol HCl crystals are reportedly yellow.

So the source of the red color in the HCl needles remains an open question in my mind. We could analyze red needles and yellow needles if endlessness graciously helps. We can check if the red ones have a small harmalol peak that the yellow ones don't.

Another test would be to not base strongly during red needle clean up (e.g. only use baking soda). If the red is more difficult to clean out that would be consistent with harmalol not separating out as well due to pH conditions (see pH solubility chart below) - a random contaminant would not behave like that.

I almost forgot another piece of the puzzle. There was the experiment in this thread where we centrifuged at different pHs. Seems like the picture is gone (it is a link to the dmt-nexus chat). Picture is attached below. Coupled with the solubility plot it seems to support harmalol. It also suggests that soda wash will also make it harder to get rid of the red, or alternatively, that red manske crystals have a harmalol bonus (hypothetically).

Thanks for the many words, checking out all these possibilities.
Are you going to continue hunting down the harmalol?
It feels like you are really bitten into this

I do want to pursue harmalol more but that will be in the future. My wife got fed up with all the rue extracts I had going and said "no more diarrhea water!!!". So I had to take a pause...

While only 0.6% of the seed mass, harmalol is ~6% of the betacarboline mass (using these numbers). It may give the harmala experience a more rounded feeling if we find a way to keep harmalol. It may be reducible to tetrahydroharmal too.

Right now I'm having fun with mushroom extracts. Wife has not complained to much about that (yet...).