Sorry for my somewhat belated joining of the party.

First, a few words to Loveall.

Regarding the cleaving of the methoxy group from harmala alkaloids, I would like to reference the horse's mouth, R.H.F. Manske himself.

From page 50 of "The Alkaloids" @ google books :


From the above description, it seems reasonable to conclude that attemting to convert harmine or harmaline into harmol or harmalol is certainly not a kitchen-safe procedure. Refluxing fuming hydriochloric acid @140C would not be the worst part, the methyl chloride would. Methyl chloride is a dangerous carcinogen, and furthermore has considerable acute toxicity.

Methylating the phenolic oxygen of harmol and harmalol is not as easy as simply adding a methylating agent (almost all of which are dangerous carcionogens, btw). Apart from the practical aspects of actually getting the methylating agent to react at all, how will you prevent it from methylating either of the two nitrogens on the molecule instead?

Oh, and Loveall, don't attempt to methylate mercuric chloride with vitamin B12, not in your kitchen, not anywhere. At least not before you have read about the fate of Karen Wetterhahn, a highly experienced organometallic chemist, how accidentally spilled a few drops of dimethylmercury onto her glove.

Regarding 69ron, he was certainly a trove of useful knowledge on various matters relating to extractions of alkaloids. Yet he also had a quite a few run ins with some of the more esteemed chemists on this forum, who occasionally took issue with 69ron making bold claims without providing proper proofs or references to back up his assumptions.

Now, onto the meat of the original topic.

My rough guess is that the "manske" salting out of reasonably pure harmala hydrochlorides has a yield of 95%, meaning that 5% will be left in solution. From the filtrate, these 5% can be freebased, collected and acidified to be re-salted in an appropriately smaller volume in order to collect most of the lost yield.

Having said that, I want to re-emphasize the "reasonably pure" part in the above paragraph. Meaning, that on performing the initial "maske" salting, the solution is not likely to contain only harmala alkaloids, but many other substances, both in solution and in suspension. To assume that the only other substance present would be the quinazoline vasica alkaloids, is only an assumption, not supported by any analytical evidence. Likewise, there is not factual basis for assuming that the non-precipitated harmalas from salting are be harmol and harmalol.

I suspect that many other plant derived substances will still be present, even after a few acid-base cycles. The slimyness of the observed precipitates suggests the presence of proteins. That is, admittedly, an assumption. My personal observation is that basing the filtrate from a salting of refined harmala alkaloids begets a far less slimy precipitate than that which is obtained from basing the filtrate of the first saltings of a rue extract.

Welcome to our betacarboline party pitubo!

Many thanks for the insight and precautions regarding active methylation/demethylation, very good points and I will be more careful with bringing these up in the future.

Regarding the clues for possible harmalol/harmol content of rue extracts, the literature seems to be all over the place. I have attached 3 papers. One found harmine/harmaline only (they had an ammonia basification step), another found harmine/harmaline/harmalol (methanol extraction), and another found harmine/harmaline/harmol (warm water infusion). Finally from the horse's mouth it seems there should be harmine/harmaline/harmalol (page 393),



Also, while demethylation with HCl can be very dangerous, what about accidental passive demethylation through background acid hydrolysis during extractions that have the seeds in a low pH environment under heat for a long time? The paper that found harmol mentions that incidental harmine conversion could in part be the source of the harmol.

At this point rue seems like a beautiful enigma with different folks reporting final results that seem to depend on the method of extraction along with whatever is in the seeds to begin with. Who knows, one may even soak live seeds in water and the different betacarboline contents may change as the seeds prepare to sprout...

All that being said, we have a solid rue extraction technique at the Nexus that reliably gives 6% yields of harmine/harmaline. However, Could rue still have something left to give? Maybe not, but I think it is worth looking into. Likely there won't be anything new found by a beginner kitchen alchemist, but the investigation is the reward itself, and I catch myself smiling while doing this work. I'm just a silly monkey trying to understand an amazing plant.

as i mentioned before i did a manske on the slime and got some crystals, which are most probably the harmine/harmaline that didn't crash out in the first manske. i basified the new manske water and the slime is still present as expected.

the crystals i got were freebased with NaOH and i got 220 mg freebase harmalas, compared to the 4g i got originally this fits nicely with the 5% pitubo found.

the freebase i got was more crystalline than powdery and had a light brown color, this is not the first time i get it in crystalline form when basing a still hot small volume harmala solution but i don't know why it is brown, when i got it before it was very white and pure. i could not have introduced any impurities from first manske till this and on the contrary i did many acid base cycles and water washes so it should be more pure...
and as you can see in the pic there are 2 layers one has a lighter color than the other could these be mostly harmaline(top) and mostly harmine(below) and they separated somehow. maybe the brown color can be attributed to more harmine than harmaline? you can see the color difference in this thread (lucky that we didn't lose phulx- pics in this thread too) so maybe harmaline crashes out more easily in manske.

with the slime i'm doing a last water wash with it and i'm going to decant as much as possible water and dry it weigh it and save it, i don't know what to with it yet, if we find an easy way to test it then i will..

and now i figured what those crystals were in settled slime pic, they are harmine crystals

Sakkadelic, IPA may get a some of the mamske salt out of the slime. Maybe getting the salt out is important to change it's sliminess. From Wikipedia:

"Unlike ethanol or methanol, isopropyl alcohol is not miscible with salt solutions and can be separated from aqueous solutions by adding a salt such as sodium chloride. The process is colloquially called salting out, and causes concentrated isopropyl alcohol to separate into a distinct layer.[7]"

So you could do some tests on some the slime with 99% IPA, hope would be that the IPA picks up the slime and leaves the salt (and a small amount of wwate begin). Decant IPA, evap, and see if new unknown xtals form. Sorry if this suggestion has already been tried before without success.

I have no idea if this would work. Just a thought. Also, expect a small amount of salt to go into the IPA so a small IPA volume would minimize potential salt residue after evap.

I've gotten to the point where I have what I think is Na2CO3 freebase
broad-spectrum brown powder in centrifuge test tubes (images below in order post basing ready for fuge, loaded in fuge, fuge running at 4000rpm for 10 minutes, and separated centrifugate freebase which stays in tube when upside down). No manske done, using centrifuge only for clean up (for fun, I did not do any filtering or Mason jar decanting either, only repeat separations with acid (keep supernatant) and base (keep centrifugate) with the $50 4000rpm fuge, I'm very happy with it). Next I'm going to centrifuge wash the centrifugate freebase in water (to clean Na2CO3 will monitor pH as we want the wash somewhat basic but low in Na2CO3 so potential harmalol does not go into water), and then washes/evap with 3% ammonia to see if the ammonia picks up anything such as a harmalol candidate. Should have results in a couple days.

interesting idea, if i understand you correctly i don't think what you are suggesting is practical, the slime appears after you base the manske solution and a couple of water washes will remove most of the salt i believe (solution doesn't taste salty ) and harmalas alkaloids are not very soluble in alcohol.. i think this idea might work better in a DMT extraction using ISO
waiting to hear your results

Sorry, I meant to suggest to dry the basified manske slime, it should be a nasty goo I think. The salt content will be high. Can't do water washes because they would pick up our harmalol candidate I think. So instead, do an IPA wash to remove salt and base (?) and only pick up harmalol, then dry to check for what is left. A long shot and the biggest concern is harmalol solubility in IPA like you mention...

Reporting results of the following test proposed earlier:

1) 60g rue seed extract done with diluted vinegar heated in microwave and with sakkadelic's squeeze method with seeds in a fine nylon hop bag (used by homebrewers) and by squeezing the bag with a pestle in a mason jar (pestle, seed bag, and jar went in and out of the microwave all together). Did not boil for long (a minute or so) due to acid hydrolysis concerns (which many not be a real thing). After 8 boils extract was coming out semitransparent (pestle could be seen) and moved on to next phase.

2)Extracts we're combined and reduced to ~200ml.

3)Without filtering or decanting, 4000rpm centrifuge was used to keep or discard the supernatant or centrifugate for 5x acid/base conversions using 10% x 5% vinegar and 5% Na2CO3. Very clean clear liquids were seen after a couple conversions (but the first base conversion had very dark supernatant so next time I'll filter some). In this case maybe 5x conversion was overkill, bit work proceeded quickly and easily as the centrifuge did all the work and waiting for settling was not needed.

Note: No manske done. Concern is that salt introduction could make harmalol separation a slimy business (this has not been verified at this point, just a concern which could be incorrect).

4) After the last base centrifuge, the centrifugate was washed with water to remove Na2CO3. Wash had pH of 9.5, considered low enough to continue to the next step after collection a sample of this wash (W1).

5) A second wash was performed with 3% Ammonia and collected in W2. pH was verified to be above 10 during all ammonia washes.

6) A 3rd and 4th ammonia wash we're collected (W3 and W4).
Bellow is a picture of W1, W2, W3, and W4 from left to right in normal (top) and UV (bottom) light.




First picture indicates that something was picked up during W2 and exhausted during W3 and W4. The UV fluorescence from W1 was strong, maybe because some harmaline was picked up in the lower pH wash? Also, there was no fluorescence in W2 which could be due to depressed harmalol fluorescence in a strong bases as described in the attached paper (if I understand it correctly).


7) W2 was dried. A dark reddish/brown powdery residue formed upon scraping a small section of the dried material, pictures below.





Could this be harmalol (or part harmalol)? Or are we just looking at something else such as standard harmaline/harmine which is everywhere (but I was careful to check for no precipitate before drying, and there was no obvious fluorescence in W2). So now we have the red/brown powder as a possible harmalol candidate. More tests to follow, any ideas? One thing to try is to dissolve in warm water and then try to reX by cooling down, as mention in this book (page 469). At the top of the page the water re-x is mentioned. In the middle of the page harmalol isolation is discussed (where KOH is used instead of ammonia in combination with Na2CO3 to separate harmalol from harmine/harmaline).

Also, per the attached paper in not very basic solutions harmalol should have a green fluorescence which can be checked for. One piece of the puzzle that does not fit is that harmalol should be a tumeric yellow (when pure), so we need to investigate further. Will update again after further experiments. As Jees has mentioned sending out for lab analysis would be the real test, but for now we can try to understand further before sending something out (and I can't get prices online for GC-MS, need to contact the companies for a quote).

UV fluorescence of harmala alkaloids is pH dependent. Roughly speaking, the salts do fluoresce, the bases don't.

If you want to have your results tested, contact endlessness. Although any testing of your results would be interesting in itself, I still remain unconvinced of your harmalol hypothesis.

BTW I don't always find it easy to follow your procedures. I wonder if you may be meaning to speak of Na2CO3 where you write Ca2CO3 (which does not exist, calcium carbonate is CaCO3). CaCO3 is quite insoluble and it would make no sense to try to wash it out with water.

Oops, yes it was Na2CO3, sorry bad typo. Fixed in the previous post to avoid confusion. Also, if you look at the paper attached in that post it describes the fluorecence of harmalol in KOH solutions at different KOH concentrations.

Are you also skeptical of the claim in the book referenced that harmalol can be separated with KOH from harmaline/harmine during rue extraction? I think I'm on board with that claim (made in 1901 apparently). The claim jives with the luminecense paper which keeps harmalol in solution using KOH.

On the other hand, I'm also on board with being skeptical of what the red/brown powder from post #27 is where a "separation" was done using ammonia (promted by 69ron's claim that harmalol, if present, would dissolve in ammonia). With that healthy skepticism on-hand we'll try to figure it out one hypotesis at at time.

Sorry if I'm not clear and difficult to follow. I'll do my best to be clear and typo free moving forward.

Mystery powder was tested for fluorescence after redisolving in distilled water. Green emission observed which disappeared upon basing slightly, both observations compatible with the text in the paper attached to post #27:

"..., in solutions of about pH 10, the emission spectrum of harmalol shows a very intense band at 534 nm Corresponding to the emission from zwitterionic Species [13].
The intensity of this band is reduced as the potassium hydroxide concentration increases up to 1-2 M."

Picture below of the green fluorescence in the dish from the mystery powder. Test tube has dilute Na2CO3 (pH9.5) solution which was yellow for comparison. Below that, what 534nm green looks like. Photo is not very clear, but visually the greens seemed to roughly match.





If anyone wants to try to recover this same mystery powder, use ammonia (KOH should also work) during the initial acid/basing, but don't throw away the ammonia solutions. Combine the separated ammonia liquid (which would usually be discarded) to work on them separately while the traditional manske is followed on the traditional base precipitates. On the new ammonia pulls that you have kept, try to recover the harmalol candidate. This does not seem like something new, for example from this 2013 book (page 469):



Where the last chloroform step and the KOH use is not the same as the earlier posts, but the ideas are similar (use of ammonia and vinegar should allow for a gentle oven dry for example).

So far things are checking out, so gearing up for a substance analysis. If someone sees a flaw in this logic please point it out so we don't waste outside analysis efforts. Also, would be interesting if anyone can verify that a new candidate is pulled by ammonia.

beautiful! the results so far seems promising to me, of course there is harmalol and you are isolating it here with this smart method but so far we cannot confirm that it is the mystery precipitate in the basified manske, or can we?


sodium carbonate is not enough to fully convert all the harmaline salt into freebase, you can see this by the present yellow coloration and the strong fluorescence you got, if you further basify this solution with NaOH you can see that this yellow coloration will almost disappear.

so basically the idea is that harmalol won't precipitate in ammonia solution and we are using this to isolate it right? what if instead of evaporating, we farther basify this solution with NaOH would the harmalol precipitate? maybe not the best way to recover it but we would see it and we can tell whether it is slimy or that as you say the salt is making it like that..

also about the harmalol color can you please direct me to where you found it?

Agree with the second part: we can't say if it the precipitate in the basified solution is the same as the mistery power pulled by the ammonia. Also, we can't say that the mystery powder is harmamlol, we really don't know at this point. What we can say is that based on how it is behaving it is a good candidate for outside analysis unless we can discount it somehow in upcoming kitchen tests. Endlessness is helping us in another thread for that possible outside future analysis.



That makes 100% sense. Thank you! There is a possibility that the fluorescence is being changed by the pH more than residual harmaline concentration but my money is on what you describe. There should be a fluorescence paper that can help us know for sure.



This is kind of what FISHER did in 1901 to isolate harmalol from rue (according to the book quote above). He used KOH instead of ammonia, but before basifying with a different base (he used Na2CO3), he neutralized the KOH. You may need to neutralize the ammonia in this case, otherwise precipitate may be messy since the mystery powder likes to stay in ammonia.



I searched "harmalol fluorescence" in Google Scholar and it showed up there. To get the paper I used sci-hub.cc (the site that was recommended in the Nexus to get published papers). There is a lot of info on harmala fluorescence out there

Then in the paper it said that harmalol emits in the 534nm wavelength as long as it is not in a very basic environment (<1M KOH). Then, I went to this website,

https://academo.org/demo...-to-colour-relationship/

I typed in 534nm and the green color that roughly matches the mystery powder showed up.

It strikes me that this was done because the phenolic OH group in the harmalol likely forms an anion when treated with KOH, keeping it in solution. Neutralising then re-basifying with Na2CO3 should then precipitate the remaining harmalol (based purely on chemical principles, not personal experience!!)

Stating this just in case, perhaps you grasp this already.

Interesting, but wouldn't Na2CO3 being a base also provide depronation to the pehnolic group?

I think there may be something else going on. Harmalol has both -OH and -NH3 groups, acting as both proton donor and acceptor sites. Looks like an example of a zwitterion, maybe being able to stay dissolved in acids and bases alike by choosing a side. Na2CO3 is interesting because it will have the weak carbonic acid in solution at the same time as the stronger NaOH base (Na2CO3 + 2H2O <-> H2CO3 + 2NaOH). NaOH dominates, but H2CO3 is abundant and could be a strong background element. Maybe since both are present it forces the ziwertion to stay basically non polar (if it first tries to become depronated due to the basic environment, that attracts the carbonic acid present in the background, which pronates the amine making the ziwertion very slightly internally polar after witch it precipitates?) I'm not sure about the exact details, but currently taking free chemistry courses online, so maybe I'll know more later and/or one of our wonderful nexus chemists can enlighten us.

And sorry if this is all wrong. It is mostly made up, but it is the best I can come up with.

Also, if anyone could get a hold of this paper that would be great. Trusty sci-hub can't find it:

https://www.ncbi.nlm.nih.gov/pubmed/8827246

It's more that the overall basicity of the Na2CO3 solution isn't sufficient to deprotonate a significant proportion of the harmalol, I would think.

You won't get background carbonic acid in a Na2CO3 solution - but there will be a bit of bicarbonate once any of the more labile protons have been mopped up.

I see, I like your simpler explanation better.

How about this: in acidic environments harmalol would be in solution thanks to the amine group pronating. Around a certain pH (~Na2CO3?) it will be less soluble and fall out as the O-H and N-H3 groups are "chilling" and/or balanced. At high pH, the -O-H depronates and harmalol becomes soluble again. If true, KOH, NaOH, and NH4OH should all be able to get the harmalol in solution when/if the pH is high enough (with Na2CO3 having difficulty getting there as you suggested).

For those who see the post manske basing slime, how high did the pH get? If you push the pH higher does the slime disappear? Not sure how effective NaOH would be post manske because of the common ion effect (this time on the Na+), so ammonia may be needed.

Til 9.5 with ammonia and til 12.5 with lye, it stays.

Don't feel sorry even if you turn out to be wrong or partially wrong. You're doing great work and every bit of well-documented experimental effort is an addition to the collective knowledge and a valuable contribution to the site.

I have already stated that I suspect that proteins are a major constituent of the precipitate in based manske filtrate. Depending on the analytical technique used when you get to test them, these may or may not show up. Proteins are typically large molecules and I am not sure how effectively a mass spectrometer will detect those. Benzyme would probably have some wisdom to shed about that subject, but I haven't seen him around a lot recently.

Another point to consider is that many phenolics are air and base sensitive tend to polimerize easily. Perhaps this is something that happens during protracted extraction procedures. It could explain the reddish brown crud that gums up filter papers and that also keeps appearing as an oily scum during the boilings with NaCl?

Thanks for the words of encouragement pitubo.

Reporting on a new round of experiments around our mystery powder (harmalol candidate). Equal amounts of harmalol candidate from post #27 were dissolved in (a) vinegar, (b) water, (c) NaHCO3, (d) Na2CO3, (e) Ammonia, (f) NaOH.

Here is the starting situation after shaking the test tubes (a->e order is left to right). They all looked similar at this point.



After allowing to settle the for a few hours observation was:
- minor precipitate in a
- medium precipitate in b, c, and d
- very minor precipitate in e
- no precipitate in f
Image below (not all precipitates are easy to see, sorry about that),


After re-shaking test tubes and a 10 minute 4000 rpm centrifuge, observation was:
- major precipitate in a
- virtually complete precipitate in b, c, and d
- minor precipitate in e
- very minor precipitate in f
Image below:




- For the tested pH conditions, the Harmalol candidate is more soluble in strong bases and somewhat soluble in acid. It precipitates out best in neutral to slightly basic pH, shows some precipitation tendency at low pH, and can only be somewhat precipitated with a centrifuge at very high pH.
- Due to the (hypothetical) phenolic group, the harmalol candidate does not behave like harmine and harmaline. It goes through a solubility (or suspension?) minima around neutral pH, increasing solubility in acids (due to the amine group it shares with harmime/harmalime) and bases (due to the phenolic group as downwardsfromzero astutely pointed out).
-This harmalol candidate can be lost during the "classic" rue acid/base steps, especially if the acid steps are not very acidic (splash of vinegar), or if the base steps are very basic (boosted with NaOH).
-The harmalol candidate could also be removed during manske. Strong acid would be advantageous if removal is desired to keep more of the red/brown substance in suspension/solution (assuming here that the mystery compound itself will not start crystalizing at very low pH in salt which needs to be checked).



- To test for quicker removal of this red/brown candidate, use weak acid and strong base during A/B, then move to a strong acid during manske. If red disappears faster than usual, the observations above are somewhat corroborated.

- To isolate the harmalol candidate in large quantity for more testing, a new strategy is required. The observed 0.25% yield was reported on a procedure that discarded a lot of the harmalol candidate during the A/B centrifuges. Possible strategies:
1)
- Extract seeds in a strong base first. Keep the seeds for a later acid extraction.
- Filter and/or use centrifuge at low power -> keep supernatant.
- Bring pH to 8 and allow to settle or use centrifuge at high power -> keep sediment.
- For final cleanup, redissolve in water and settle or centrifuge keeping sediment.
- Note that if not using a centrifuge, settling is slow and losses may occur.
2)
- Extract seeds in strong acid.
- Without filtering go to a strong base.
- Settle/centrifuge.
-Supernatant treated like the base extract in 1) (to collect candidate harmalol)
-Filtrate/Centrifugate to be treated per usual rue tecks to obtain harmine/harmaline.

-Test isolates with kitchen TLC

-Attempt to convert harmalol candidate to tetrahydroharmol in acidid Zn solution and check for luminescence change.

-If experiments cannot discount that candidate is harmalol, send mystery substance out for testing.

Any thoughts? One question that pops up is has anyone tried an Na2CO3 pre rinse/soak/wash on the seeds to clean them before extracting? Of course this could backfire if the harmalas come out of the seeds and precipitate, but if they stay in the seeds (since they don't like to move into this level of pH) such a pre-wash may make the extract process simpler/cleaner.

More experiments and reports to follow

Also, (expanding on the passing manske comment in post #39, planning on using a stronger acid (Phosphoric acid from the brew shop). Should give a pH of 1.5. This may be low enough to pronate the amine group, after overcoming the internal repulsion from the phenolic -OH group. If the harmalol candidate does pronate and goes into solution in this condition, a manske will be attempted (the pronated amine may now attract the Cl-, along with the phenolic group too?).
Will also manske the strong base just to see what happens (chance of forming a sodium phenoxide?). Just for curiosity, this would be discarded.