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Simplified Column Chromatography

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member for the trees
Senior Member
..this was influenced by a short article in one of the very first editions of Entheogen Review, in the early 90s...i've slightly adapted it, from experience, into what i'll present below...this is to help those pioneering independent research at the Nexus..

Simplified Column Chromatography

..This can also be called Planar Chromatography, or also (modified) Paper Chromatography. It was a valid chemistry method prior to more complex modern systems. And still is.
It has similarities to TLC (Thin Layer Chromatography), but doesn't require silica gel plates. [there is much great information on TLC on the nexus that endlessness has posted]

Please note this method is intended/tailored to be performed on already extracted alkaloids, of a full spectrum nature, as the freebase(s). It will not be suitable for compounds like flavonoids for instance, which remain in the water soluble fraction of a non-polar solvent extraction.

The question often arises on this site of: 'How does one separate this tryptamine from that?'
This is a simple solution (once perfected) , particularly if one has an idea of what they're trying to separate. Also when a bioassayer suspects a plant of containing multiple alkaloids, this allows one to get an idea of how many there are (to within perhaps 3-5% range of total content, not traces). And also, with the use of reagents, an idea if what they may be. Only 2-30mg of alkaloid mix are required, although it can be performed on larger amounts (up to 150-200mg). It is designed for small, but still utilisable, amounts of alkaloid.

The principal is simple.

The extract/crude alkaloid is dissolved/made soluble in either simply a non-polar solvent, or preferably (for clearer results) a Solvent System (Mix) (see below) - 100-250ml. This is left to stand in a bowl or flat walled tray.

A rectangular strip of blotting paper/thick Paper is selected, at least 12cm x 5cm (it can be longer or wider). It should be preferably acid-free paper.

..the paper is placed between 2 panes of glass which approximate or are slightly larger than the paper. The panes of glass with paper in middle are clamped together (or fastned tight and flat with rubber bands) as tightly as possible, leaving approx 1cm of paper extending from the glass panes on one of the shorter sides. This is the 'simplified column' (rather than straight paper chromatography, in which there isn't the glass..but it can also work).

The paper+panes are then placed with the 1cm of extending paper sitting in the solvent mix, and standing upright at a 45°-65° angle. It must be either suspended or leaning upright. It is suggested to start with a 45° angle. Steeper angles all the way through to vertical can be experimented with as one perfects the process.

The solvent (mix) is then left to evaporate at room temperature. As it does, the solvent will wick up the paper column along with the alkaloids...When it has completely evaporated, the alkaloids are left deposited as bands in horizontal lines on the paper, rising vertically, according to molecular weight or polarity. [NB perhaps a chemist can give the technical explanation, and whether there's a connection between the two]. Each band is a separate alkaloid, and will vary in width depending on amount present.

There can be some smear between the bands (overlap) depending on how successful the process has been. If the smear is small, one can then cut each band as a strip of paper (avoiding the overlap/cross-over if there is any). Some bands such as betacarbolines are made visible with UV light. Some bands, where the amount present is small or trace, may be too narrow to see. Hence this cannot replace systems like GCMS for fine detail, and wavelength or fragment confirmation. But it can be very handy.

If sufficient mg have been used, the strips can then be redissolved in a solvent and individually crystalized, permitting each individual alkaloid to be independently bioassayed.

Examples of Solvent Systems

You can initially just use a non-polar solvent, of the ‘broad spectrum’ variety.

Mixtures are used to help various alkaloids of differing polarity separate/partition better. Mixing a more polar solvent like ethanol, with an NP solvent can allow a greater separation, and cover a wider range of compounds.
Some commonly used examples include

Ether/Petroleum Ether, Ether/Hexane, Ether/Pentane
(general purpose)

10 percent Ammonia in Methanol Solution/Dichloromethane
(helpful for amines separation)

..these are commonly used mixture for separation of alkaloids, various examples of actual ratios , as well as many other systems can be found here: Solvent Systems for Silica Gel Column Chromatography

Or, my specific starter example used for tryptamines/beta-carbolines and other alkaloids in experiment is
• Aqueous Methanol or Ethanol (10%)/Dichloromethane 1:9
..meaning 1 part of 10% water/90% methanol (or ethanol) to 9 parts dichloromethane (other NP solvent can be substituted) ..this was the system that was used to successfully separate NMT from DMT and a betacarboline in 2001 experiments, and in fact also provide material for test bio-assays of nmt...

Optimal Performance

It can require some experimenting to get it to work optimally (i.e. clear and clean separation) ..But once working it's incredibly powerful as a glimpse into multiple components in plants. The main parameters to experiment with are -

- Blotter paper thickness/absorption
- Column angle - 45° is a good starting point.
- Solvent system - this may benefit from adjustment depending on the kinds of substances one is dealing with, or looking for...

As far as identifying tentatively what the alkaloids may be, one will need:

Reagents - there has been much on these in the nexus, in TLC threads

Colorimetric test results (Marquis, Mecke, Ehrlich, etc) for different alkaloids

How do analytical methods work? (TLC, UV-Vis Spectrophotometry, GC-MS, LC-MS, etc)


..some will better tell the difference between tryptamines than others, most at least allow for between classes of alkaloid (eg tryptamines vs phenethylamines)

rf value/height lists - these can be found in various places, and say what distance or height and time to expect between bands, however these values vary according to the specific Solvent System used.

..Good resources for further instructions and information on reagents/rf values include the Nexus, and Trout's Notes/'Some Simple Tryptamines'. It is best procedure to have known references available, of things known for example to contain mainly just dmt, or 5meo. Then you will have a clear idea how these change colour , and what height they are deposited at, in your specific solvent system/setup.

This method is also 'simplified' here, in that this isn't dealing with very polar compounds, which would stay in the polar phase of an NP extraction. That can be done, but it's more complicated.

So there you have it, simple and potentially powerful (in the right hands).

Comments, suggested modifications, systems, and reports absolutely welcome...

Now, onwards with future research adventurous nexians!

This is fantastic, I've been hoarding a picture frame - made with two glass sheets and a convenient clamp - for exactly a situation like this. Thanks for posting and reminding me of the fact.

It does make me wonder, do the edges of the paper have to be an optimal distance from the edges of the glass? It looks like an area where there could be problems with uneven separation. Perhaps some kind of sealant band along the vertical edges might prove necessary, but first a few experiments are in order. I'm getting the blotting paper lined up right now :)
thanks acacian..i'm sure you'll put it to good use..

and thanks also downwardsfromzero...i appreciate the modification suggestion
It does make me wonder, do the edges of the paper have to be an optimal distance from the edges of the glass? It looks like an area where there could be problems with uneven separation. Perhaps some kind of sealant band along the vertical edges might prove necessary
..i've observed it work where the glass was around 5-7mm wider on the sides, and a few cm taller at the top..there just happened to be nearby some thin strips of glass that suited, and paper was cut accordingly. Also it was thin glass, not as thick as window glass for instance, i don't know if this helps or not.The glass was clamped and rubber band fastened tight, but no it probably wasn't like vacuum sealed. The glass should be ok if it extends further beyond the paper, but getting a tight clamp may be more difficult. So yes allowing enough room at the edge of the glass for some kind of sealant is a very good idea for optimisation. As long as the sealant doesn't touch the paper it should be good..i've seen it work with just paper leaning, no glass, but the 'simplified column' helps with clearer/finer partitioning..

i should also add that, as i said in the nmt thread, it took a fair bit of experimenting and trial and error to arrive at the right parameters, and this will vary with different set-ups...but once it was working it worked well..

..my hope is that, as the mainstream scientific world is currently so slow and/or caged about new plant entheogen discoveries, the more enlightened and astute at the nexus can use this and similar techniques to bring in some more solid data on some of these plants, without the need for full laboratory facilities..
I've located the picture frame, and found that it pairs with an old glass container and a lampshade, both of which I dug out of the attic a couple of weeks ago.

The shop didn't have any blotting paper - but I bumped into a friend there (right in the stationery section) who will give me some! I did buy some other thick paper just in case, but the stars seem to be lining up for this one in a special way :)

Pics of my forthcoming chroma tank attached - glass plates are 175×253mm, effective tank depth 200mm, with diameter the same. Lamp shade takes care of the headspace, slotting almost perfectly into the tank while providing extra room for the plates to project into a vapourspace. The integral stand/clamp also fits into the setup but it clips on at what are the top and the bottom of the plates in this orientation and I'm not sure if that's a good thing.


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Here are the glass plates after cleaning, plus a piece of paper trimmed to size and loaded. I'm contemplating using the fold that is forced by the orientation of the frame clip as a convenient way of loading a single stripe of sample. The paper also protrudes somewhat from what will be the top end since this makes it easier to align in the plates, and keep it aligned while sliding it all into the frame clips.

If I had to describe it, it would be "ghetto, but elegant". 100% found items, except for the IPA I just cleaned it with. I'll be wiping down the tank in the morning and with any luck I'll know what sample to run through it by then too. It won't be anything precious until I've completed some optimization tests, in particular regarding the paper thickness as well as the aforementioned protruberance question.


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Bookmarking this.. I intend to mess around with wild phalaris grasses the coming spring / summer for the benefit of the community (and my own, there's just no way in heck I'm buying another batch of dead plant matter), and hope to develop some analytical muscle so I can at least tell 5-MEO and N,N apart..
advantages - When this technique is working properly, or well, the bands of separation are very clear, with less than 1mm crossover...this is clearer than the majority of TLC's i have seen. Also the section of paper is very easy to then cut with scissors, in order to re-crystalize an alkaloid separately. This is why it was chosen as the method to do (successful) experiments on NMT..TLC/silica gel methods on the other hand have their own advantages (such as for polar substances)

The Art of Chromatography is much like that of extraction and crystalization, only the next level up


like your ghetto style BTW DFZ 8)
nen888 said:
like your ghetto style BTW DFZ 8)
Thanks, nen - I just need to get it into action now that I have a fair-size pile of sea buckthorn offcuts; I'm planning to compare the bark with the heartwood as hinted in the sea buckthorn and russian olive threads. There have been delays what with preparations for the festive season, family life and all that that entails - on top of being a general scatterbrain - so cross your fingers but don't hold your breath 😁 The blotting paper also turned out to be a bit harder to get hold of than I'd otherwise have hoped but I'll get there soon enough.

I also found some 1-methoxy-2-propyl acetate which is expensive but interesting so I'd like to establish what kind of things that dissolves best, besides the intended polyurethane yacht varnish. One thing worthy of note regarding this particular solvent is that it is chiral - in principle, its two enantiomers will interact differently with naturally enantiomerically pure, chiral cellulose - could this open up chiral separation of THH?

There's also the possibility that enhanced paper chromatography could be used with a standard mixture of analytes in order to test various eluants and eluant mixtures. Add that to the aforementioned chiral solvents along with the opportunity for simplified quantifications and we're looking at an exciting project here.
Loving the setup downwardsfromzero.. I’d say it tends towards boujee rather than ghetto

Nen with the solvent system.. since ethanol is not miscible with other commonly used NP solvents does this create issue? Or is it fine to have the separation and alkaloids sitting in each layer?
hey acacian, with most of the solvents recommend for especially amine separation there is no absolute soluble or not soluble line...dcm for example is slightly miscible with water and ethanol..but the point is that there are polarity differences in the solvent system ..that's precisely what causes the separation of alkaloids into bands, along with the planar aspect..it's a gradient..
also, note i didn't recommend any base in my starter formula, but when other systems are talking about using naoh or ammonium (to create greater separation) it is very small amounts..this isn't an extraction..
there will be no 'separation and alkaloids sitting in each layer' regardless of miscibility..each thing will arrange itself according to polarity
..if we're talking 'commonly used' solvents, something like naptha (limited spectrum) will probably not work as well as something like dcm (broad spectrum) if wanting to truly test the alkaloid range...

as I've said this method is simple, the actual molecular physics may not be, but don't worry about that..just look for the patterns
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