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Art of Liquid-Liquid ext. The Basics Options
 
Dozuki
#1 Posted : 1/13/2012 10:45:46 PM

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Acid Base extraction is a liquid - liquid extraction technique for isolating compounds from various sources. It is often used with plants to isolate alkaloids. While not a complicated procedure, some understand of how the procedure works and how to deal with issues that arise could be helpful to the beginning extractor. So, here is 'The Art of Liquid - Liquid Extraction' or How I came to love A/B.

The Acid/Base extraction procedure works on the principal of the solubility of the different states of a compound in various solvents. Specially here I will be referencing alkaloids found in plants as the compound of interest. The alkaloids can be in a couple of different states such as a salt form and a freebase form. These forms have different solubility properties. The salt form is typically soluble in aqueous or polar solutions and not in non-polar organic solvents like chloroform or xylene. The freebase form is just the opposite; soluble in non-polar organic solvents and not soluble in a polar aqueous solution. This is the basic theory in a nutshell, and it should be noted that there are some exceptions to this. In A/B extractions, we take advantage of the solubility characteristics of alkaloids to both isolate the alkaloid and rid the extraction of unwanted compounds. In plants, these unwanted compounds are things like chlorophyll, lipids, fats, oils, cell walls, etc.

Another thing to know at this point is that solvents cover a range of polarity and some solvents are miscible with each other. If two solutions, one very polar and one very non-polar are tried to mix together they will separate from each other to form two layers. Oil and water are a great example of this. Water being very polar and oil being very non-polar. The separation into two layers is also key here as it allows us easily separate the solvents.

Some solvents are very good at dissolving things including plastic! Please use glass for these procedures, chemistry glassware being ideal. For this hypothetical extraction we will be using the following glassware items: A separatory funnel, 3 or so glass beakers or flasks, a buchner funnel, filter paper, a filter flask, a vacuum source such as a hand pump or a water aspirator, a glass rod, and a stand for the separatory funnel. The sizes should match the size of the extraction. For 10 gm of plant material a 250 ml size should be sufficient.

So with this (limited) knowledge, lets run through a typical extraction of an alkaloid from some leaves of a plant:

The starting material can be either wet (fresh) or dried depending on the sample. If fresh, we could dry them to make things easier, however this may or may not affect the alkaloid we are wanting to extract. Either way, grind, chop, or cut the sample into small pieces. The finer the better typically.

Extract: We put the plant material into a beaker or flask with an acid solution which is adjusted with distilled H2O to a pH of around 3. Stir and or heat this solution with the plant material gently for at least an hour. Here we using the acid to extract compounds out of the plant material and form the salts of the alkaloids. Remember, the salt forms are soluble in polar solvents like an acid and they will go into this solution. So will other things like chlorophyll and some of the other compounds mentioned above. Our solution will turn different colors depending on the compounds that are pulled into the solution. Take note of this. The type of acid that we use here will determine the type of alkaloid salt that is formed. If we use hydrochloric acid, then we are forming the hydrochloride salts of the alkaloids.

We decant the acid off of the plant material into another beaker trying to keep the plant material in the original beaker. If some of the plant material spills over its no big deal as we will deal with this when we filter.

Now we repeat the acid extraction of the plant material again with fresh acid at the correct pH. When done, we combine this acid with the first. Ideally this would be done a minimum of 3 times with all the acids from the extractions combined. With fresh leaves, they should now look quite a bit paler as we've extracted out most of the colored compounds from them.

Filter: Now it's time to filter. This is important as it effects the rest of the extraction procedure. The idea here is that we want to filter out ALL of the insolubles, even the tiny particles. If the acid solution has lots of different sized particles in it, it is a good idea to rough filter the solution first. This can be done with a cotton ball pushed into the spout of a funnel. We could also use a coffee filter, but this can be a slow and messy process. Next comes fine filtering. This is best done through filter paper made for the job. Vacuum filtration is the way to go. A buchner funnel is fitted to the top of a filter flask with the appropriate adapter and suction is applied to the flask with a water aspirator or a hand pump as the solution is poured into the funnel and caught in the filter flask. The filter paper with the insolubles is discarded.

De-fat: Now that our solution is filtered and we dealt with the unwanted particles it's time to address the unwanted compounds that are dissolved in the acidic solution. Most of these compounds, not forming salts, will be soluble in a non-polar solvent. Our alkaloids, being the salt form, are not soluble in it. Taking advantage of this, we add our acid solution to a separatory funnel and then a smaller amount of a non-polar solvent such as xylene to the funnel as well. Cap the funnel and then gently invert the funnel while holding onto the cap so that it doesn't fall off. Do this a few times and note the separation of the layers. Depending on the solvent used, the non-polar layer will either be at the top or the bottom. Because less of the non-polar solvent was added it should be easy enough to determine which is which. Also note after a minute or so of these inversions if an emulsion layer is starting to form. This can look like a cloudy layer that sits at the interface of the two layers. With good filtration we should not have much of an emulsion and we can invert the funnel a little more quickly. What happens here is that as the two layers mix around the fats, oils, and such move into the non-polar layer while the acid solubles (alkaloid salts) stay behind. After some minutes of aggitation, the funnel is put on a stand, the layers allowed to separate, the lid removed, and the aqueous layer drained off into a clean beaker. If there is an emulsion layer, drain this into the beaker as well. Discard the non-polar solvent (contains unwanted compounds) and add the aqueous layer (emulsion and all) back to the funnel. Repeat this process at least 3 times, at which point the non-polar solvent should be free of most any coloration. If not, repeat this de-fatting procedure till it is. The very last time that the solution is de-fatted leave the emulsion layer in with the non-polar solvent and discard it. It should not be very big. If the emulsion layer is large use one of the strategies below to reduce it before discarding it with the aqueous layer.

Filter: If any solids have precipitated out of the solution at this point, then the solution should be fine filtered again to remove them. This is the usual cause of emulsion layers and fine filtering is one way of dealing with them.

Making Basic: Now that we have the majority of the unwanted compounds out of the aqueous solution, we want to get the alkaloids out. To do this we want to add a base to the acid solution till the solution reaches a pH of about 12 or 13. There are many bases that can be used. Some common ones are ammonia, sodium hydroxide, calcium hydroxide, etc. Note after doing this if any more solids have precipitated out of the solution. If they have, fine filter to get rid of them as long as the pH is in the desired range. What we are doing here is to make the salts of the alkaloids into the freebase alkaloids. Now the alkaloids in the freebase form are more soluble in non-polar solvents than they are in solutions of higher pH. As a general rule, the alkaloids are more soluble in basic solution the higher the pH is. We also want the pH to be high enough to convert all of the salts and to not have the free base alkaloids suspended as particles in the solution as might happen at a lower pH.

Extraction: This basic aqueous layer now goes back into the separatory funnel and a smaller amount of the non-polar solvent is again added to the funnel. The procedure here is exactly the same, however we want to keep the non-polar layer this time as it will contain the freebase alkaloids. If we have an emulsion layer here (did we filter enough?) now is the time to deal with it. Lets assume there isn't one. After we have done this extraction portion a minimum of three times and combined all of the non-polar solvent layers we should have a pretty clean solvent containing our alkaloids (it may have color, this is okay). The easiest thing to do now is to evaporate the solvent which will leave the alkaloids in the evaporating dish. If we were really careful about filtering and separations, we may be left with crystalline or powdery alkaloids. Chances are pretty good at this point that we will end up with a sticky and gummy residue. There is nothing wrong with this, the alkaloids might have some impurities in them. At least they are much more isolated from the starting material and we can work on cleaning them up.

Before we get to cleaning up the extraction, lets consider some points to work on to get the cleanest possible extraction at this point:

1) Filter. I know I'm harping on this. The more well filtered the solutions, the easier the process is.

2) Separation of layers. When using polar/non-polar combinations in a separatory funnel, one of the layers may stick to the sides of the funnel. When separating the layers it is easy to contaminate the layer that you want to keep with the layer to be discarded. We can gently roll and or swirl the funnel to get the parts of the layers to go back together. Next, let the bottom layer out of the funnel slowly. Leave a bit of this bottom layer and close the stopcock. Now, now gently roll and or swirl the funnel to get the layers to separate out more and finish draining the bottom layer.The Top layer should be more clean.

3) Use clean equipment. This is pretty obvious, but the more clean the better. Clean all the glassware after each use. Ethanol is a good cleaning solvent for extractions. Clean equipment first with ethanol and then light soap and water, rinsing well to get rid of soap. (The ethanol can be everclear or vodka)

4) Emulsions. Deal with them as soon as they appear. Most of them are formed from particles in one of the layers. We can try filtering the extraction through filter paper to break up the emulsion. We can also try a thin layer of very clean sand on top of the filter paper in the vacuum set up. Some people suggest poking at it with a glass rod to break it up. Heating the extraction up works sometimes as does adding more base if you are at the alkaloid extraction portion of the process. Adding non-iodized table salt to the acid solution may help in the de-fat portion. Sometimes walking away from it and leaving it to do its thing overnight or longer will resolve the emulsion or at least condense it into something manageable. If all of the above have been tried and its still there, we can very carefully isolate it out of the extraction using the separatory funnel by slowly letting the bottom layer out till we get to the emulsion. Close the funnel and switch containers to catch the emulsion with a touch of the bottom and then a touch of the top layer. If the emulsion layer is small, we may want to discard it. If it is large we may want to clean it up separately from the extraction to get any alkaloids that may be locked up in it. We'll talk about that a bit later.

5) Drying. Dry the final solvent extraction using a sulfate salt such as magnesium sulfate or sodium sulfate. This will absorb any of the aqueous portion that may have come over with the non-polar solvent.

6) Powdered material. Powdering dry material can help to get compounds out of the starting material by increasing surface are. The down side to this is that the powder can be very fine and very difficult to filter completely out. These fine particles can be the cause of emulsions.

Now what do we do with this gummy material to clean it up? The first option is to re-dissolve the gum in an acid solution and repeat the A/B procedure once more on a much smaller scale. If we are as careful as we can be this just might leave a dry material in the end. We could also run chromatography on the gum to separate all the compounds, but this can be even more complicated than an A/B procedure. We can also try to clean it up using re-crystaliization. If we have a mixture of alkaloids, re-crystallization might lose some of these alkaloids. This can be a good or bad thing depending on what we are trying to achieve.

Re-crystallization. So, here we go... First we need to know how soluble our alkaloid is in various solvents. We pick a solvent that it has a moderate solubility in. What we are looking for is a solvent that will readily dissolve the alkaloid when heated but not so much when cool. The idea then is that we heat the solvent to a point that is below its boiling point and add just enough to our gummy material to dissolve it completely, but not more. Hopefully at this point there are no particles in the warm solution. If there are then we might want to think of a way to filter them and not cool the solution very much. We probably should have done another A/B to further clean it if this is the case. If all of the gum does go into solution, then great! No filtering needed, warm filtering is a pain that I'm not going to go into here. Now we slowly let the solution cool to room temperature. The key here is slow. Once its been at room temperature for a while we put our beaker of solution in a cold water bath. Some people would put it in the fridge, but we don't want our fridge contaminated. Maybe one day we will think about getting a fridge for the lab. Now, periodically we are going to add ice to the water to further cool the water and the solution in the beaker. We are going to try and do this as slowly as possible and it might just try our patience. We should see the alkaloids precipitating out of the solution as it cools. With enough patience we can let these settle to the bottom of our beaker and then decant off the solvent to collect wet crystals. The other option is to chill our vacuum filter set up in a cooler of ice to chill it and then vacuum filter the cold crystals. Now we need to wait for them to dry and then store them in a safe place.

Cleaning up large emulsion layers. What we do here depends on whether the alkaloids are in the salt form or the freebase form in the emulsion, so we will look at these separately:

For the salt form we will add our acid at the correct pH to the emulsion. This should take up the salts in the acid and separate out any of the non-polar solvent that is in the emulsion. This is then run through our vacuum set up to remove any particles and then into the separatory funnel. We now add a smaller amount of our non-polar solvent, agitate, and see if we still have the emulsion or not. If so, we can try one of the above methods to further reduce it. Once the emulsion is cleared we run through the rest of the A/B procedure starting with the de-fat to recover the alkaloids.

For the alkaloid form, we want to add a solution of non-iodized table salt in distilled H2O to the emulsion. This should separate out the non-polar solvent and draw any contaminations into the salt water. Run this through the vacuum filter set up as above and into the separatory funnel. If there is no emulsion, add a little more non-polar solvent to the funnel, agitate and then separate the layers. See the above methods for further work on the emulsion layer if it still forms. The alkaloids should now be in the non-polar solvent. This can be evaporated off to leave the alkaloids.

Don't worry about messing up. If we make a mistake along the way, we should be able to figure out how to fix it by thinking about how the process works. It works both forward and backward allowing a good deal of manipulation of the desired as well as the unwanted compounds.

Please be aware that solvents can be flammable and dangerous to your health. Never work near any open flame, spark, etc. Always wear gloves and safety glasses at the bare minimum. Always practice your extraction in an area with adequate ventilation. Strong acids AND strong bases will cause burns. Never forget about safety.

Dozuki
 

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nen888
#2 Posted : 2/8/2012 2:41:46 AM
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Acacia expert | Skills: Acacia, Botany, Tryptamines, CounsellingExtraordinary knowledge | Skills: Acacia, Botany, Tryptamines, CounsellingSenior Member | Skills: Acacia, Botany, Tryptamines, Counselling

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..this character knows what he's talking about! Smile ..can't find much to add to that, except most acacias in experience usually don't need defatting..
great summary of the A/B principle Dozuki..
 
endlessness
#3 Posted : 2/8/2012 11:15:36 AM

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Good work Smile

This was added as a link at the end of this faq entry:
https://wiki.dmt-nexus.m..._Guide#Where_to_start.3F
 
DidMyTest
#4 Posted : 2/8/2012 11:43:47 AM

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Good instruction, well done. I would add a point I think it is easier when the solutions gets reduced first before adding the solvent to get the alkaloids out, so it is less effort in the seperation process. For that step I got a questions: The DMT handbook tells us we should reduce the acidic solution, I asked myself if it's possible:

1) To neutralize the solution first before basifying it.
2) To reduce it with PH around 7
3) Then basify it to PH 12 or higher

The advanatge would be that the process would be less dangerous (so that one could do it in the home kitchen) and also there wouldn't be a need for a very good ventilation. Or are the freebase molecules not stable to 100 degrees (+ some more degrees on the bottom of the pot)?
Any spelling or grammar mistakes? Please help me to improve my English and write me a PM. Just write what is wrong and how the rule is.
 
endlessness
#5 Posted : 2/8/2012 11:53:20 AM

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At pH 7, 98% of DMT will be in salt form, so it will be mostly stable. If you want to be safer, go for pH 6 for reduction so 99.8% is in salt form.

But if you're using vinegar as an acid, there is no danger reducing your solution when its acidic.

I would not boil a solution containing freebase dmt (though I know people here in the forum do water baths, which should b a bit under 100c, with dmt freebase and it doesnt boil away, it just melts)
 
Oneiroi
#6 Posted : 2/12/2012 11:04:15 PM

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Excellent post man! Very well written, this could be a lab for the organic chem class I'm taking haha. Anyways, I had a few questions:

Quote:
As a general rule, the alkaloids are more soluble in basic solution the higher the pH is. We also want the pH to be high enough to convert all of the salts and to not have the free base alkaloids suspended as particles in the solution as might happen at a lower pH.

I thought once the alkaloids are converted to salt form, they are more soluble in the organic non polar solvent. You make it sound like the alkaloids are more soluble in the basic aqueous layer when the pH is greater.

Once the alkaloids are in the freebase form, they will remain relatively insoluble in the aqueous layer so long as its not strongly acidic, correct? I am doing an extraction where I wash freebase alkaloids in organic solvent with some tap water to remove impurities. I just want to make sure I'm not accidentally washing away my alkaloids.

Also, I was wondering if you are familiar with the STB methods. If you are, what do you consider to be the benefits of doing the A/B extraction over the STB extraction (and vice versa)? I'm assuming you get a more pure product with an A/B extraction but probably a lower yield.
Anything I say is completely false and should not be taken seriously.
 
Dozuki
#7 Posted : 2/13/2012 12:15:06 AM

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DidMyTest wrote:
I would add a point I think it is easier when the solutions gets reduced first before adding the solvent to get the alkaloids out, so it is less effort in the seperation process.


This is true for larger extractions. Since I do mostly alkaloid determination testing I don't usually extract more than 10 grams of material. Most of my partitioning can then be done in a 250 ml separatory funnel.

Another method to help reduce the amount of solvents is to extract first with EtOH, MeOH, ammoniacal CHCl3, or ammoniacal DCM. Evaporate this to dryness and take up the residue in the acid of your choice and proceed normally.

DidMyTest wrote:
2) To reduce it with PH around 7


This probably isn't a bad idea to do before reduction to avoid extreme pH levels. However, I have accidentally acidified a grass extraction to pH .5 before de-fating with no ill effect that I could observe. But, pH 2-3 seems safer.

Oneiroi wrote:
I thought once the alkaloids are converted to salt form, they are more soluble in the organic non polar solvent.


Once in salt form, the alkaloids are more soluble in the acid aqueous layer. IIRC this is typically about 2-3 pH lower than their pKa. pH 2-3 is a safe pH to shoot for with tryptamines to get them into their salt form. Here they stay in the aqueous acidic layer when partitioning with a non-polar solvent. This is the basis of a de-fat. The NPS here rids the extraction of oils, lipids, and other things so that they don't get extracted in the NPS with the alkaloids after conversion to freebase.

Oneiroi wrote:
You make it sound like the alkaloids are more soluble in the basic aqueous layer when the pH is greater.


There will be a point when increasing the pH after the de-fat that the alkaloids are less soluble in the aqueous layer if the pH is increased slowly. Once the pH is raised above this point, they tend to go back into solution. Here, we are only talking about the aqueous layer without a NPS layer prior to partition. Harmalas are a case in point here as they will readily drop out of solution at specific pH. This is a technique use to separate them. You can also observe this with MHRB if you increase the pH slowly. In this case some of the precipitation is probably due to tannins, but as you raise the pH you can watch the cloudiness (precipitation) go back into solution. Once they are back in solution then we extract them with a NPS.

Oneiori wrote:
I am doing an extraction where I wash freebase alkaloids in organic solvent with some tap water to remove impurities.


This is called back washing. The alkaloids in the NPS are freebase form, so they are more soluble in this solvent than in neutral H2O. This is essentially the reverse of a de-fat and is an often used method for further purification. You may lose a small amount of the alkaloids to the H2O, but it should be negligible. If you want to look into this further, look into partition coefficients.

The benefit of A/B over STB is a cleaner extraction. I do a lot of extractions on leaf material and there are more compounds that need to be dealt with than root bark. One is chlorophyll. Doing a STB on leaf material would extract the chlorophyll, oils, lipids, etc. into the final extraction. STB is used because MHRB tends to be fairly free of these other impurities. Re-crystallization after the STB essentially eliminates any of these impurities if they do cross over. With the small amounts of samples I work with, any sort of re-crystallization would be very tedious, so I try and shoot for a clean initial extraction. Both methods should produce very similar yields.
 
Dozuki
#8 Posted : 2/13/2012 12:18:53 AM

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nen888 wrote:
..can't find much to add to that, except most acacias in experience usually don't need defatting..


What about when extracting phyllodes?
 
Oneiroi
#9 Posted : 2/13/2012 7:43:40 AM

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Great thanks for the information Dozuki! Smile You really seem to be quite the expert.
Anything I say is completely false and should not be taken seriously.
 
Dozuki
#10 Posted : 2/13/2012 9:37:44 PM

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I wouldn't say I'm an expert. I've just done a number of extractions and like to understand what's going on. Thank you for the compliment tho.
 
nen888
#11 Posted : 2/14/2012 2:20:47 AM
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Dozuki wrote:
Quote:
nen888 wrote:
..can't find much to add to that, except most acacias in experience usually don't need defatting..


What about when extracting phyllodes?


..i'm still eagerly awaiting endlessness GCMS on an extremely crude leaf extract, but from a little advance word, there doesn't seem to be a lot else in there..

the obtusifolia x maidenii extract (almost pure DMT, crystalized) of leaf and twig was not defatted..i've never gotten very much of anything out when defatting acacias and don't bother anymore, though in the case of leaf extracts, maybe removing trace fatty acids will lead to a cleaner product..
 
phyllode
#12 Posted : 11/11/2012 1:16:38 AM

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I've bumped this (thanks nen for link, and tips in the past) because:

a) It's a very good summary of Extraction principles.
b) There are an increasing number of extraction question threads lately in which people obviously haven't quite grasped the concept of A/B Extraction. It's more important to understand why you're doing something than following some step by step instructions that make no sense to you.

Thank you Dozuki for your Art of Explanation.

I would suggest no one undertake an extraction unless they understand the following words/concepts:

1) pH; 2) SALT; 3) FREEBASE; 4) SOLUBILITY; 5) NON-POLAR.

Also, and Nen has suggested this a few times, if inexperienced, it is better to practice on material known to contain what you're looking for, than on unknown plants.

Now, the aspect I myself would like to know more about and explore further is SALTING. Any tips?
 
 
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