Iv been very interested in solid phase extraction of c. paspali fermentation broth.
There are many ways to do this

US patent 4237291

EXAMPLE 5

30 l Culture solution of Clavicepts purpurea IMET PA 134 at its natural pH value (5-6) is stirred for 30 minutes with 1.2 kg bentonite. It is then filtered through a layer of calcium sulfate dihydrate and the filtrate discarded. Drying of the mycella-adsorbent mixture according to Example 1 yields 2.8 kg of the dry mixture, which contains practically all of the alkaloid content of the culture suspension (ergosine, ergosinine, traces of chanoclavine). Rotary extraction of the alkaloids with a 10 to 12 fold volume of ethyl acetate yields up to 90% of the alkaloid content of the dry mycella-bentonite mixture.

This method works with most montmorillonite compoud.  Calcium bentonite is used because its easier to filter and doesn't make a colloid like Sodium bentonite.  Fullers earth I think will work better because it does not form clay as easily, and comes in granules, even as cat litter.  Also it works without any type of acid activate, apparently that helps with the bentonite.  The drawback with the bentonite method is the fact that you have to dry it or extract from it, which is messy and slow to filter.  Fullers earth in a flow-through adsorption column would probably work better.  These types of materials adsorp best at the natural pH of the claviceps broth.  Eluting is done with methanol, or water with base or just a mixture.  Eluting with methanol and or base is more efficient.  I think eluting with base is more prone to cause epimerization, especially with methanol involved tho.

Czech Patent 269105
This uses c18 silica

Partial Translation:
1L filtered fermentation liquid containing [peptide alkaloids] was adjusted with aqueous ammonia solution at pH 6.8 This solution was added 35 g of silica gel 2 x and the bound oktadecylovou velikosticastic? group of 50um (Separon SGX C18) and mixed 15 min on a magnetic stirrer. The mixture was filtered, washed filter cake 100 ml of distilled water and drained. Filter cake was extracted 2 x 150 ml of methanol containing 5% aqueous ammonia. Extracts were combined and concentrated in evaporators. 70% yield

This is very interesting because silica gel is cheap, and is a good flow-through method unlike the clay methods.  Scaled up of course there will be less solvent required.I believe this method is not optimized for c. paspali which contains mostly water soluble ergoamides, whereas c. purpurea methods will contain mostly water insoluble ergopeptides and methods will differ, especially type of optimal solid phase adsorbant used.


Japanese Patent 2-167051

Full translation
Specification

1. Name of the invention
     Separation and purification methods of ergot Alkaloid

2. Scope of Patent Request
   1.  Separation and purification methods of ergot Alkaloid which has a characteristic like dissolving the Alkaloid by Dissolving agent after adsorption of ergot Alkaloid by having Fermentation liquor possessing ergot Alkaloid that can be obtained by culturing Ergot Claviceps paspali contacting a kind of Synthetic adsorbent porous polymer that can be selected from Styrene-Divinylbenzene Copolymer and Phenolic resin.

3. Detailed explanation on the invention
   
Application area in the industry

This invention is regarding how to achieve separation and purification of ergot Alkaloid out of Fermentation liquor that can be obtained by culturing Ergot Claviceps paspali.
Ergot Alkaloid is a useful compound known for a variety of efficacy as a medical product.
   
Traditional Technology

Fermentation liquor of Ergot Claviceps paspali is an Aqueous solution that mainly contains 8:2 mixture of ?-Hydroxyethyl lysergic acid amide and lysergic acid amide as well as sugar which is culture component, ammonium salts of organic acids, metal ions and a variety of foreign substances such protein, fat, pigment, which are the components of metabolite and body fungus.
Traditionally, the standard method of separating ergot Alkaloid was to extract it by organic medium such as Chloroform, Ethyl acetate, and n-Butanol. However, in order to improve the collection rate of ergot Alkaloid, this method requires a lot of solvent. Another problem is that it was hard to avoid emulsification caused by protein, fat, and defoamer contained in fermentation liquor.
The other method that uses resin is explained in Patent-Application-Sho-45-12303 to use strongly acidic cation ion exchange resin. However, for ion exchange resin, a special processing is required for the resin to be activated or regeneration. Besides, there are many challenges for the practical application such as swelling and heating. Patent-Application-Sho-54-90198 has Separation and purification methods of ergot Alkaloid using insoluble polymer containing substituent like Alcoholic hydroxyl group. However, the content is about the method to separate mixture of peptide type ergot Alkaloid such as Ergocristine, Ergocryptine, and Ergocornine.

Challenge to be overcome by this invention

As mentioned above, up until this point, there is no practically satisfactory method established in the industry for separation and purification of ergot Alkaloid out of fermentation liquor.
This invention provides a new method of separation and purification of ergot Alkaloid.

Method to resolve the challenge

This invention is regarding how to achieve separation and purification of ergot Alkaloid out of Fermentation liquor that can be obtained by culturing Ergot Claviceps paspali.

Separation and purification methods of ergot Alkaloid which has a characteristic like dissolving the Alkaloid by Dissolving agent after adsorption of ergot Alkaloid by having Fermentation liquor possessing ergot Alkaloid that can be obtained by culturing Ergot Claviceps paspali contacting a kind of Synthetic adsorbent porous polymer that can be selected from Styrene-Divinylbenzene Copolymer and Phenolic resin. Fermentation liquor produced by Claviceps paspali has full of amide type ergot Alkaloid such as ?-Hydroxyethyl lysergic acid amide and lysergic acid amide. Even though it is also ergot fungus, the fermentation liquor of Claviceps purupurea mainly consists of peptido type ergot Alkaloid, these chemical structure as well as physical and chemical characteristics are different.
For synthetic polymer absorbent used for this invention can be Divinylbenzene-styrene Copolymer and Phenolic resin. These synthetic polymer absorbent does not have remarkable functional group in the molecule, but simply, absorbs the compound by Van der Waals force possessed by the molecule. Generally speaking, we use the ones with grain diameter 250~857 um, pore diameter about 500 A, and the surface space is 400 ~ 1000 m2/g. For example, styrene-divinylbenzene Copolymer, Sepabeads SP207, SP206, SP800, SP900 by Mistubishi Chemical, Duolite S-866 and S-861 by Rohm and Haas. For Phenolic resin, duolite S-761.

Though Synthetic polymer absorbent is able to process fermentation liquor by batch, the method like passing fermentation liquor by getting packed in a column is easier and better. The amount of resin to be used depends on the absorbent power of ergot Alkaloid 60 mg/ml - wet resin. The size of column is not specified, but the column length is better to be 2 to 5 times longer than the inner diameter of the tube.

Because of Hydrophilicity of amid type ergot Alkaloid, organic eluent that contains 5~80 weight % of water can be used for this invention. As organic eluent, water soluble alcohol type like methanol, ethanol, n-propanol, secondary propyl alcohol, and water soluble ketons like acetone, water solule ether like tetrahydrofuran and dioxane, and water soluble nitrile like acetonitrile are used, and can be used either as single solvent or compound solvent of those. Also, you may add alkalescent component such as Sodium hydrogen carbonate aqueous ammonia or weakly acidic component such as succinic acid and citric acid into the solvent system.

Next, example steps performed for this invention will be explained.

After filtered from fungus body, fermentation liquor of Claviceps paspali can be directly used for this experiment method. However, because the pH of the liquor is 5.0 ~ ., it is better to add ammonia water to increase pH to over 7.0 if you wish to improve yield of ergot Alkaloid. This way, it is possible to collect ergot Alkaloid constantly. Pack synthetic polymer absorbent into the column in advance, then, wash it by water. The passing speed of fermentation liquor to go through the column packed with synthetic polymer absorbent should be 0.5 ~ 2.0h. After fermentation liquor has been passed through, next, pack eluent into the column. the amount to be used and the speed of the eluent depends on the type of the eluent, but in general, 3 to 6 times larger than the content of the resin. You may decide whether or not the elution of the Alkaloid has been completed or not by thin-Layer Chromatography. It is easy to extract ergot Alkaloid with high purity by removing the remaining liquor including water using extracting solvent like ethyl acetate after removing elution under the decreased pressure. The used Synthetic polymer absorbent can be easily reused after being washed by secondary propyl alcohol including 1N sodium hydroxide aqueous solution or acetone, followed by water wash. Below, more details of this invention with application examples:

Example

Example1

After Fermentation liquor of Ergot Claviceps paspali 500 ml (including ergot Alkaloid 1040mg, pH5.2) went through a column packed with 20ml of styrene-divinylbenzene Copolymer, Sepabeads SP207 (Mitsubishi) by speed 5ml/min, wash it with 80ml of water, then, eluted with 50% methanol water, 80ml. Under the decreased pressure, the methanol is removed from the elution, and adjusted it to be pH9 by adding ammonia to the remaining liquid, extract by ethyl acetate, then, remove ethyl acetate, then let it dry, resulted ergot Alkaloid with 92% purity, 768mg (yield 68%).

Example2

Using ammonia water, the fermentation liquor used by Example1, 500 ml (including ergot Alkaloid 1248mg) was adjusted to pH9. Then, let it go through the column with 20ml of Sepabeads SP207 (o20x64mm) with the speed 5ml/min. This gets washed with 80ml of water, followed by eluting with 85% secondary propyl alcohol 80ml. Same process as used in example1, esulted ergot Alkaloid with 94% purity, 1314mg (yield 99%).

Example3

Using ammonia water, the same fermentation liquor used by Exampe1, 1000ml (including ergot Alkaloid 832mg), was adjusted to pH9. Then, let it go through the column packed with Duolite S-861 (Rohm and Haas) 20ml (o20x64mm) with speed 5ml/min. After washed by 80ml of water, eluted with 50% acetone solution 80ml. Same process as in Example1, resulted ergot Alkaloid with 90% purity, 915mg (yield 99%).

Example4

Using ammonia water, the same fermentation liquor used by Exampe1, 1000ml (including ergot Alkaloid 832mg), was adjusted to pH9. Then, let it go through the column packed with Duolite S-761, 20ml (o20x64mm) with speed 5ml/min. After washed by 80ml of water, eluted with 50% acetone solution 80ml. Same process as in Example1, resulted ergot Alkaloid with 92% purity, 859mg (yield 95%).

Effect of the invention

This invention enabled us to easily collect high purity ergot Alkaloid with high yield out of fermentation liquid containing ergot Alkaloid.

Intersting note that the resin they use in the first few examples has a trade name in the US of XAD-4.  This patent is optimized to the alkaloids in C. paspali and is as good as it gets.  I think eluting with mostly methanol would be awesome because it would be easy to remove, and base is not required with this polymer extraction.  Ph of the broth needs to be adjusted more basic to optimize extraction, I have some references which explain why this is so, will attach later on.  It may be possible elute with tartaric methanol/h2o which would stabilize the alkaloids as they come out to prevent epimerization.  This other xad-4 patent says lowering ph is not optimal for this but they refer to other alkaloids.  As usual it might help elute with somewhat basic solution but may not be necessary. 

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If you don't want to work this all up with a/b and solvents after evaping the methanol I see two options:

1)
Elute using mostly methanol and then add excess tartaric acid, remove most solvent then add 10x ether or maybe hexane to precip the tartaric salt of LSA's.  All this work will probably convert LSH to LSA by the time the tartaric hits it.  I assume the 5% water which must be present for elution may disrupt crystallization using this method?

2)
Elute using mostly methanol directly into a nitrogen purged boiling KOH solution with a distillation head on it.  Most of the methanol should boil off as it is eluted during the 2hr+ hydrolysis.  Once all methanol is added and hydrolysis no longer off gasses ammonia then lower temperature add sulfuric acid to low pH and lysergic acid sulfate will precipitate.


This makes it possible to logistically do huge extractions.

This may be of interest

United States Patent Application 20110137035
...

For the processing according to the invention of the ergot alkaloids obtained in the microbiological process, in particular paspalic acid, the entire fermentation broth, without separation of the biomass from the fermentation filtrate, can be used as the fermentation product for further processing. This entire fermentation broth contains as ergot alkaloids essentially paspalic acid as the main metabolism product of the microbiological process, in addition to smaller amounts of lysergic acid and where appropriate lysergic acid derivatives. The entire fermentation broth conventionally has an ergot alkaloid titer of between 0.5-40 g/kg of entire fermentation broth. The fermentation filtrate can also be employed as the fermentation product used for the extraction as long as the additional outlay for separating off the biomass from the fermentation filtrate is not shied away from. Preferably, according to the invention the entire fermentation broth without separation of the biomass from the filtrate is employed as the fermentation product.

...

EXAMPLE 1
1.25 kg of an entire fermentation broth which had a titer of 7.98 g of ergot alkaloid of the formula I/kg was adjusted to a pH of 10 by addition of NaOH and extracted twice with 1.25 l of n-butanol each time at this pH. The yield of ergot alkaloid of the formula I after the two extraction steps was 83%. The combined extracts were concentrated to about 30 g of ergot alkaloid of the formula I/kg of extract and about the same amount of water was added. Thereafter, the pH was adjusted to a value of 5.6 by addition of sulfuric acid. Paspalic acid passed from the organic into the aqueous phase and crystallized out. 7.2 g of paspalic acid with a purity of 93% were obtained in this way. This corresponds to a total yield of 67%.

EXAMPLE 2
878 g of a fermentation broth which contained 2.1 g/kg of lysergic acid and 8.3 g/kg of paspalic acid were kept at pH 12.5 and 50° C. for 4 hours. After 4 hours, the broth contained 7.96 g/kg of lysergic acid and 0.37 g/kg of paspalic acid. The active compound was then extracted from the broth twice with 900 ml of n-BuOH each time and concentrated in vacuo to a content of 25 g/kg. Thereafter, the pH was adjusted to a value of 5.6 by addition of sulfuric acid, as a result of which the lysergic acid crystallized out.

The crude product obtained in this manner had a purity of 75%, and the yield, including the conversion, was 50%. By adding in the next cycle the butyl acetate mother liquor to the broth before the next conversion, it was possible to increase the yield to 70%.

Comparison Process
Active charcoal is added to harvested broth containing paspalic and lysergic acid. At a neutral pH, paspalic and lysergic acid bind to the broth or to the active charcoal. The solid constituents are centrifuged off and the supernatant is discarded. The biomass isolated in this manner together with the active charcoal is then suspended with an ammoniacal alcohol solution, the two acids becoming detached from the solids. The two acids can finally be obtained in this way in the supernatant after a renewed centrifugation of the biomass and the active charcoal. When the supernatant is concentrated, paspalic and lysergic acid precipitate out as the crude product.

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