GB2180538A - Process for the separation of corrinoids - Google Patents

Abstract

The invention relates to the separation of corrinoids, more particularly to the separation of corrinoids by selective adsorption and/or selective elution on a polymeric absorbent having a large specific surface. The process according to the invention is equally suitable for the separation of incomplete corrinoids from complete corrinoids and various complete corrinoids, preferably vitamin B12 and factor III from each other.

Description

SPECIFICATION Process for the separation of corrinoids The present invention relates to a new process for the separation of corrinoids. More particularly, the invention concerns a process for the separation of incomplete corrinoids from complete corrinoids or for the further separation of various complete corrinoids, by means of a polymeric adsorbent having a large specific surface.

Compounds having a "corrin" skeleton, such as vitamin B12 (5,6-dimethylbenzimidazolyl cyanocobamide), the first and biologically most important member of this family, are well known pharmacologically active compounds. They are conventionally prepared by fermentation procedures. The various microorganisms used for fermentation may produce different "corrin" compounds and the fermentation medium may contain corrinoids which are in different stages of biosynthesis when fermentation is terminated. The cells present in the fermentation medium contain simultaneously both complete (nucleotide-containing) and incomplete (devoid of nucleotides) corrinoids.

Incomplete corrinoids, e.g. combinamide and cobinic acids, are biologically inactive and, in certain cases, biologically detrimental. Their presence makes the isolation and crystallization of the valuable complete corrinoids (cobalamines) difficult. Moreover, incomplete corrinoids should be avoided even in the crude vitamin B,2 products (containing a mixture of different "corrin" compounds), which are generally used as fodder additives, since it is known that e.g. cobinamide fed to chickens as a fodder additive, acts as a vitamin B12 antagonist (vide E.L. Smith; Vitamin B12, Methnen, London, 1960, p.84).

In certain cases the separation of various complete corrinoids from each other may also be necessary. For example, if the fermentation is carried out by means of microorganisms having a mixed population (see e.g. the semicontinuous process described in Hungarian Patent Specification 159,356), in addition to the incomplete corrinoids, mainly so called factor Ill (5-hydroxybenzimidazolyl cyanocobamide) contaminates the vitamin B12 product. When used as a fodder additive, factor Ill has essentially the same activity as vitamin B,2; accordingly when used as a fodder additive they need not be separated from each other. For human purposes, however, only pure crystalline vitamin B,2 may be employed. Thus for human appiication vitamin B,2 and factor Ill must be separated from each other.Factor Ill separated from vitamin B,2 may be further purified, if desired, and used as a fodder additive per se.

There are various processes known in the art for the separation of complete corrinoids from incomplete corrinoids, i.e. for the separation of combinamide and cobinic acids from cobalamines. There are also known processes for the separation of vitamin B,2 and factor Ill.

According to German Patent Specification 930,651 the various corrinoids are separated by partition chromatography with a mixture of water and n-butanol containing cyanide ions, on a column filled with cellulose powder. The purified product obtained from waste water, which contains factor Ill and incomplete corrinoids in addition to vitamin B,2, is precipitated on silica gel, dried and the product obtained is applied to the above-mentioned column. The whole procedure is essentially an analytical method, which is not suitable for the separation of industrial quantities. Moreover, while the prior art discloses how to separate factor Ill from incomplete corrinoids, there is no disclosure as to the separation of these components from vitamin B,2.

According to British Patent Specification 789,019 vitamin B,2 is isolated from a mixture containing other corrinoids by extracting the concentrate with a mixture of an apolar solvent and a compound containing a phenolic hydroxyl, adsorbing vitamin B,2 on alumina from the solvent phase and dissolving it using acetone.

An improved version of the above process is disclosed in Hungarian Patent Specification 147,627, in which cobinamide is separated from cobalamines by extraction with a 1:6 mixture of phenol and chloroform, from a medium having a pH of 4 to 5, whereupon the aqueous phase is made alkaline to pH 8-9 and the cobalamines are extracted with a 1:5 mixture of phenol and chloroform. Thereafter, vitamin B12 and factor Ill are separated from each other in an acetone medium, by chromatography on an alumina column.

The process disclosed in the above two patent specifications is disadvantageous in that during the extraction with a mixture of phenol and chloroform there is a high susceptibility to emulsion formation. Thus, on a large scale the solvent phases can only be separated using a centrifugal separator. Naturally, the formation of emulsion decreases the efficiency of extraction. A further drawback is that there is no economic solution for the regeneration of the solvent mixtures. In addition, since the extraction of cobinamide under these conditions is not selective, there is a great loss of cobalamines.

According to the procedure described in Hungarian Patent Specification 150,555 incomplete corrinoids are separated from a mixture of vitamin B12, factor lil and incomplete corrinoids from an aqueous or an aqueous/alcoholic solution by adsorption on oxycellulose, whereupon vitamin B12 is extracted from the purified solution obtained using a 1:7-1:8 mixture of phenol and chloroform, containing thymol. The organic phase is washed with water, acetone is added and the aqueous phase containing vitamin B,2 is released from the phenolic phase by extraction with chloroform. The solution is evaporated and vitamin B12 is crystallized. Factor Ill is obtained in crystalline form after a purification from an aqueous carried out with a suitable solvent. The latter purification step is not described in detail in the cited patent specification.

The industrial application of this process is limited by the weak specific adsorption capacity of oxycellulose. A further disadvantage is that the regeneration of the oxycellulose is cumbersome and rather expensive on a large scale, since it has to be performed at a temperature of 60 to 70"C, with a 0.1N hydrochloric acid solution. Furthermore, the separation of vitamin B12 and factor Ill involves the use of large volumes. To isolate 1 kg of vitamin B12 crystals, 2.4 m3 of the phenol/chloroform mixture are used in this procedure, and supposedly a similar volume of solvent is required for the isolation of factor Ill from the aqueous phase. Since there is no economic method for the regeneration of the large volume of phenol/chloroform/acetone mixture, this is a great disadvantage of the process.

None of the above-cited processes is suitable for the selective, economic separation of corrinoids formed during the fermentation carried out with microorganism cultures of mixed population, on an industrial scale.

The purpose of the present invention is to provide an industrially applicable, economic method for the selective separation of incomplete corrinoids from complete corrinoids and complete corrinoids, primarily vitamin B12 and factor Ill, from each other.

We have experimentally found that the cobalamines can be efficiently separated from cobinamide and cobinic acids in a single step, in satisfactory purity by selective adsorption or elution on a polymeric adsorbent having a large specific surface and if desired, factor Ill can be removed from vitamin B12 in a further step, by selective elution and vitamin B12 can be isolated in a known manner.

In one aspect of process according to the invention complete corrinoids are adsorbed from an aqueous solution or from an aqueous solvent mixture containing at most 20% by vol. of a water-miscibe lower aliphatic alcohol (preferably methanol or ethanol) or at most 5% by vol. of a water-miscible lower aliphatic ketone (preferably acetone) containing a mixture of complete and incomplete corrinoids, e.g. cobalamines, cobinamide and cobinic acids, on a polymeric adsorbent having a large specific surface, in the presence of cyanide or sulfite ions or compounds capable of providing these ions, at pH 8 to 11, preferably 9 to 10, when incomplete corrinoids remain in the solution.

Alternatively, both complete and incomplete corrinoids may be adsorbed on the adsorbent from a dilute aqueous or aqueous/organic solution, at pH 2 to 8 in a known manner, whereupon the incomplete corrinoids may be eluted with an aqueous solution made alkaline to pH 8 to 11, preferably 9 to 10 or with an aqueous solvent mixture containing at most 20% by vol. of a water-miscible lower aliphatic alcohol (preferably methanol or ethanol) or at most 5% by vol. of a water-miscible lower aliphatic ketone (preferably acetone), in the presence of cyanide or sulfite ions or compounds capable of providing such ions, at room temperature, while complete corrinoids remain bound on the column.

Complete corrinoids may then be eluted from the adsorbent using generally known methods preferably by using a water-miscible organic solvent, optionally containing water, such as an alkanol.

If vitamin B12 and factor Ill are to be separated from each other, in both process variants factor Ill is eluted from the column with an aqueous solvent mixture adjusted to pH 8 to 11, preferably 9 to 10, containing 20 to 40% by vol. of a water-miscible lower aliphatic alcohol (preferably methanol or ethanol) or 5 to 20% by vol. of a lower aliphatic ketone (preferably acetone), in the presence of cyanide or sulfite ions or compounds capable of providing such ions, at room temperature, whereupon Vitamin B,2 is eluted from the column in a known manner preferably with a water-miscible organic solvent optionally containing water e.g. with methanol or aqueous acetone.

If vitamin B12 and factor Ill do not need to be separated from each other, the complete corrinoids are eluted from the solution in a known manner, e.g. with methanol or aqueous acetone as described above.

As a polymeric adsorbent having a large specific surface for example a non-ionic, nonpolar or slightly polar macroreticular adsorption resin may be employed, which has a pore size of 10 8 to 10 7 m and a specific surface of at least 200 m2/g. Such resins include e.g.

Amberlite XAD 2, XAS 4, XAD 7, XAD 8 (Rohm and Haas), Diaion HP 20, HP 21, SP 207, HP 2 MG (Mitsubishi).

In the process according to the invention, as compounds capable of providing sulfite ions e.g. sodium or potassium bisulfite, sodium or potassium metabisulfite, sodium or potassium sulfite, aqueous sulfur dioxide solution, dialkyl sulfites, such as dimethyl sulfite may be used.

Compounds capable of providing cyanide ions include e.g. metal cyanides, such as potassium cyanide, sodium cyanide, and ammonium cyanide, and hydrogen cyanide. Compounds containing the cyanide group in a complexed form (e.g. potassium ferrocyanide) or bound to a carbon atom (e.g. benzylcyanide, benzonitrile) are also suitable for this purpose.

The adsorption can be carried out by admixing the adsorbent employed with the solution containing the corrinoids. The adsorbent may be filled into a column, fixed bed or fluid technologies can be employed and the solutions can be passed through the column either downstream or upstream.

The pH can be adjusted with an appropriate base, preferably ammonium hydroxide or sodium hydroxide.

The separation technique according to the invention can be employed starting from crude fermentation media, but is equally suitable for use on a purified solution.

The main advantages of the process according to the invention include the following: 1. The separation is selective and there is no loss of cobalamines.

2. The process is equally suitable for the separation or corrinoids from crude fermentation media after digestion of cells and for the treatment of corrinoid-containing solutions pre viousiy purified in one or more steps.

3. The method provides excellent industrial results since the polymeric adsorbents having a large specific surface which are used have a high adsorption capacity, good physical and chemical stability and the adsorbent is easy to regenerate after separation in the adsorption equipment.

4. An additional advantage is that the process can be carried out in the presence of sulfite ions or compounds providing sulfite ions only, i.e. if desired, the use of cyanide ions and the consequential investments due to safety and environmental problems can be avoided.

5. If vitamin B12 is eluted with acetone, according to the invention vitamin B12 can easily be crystallized by the addition of a further portion of acetone. If other solvents, disclosed in the prior art methods, are employed, vitamin B12 can be precipitated in a crystalline form only after a further evaporation step.

6. The factor Ill can be utilized after separation and if desired, after elimination of salts, evaporation and drying as a fodder additive concentrate.

The invention will be illustrated in greater detail by the aid of the following non-limiting Examples.

Preparation of the starting materials (a) The digestion of the fermentation medium containing the strains Corynebacterium sp. (24AI), Corynebacterium sp. (244Cl), Lactobacillus sp. (244 B Cl) and Propionibacterium sp. (239 Al/6) deposited in the Hungarian National Collection of Medical Bacteria, National Institute of Health (OKI) under Nos. 76, 77, 78 and 79, respectively (a methane producing, mixed population fermentation medium prepared by the process disclosed in Hungarian Patent Specification 159,356), i.e. the dissolution of the corrinoids present inside the cells is carried out according to Hungarian Patent Specification 1 71 339 as follows:: To 1 m3 of fermentation medium containing 24.2 g of cobalamines and 95.6 g of incomplete corrinoids 1 lit. of a 10% sodium cyanide solution is added, the solution is made alkaline to pH 9 with a 20% sodium hydroxide solution, the mixture is heated up to 70"C under stirring, and kept at this temperature for 30 minutes. Thereafter 50 lit. of a 30% Fe(lll)chloride solution (17,4 kg of FeCI3.6H20) are added to the mixture, which is stirred for 10 minutes. The pH of the mixture is then adjusted to 8 with a 20% sodium hydroxide solution. After a further 10 minutes stirring the precipitate is eliminated from the mixture by sedimentation centrifuge. The solution obtained contains 23 g of cobalamines and 88 g of incomplete corrinoids per lit of solution.

(b) The fermentation medium prepared according to step (a) above is purified as described in Example 1 of our British Patent Application 2,137,207, by separation of the impurities present in the fermentation medium from the intact cells containing corrinoids by adsorption on a macroreticular adsorption resin, and subsequently digesting the cells and dissolving the corrinoids. The selective adsorption or selective elution of the corrinoids according to the invention is carried out starting from the precipituous solution obtained.

The fermentation medium is purified as follows: 1 m3 of a fermentation medium obtained as described in step (a) above, which contains 28 mg of vitamin BI2 and 5.2 mg of factor Ill together with 129 mg of incomplete corrinoids per liter is purified in a two-member, fluid bed adsorption system, in which the members are connected in series. The unfiltered fermentation medium is continuously passed through two columns each having a useful volume of 25 lit. and filled with 10 lit. of Diaion SP207 adsorption resin, at a rate of 100 lit./hour.

During this the majority of the extracellular impurities present in the fermentation medium will be adsorbed on the resin. Thereafter the columns are washed with 100 lit. of water.

2500 ml of a 10% potassium metabisulfite solution are added to the partially purified fermentation medium obtained, and the pH is adjusted to 4 by continuous addition of a 50% aqueous sulfuric acid solution. The fermentation medium is then heated up to 110"C in a continuous system, with a residential time of 10 min, where the cells will be digested and the corrinoids are dissolved into the fermentation medium. The fermentation medium is then cooled to a temperature below 30"C by passing through a heat exchanger. The solution obtained contains 27 mg of vitamin Ba2, 5 mg of factor Ill and 118 mg of incomplete corrinoids per liter.

(c) From the fermentation medium obtained in step (a) above an aqueous concentrate containing both complete and incomplete corrinoids may be obtained following the procedures described in our British Patent Applications 2,137,207 and 2,149,407 in the following manner: The fermentation medium purified and digested as described in step (b) is passed through two series-connected columns containing 10 lit. of Diaion SP 207 resin each, in an upwards direction, using fluid bed technology. In this step the complete and incomplete corrinoids are adsorbed on the resin. The cell debris and the organic and inorganic impurities are washed off the columns with water, in the same direction as the adsorption has taken place.Thereafter 20 lit. of aqueous solution containing altogether 3 g of potassium cyanide are applied to the columns in an upwards direction to form the cyano form of the corrinoids. After standing for 10 minutes the potassium cyanide-containing water is discharged from the columns, which are then washed with 100 lit. of water. The cyanocorrinoids are dissolved from the resin by 100 lit. of a 90% by vol. methanol solution, which is passed through the columns continuously, downwards. From the methanolic eluate methanol is eliminated by evaporation at 50"C. 10 lit. of an aqueous concentrate are obtained, containing 23 g of vitamin B12, 4 g of factor Ill and 99.7 g of incomplete corrinoids.

The process according to the invention is further illustrated by the following working non-limiting examples.

Example I An aqueous concentrate prepared from 1 m3 of fermentation medium according to step (c) above, which contains 27 g of cobalamines and 99.7 g of incomplete corrinoids is adjusted to pH 9.5 with a concentrated ammonium hydroxide solution, whereupon 50 g of potassium metabisulfite are added to the solution. The solution is then passed through a 100X1000 mm adsorption column prepared from 5 lit. of Diaion HP-20 resin, at a rate of 5 lit./hour, the column is washed with 5 lit. of water made alkaline to pH 9.5 with a concentrated ammonium hydroxide solution and containing 1g/lit. of potassium metabisulfite. The column is then washed neutral with water.

The solution passed through the resin and the aqueous washing solutions contain the total amount of incomplete corrinoids present in the starting solution, while the cobalamines are adsorbed on the resin. The cobalamines are eluted with 2 lit. of 90% by vol. of aqueous methanol in a known manner. After evaporation of the methanol the aqueous solution contains 25 g of cobalamine. Yield: 92.6% related to the cobalamine content of the starting concentrate.

Example 2 To 15 lit. of an aqueous concentrate obtained from 1.5 m3 of a fermentation medium according to step (a) described in connection with the preparation of starting materials, which contain 40.5 g of cobalamines and 177 g of incomplete corrinoids as well as 5% of residual methanol (after evaporating off the main volume of methanol) 150 ml of a 10% potassium cyanide solution are added. The pH is adjusted to 10.0 with a 2 N sodium hydroxide solution. The solution obtained is passed through a column prepared from 6 lit.

of Amberlite XAD-2 adsorption resin at a rate of 6 lit./hour, whereupon the column is washed through with 10 lit. of water. The solution passed through the column and the washing liquor contain the total amount of the incomplete corrinoids. The cobalamines adsorbed on the resin are eluted with 6 lit. of an 80% by vol. aqueous methanol solution in a known manner. Methanol is evaporated off to yield an aqueous solution containing 36.45 g of cobalamine. Yield (based on the cobalamine-content of the starting aqueous concentrate): 90%.

Example 3 An aqueous mixture containing corrinoids, other materials dissolved during the digestion of cells and cell debris obtained as described in step (b) hereinabove from 1000 lit. of fermentation medium, which contains 32 g of cobalamines, 118 g of incomplete corrinoids and has a pH of 4.0 is passed through a column filled with 20 lit. of Diaion SP 207 adsorption resin upwards, at a rate of 200 lit./hour. In this step all corrinoids are adsorbed on the resin. The cell debris, the organic and inorganic impurities are washed off the resin column in the same direction as their adsorption has taken place. Thereafter 200 lit.

of an aqueous solution are passed through the column downwards, which contain 0.1% of potassium cyanide, 4% by weight of acetone and has a pH of 9.5. The column is then washed with 200 lit. of water. The total amount of incomplete corrinoids present in the starting mixture will be present in the potassium cyanide-containing alkaline washing liquor and in the subsequent aqueous washing liquor.

The cyanocobalamines are eluted from the adsorption resin in a known manner, with a 60% by vol. aqueous acetone solution. After the evaporation of acetone an aqueous solution containing 28.5 g of cobalamine is obtained.

Yield (based on the cobalamine content of the starting purified and digested fermentation medium): 89%.

Example 4 The pH of 1000 lit. of the fermentation medium obtained as described in connection with the preparation of starting materials in step (a), which contains 23 g of cobalamines and 88 g of incomplete corrinoids, and has been digested and released from the precipitate by centrifuging, is adjusted to 9.0 with sodium hydroxide. 50 g. of sodium cyanide are dis solved in the medium, which is then passed upstream through a column filled with 40 lit.

of Diaion SP 207 adsorption resin, using fluid bed technology, to bind cobalamines. Under these conditions the incomplete corrinoids are not adsorbed on the resin. The adsorbent is washed with water, again upstream, using fluid bed technology, whereupon the cyanocobalamins are eluted with a 90% methanol solution in a known manner. The eluate contains 20.24 g of cyanocobalamines. Yield (based on the cobalamine content of the starting digested fermentation medium): 88%.

Example 5 10 lit. of an aqueous solution purified and converted into cyano-compound as described in connection with the preparation of starting materials in step (c), containing 27 g of cobalamines and 99.7 g of incomplete corrinoids are made alkaline to pH 9.5 with a 1 N aqueous sodium hydroxide solution. 50 g of potassium metabisulfite are dissolved in the solution, which is then passed through 4 lit. of a slightly polar acrylic ester-polymer Diaion HP2MG resin column. The column is then washed neutral with water. The solution passed through the column and the alkaline washing liquor contain the total amount of the incomplete corrinoids present in the starting material. The cobalamines are then eluted from the adsorption resin with a 65% ethanol solution, in a known manner. The eluate contains 23,2 g of cobalamines.Yield (based on the active ingredient content of the starting aqueous concentrate): 86%.

Example 6 Essentially the procedure described in Example 1 is followed, starting from 10 lit. of an aqueous concentrate containing 23 g of vitamin BI2, 4 g of factor Ill and 99.7 g of incomplete corrinoids, except that the cobalamines adsorbed on the resin are eluted selectively and not together. This is carried out by passing 10 lit. of water containing 10% by vol. of acetone (in which 2 g of potassium cyanide are dissolved after adjusting the pH to 9.5 with ammonium hydroxide) through a column filled with 5 lit. of Diaion HP 20 adsorbent in upwards direction, at a rate of 5 li- t./hour. Thereafter the column is washed with 5 lit. of water.The eluate and the washing liquor are combined and from the mixture obtained a factor Ill-containing product suitable for use as a fodder additive is prepared in the following manner: The alkaline acetonic eluate and the aqueous washing liquor are combined, the pH is adjusted to 4 with a 10% aqueous suifuric acid solution and water is added until the acetone content is below 5% by vol. The solution is then passed though a column filled with 0.5 lit. of Diaion HP 20 resin, to adsorb factor Ill.

The adsorbent is washed with 5 lit. of water, and factor Ill is eluted with 1 lit. of a 90% methanol solution. The eluate contains 3.7 g of factor Ill. Yield: 92.5%. After evaporation of methanol and drying the aqueous solution by spraying, the product is utilized as a fodder additive.

After the elution of factor Ill and aqueous washing the resin column is washed with 1.5 lit. of a 0.1 N acetic acid solution and subsequently with water until it becomes neutral.

Vitamin B,2 is dissolved from the column with 2 lit. of a 70% by vol. aqueous acetone solution. From the solution obtained vitamin B,2 is crystallized in a known manner, by adding acetone. Yield: 19.3 g of vitamin B12, 84% based on the starting aqueous concentrate.

Example 7 15 lit. of an aqueous concentrate prepared from 1.5 m3 of fermentation medium as described in step (c) of the process described hereinabove in connection with the preparation of starting materials, containing 5% by vol. of methanol, 34,5 9 of vitamin B,2, 6 g of factor Ill and 149.6 g of incomplete corrinoids, are treated as described in Example 2, except that vitamin B,2 and factor Ill adsorbed on the resin are dissolved separately, in the following manner: 30 lit. of water containing 30% by vol. of methanol (pH is adjusted to 9.0 with a 1 N sodium hydroxide solution), in which there were dissolved 0.01% of potassium cyanide, are passed through 6 lit. of an Amberlite XAD 2 column, at a rate of 6 lit./hour. The column is then washed through with 6 lit. of water.

The washing water is combined with the eluate. The solution obtained contains 5.4 g of factor Ill. The pH is adjusted to 4 to 4.5 with a 10% aqueous hydrogen chioride solution, and water is added to the solution until its methanol content is below 10% by vol. The solution is then passed through 1 lit. of an Amberlite XAD 2 resin column to adsorb factor Ill, the column is washed with 5 lit. of water, whereupon factor Ill is eluted with 2 lit.

of a 90% methanol solution. Methanol is evaporated and the residual aqueous solution is spray dried to yield a product which can be utilized as a fodder additive.

After the elution of factor Ill and washing with water, the resin column is washed with further amounts of water until the discharged water is neutral. Thereafter 2.5 lit. of a 90% methanol solution is passed through the solution to dissolve vitamin B,2. After standing for 10 minutes the eluate is discharged. The above treatment is repeated twice with 1-lit.

portions of 90% by vol. of methanol each time. The combined eluate contains 31.4 g of vitamin B,2. From the aqueous solution obtained after evaporation of methanol 31 g of crude crystalline vitamin B,2 are obtained in a known manner, by adding a 7-fold amount of acetone. The crude product contains 28.9 g of vitamin B12. By crystallization of this product from water in a known manner 30.2 g of vitamin B,2 crystal are obtained, meeting the requirements of the Vlth Hungarian Pharmacopoeia having a vitamin B,2 content of 96%.

Yield, based on the vitamin B,2 present in the starting aqueous concentrate: 84%.

Example 8 A fermentation medium obtained by purification and digestion of 1000 lit. of a starting fermentation medium as described in step (b) of the process illustrated in connection with the preparation of starting materials, containing 27 g of vitamin B,2, 5 g of factor Ill and 118 g of incomplete corrinoids is treated as described in Example 3, except that after the selective elution of incomplete corrinoids, factor III is eluted from the column with 30 lit. of water adjusted to pH 9.5 with a 10% aqueous sodium hydroxide solution containing 15% by vol. of acetone and 0.01% of potassium cyanide, whereupon the column is washed with 5 lit. of water. The combined eluate and washing liquor contains 4.3 g of factor Ill. Yield, based on the factor Ill content of the starting digested fermentation medium: 86%.

After the elution of factor III and washing with water the resin column is washed with water until the discharged water becomes neutral. Thereafter vitamin BI2 adsorbed on the resin is dissolved in 30 lit. of a 70% by vol. acetone solution. To the eluate the same volume of acetone is added to obtain vitamin B,2 in a crystalline form. By recrystallization of the obtained crude crystals from water, in a known manner 21.6 g of a crystalline product are obtained, containing 96% of vitamin B,2 (20.7 g). Yield, based on the BI2 content of the purified and digested fermentation medium 76.8%.

Example 9 From 10 lit. of the aqueous solution obtained under point (a) in connection with the preparation of starting materials, containing 23 g of vitamin B,2, 4 g of factor Ill and 99.7 g of incomplete corrinoids vitamin B12 and factor Ill are selectively adsorbed as described in Example 5, using 4 lit. of a Diaion HP2MG adsorption resin column. After washing the resin with water, 5 lit. of water containing 0.02% of potassium cyanide, 25% by weight of methanol and adjusted to pH 10.0 with a 10% aqueous sodium hydroxide solution are passed through the column, which is then washed with 5 lit. of water. The combined eluate and washing liquor contain 3.4 9 of factor Ill. The resin is then washed neutral with water, and vitamin BI2 is eluted from the adsorbent in a known manner, using a 90% by vol. methanol solution. Methanol is evaporated and a seven-fold column of acetone is added to the aqueous solution, to yield 23.57 g of crude crystals containing 80% of vitamin B,2. By recrystallization from water in a known manner 17.6 9 of vitamin B,2 are obtained (B,2 content: 96.5%) in a pharmacopoeial quality.

Yield, based on the starting aqueous concentrate: 74%.

Claims (10)

1. A process for the separation of corrinoids from a solution thereof by selective adsorption and/or selective elution by means of a polymeric adsorbent having a large specific surface, which comprises either (a) selectively binding complete corrinoids on the adsorbent from an aqueous solution or an aqueous solvent mixture thereof containing at most 20% by vol. of a water-miscible lower aliphatic alcohol or at most 5% by vol.

of a water-miscible lower aliphatic ketone, at a pH of 8 to 11, in the presence of cyanide or sulfite ions, or of compounds capable of providing such ions, or (b) selectively eluting incomplete corrinoids from a mixture of corrinoids adsorbed on the adsorbent with an aqueous solution or an aqueous solvent mixture containing at most 20% by vol. of a water-miscible lower aliphatic alcohol or at most 5% by vol. of a watermiscible lower aliphatic ketone, at a pH of 8 to 11, in the presence of cyanide or sulfite ions, or of compounds capable of providing such ions, followed, if desired, by eluting the complete corrinoids bound on the adsorbent, or, if desired, eluting any factor Ill present on the adsorbent with an aqueous solvent mixture containing 20 to 40% by vol. of a water-miscible lower aliphatic alcohol or 5 to 20% by vol. of a lower aliphatic ketone, at a pH of 8 to 11, in the presence of cyanide or sulfite ions, or of compound capable of providing such ions, followed, if desired, by subsequent elution of vitamin B12 from the adsorbent.

2. A process as claimed in claim 1, wherein the pH of the solutions used in steps (a), (b) or employed for the elution of factor Ill is adjusted to 9 to 10.

3. A process as claimed in either of claims 1 and 2 wherein the water-miscible lower aliphatic alcohol used in either of steps (a) and (b) is methanol.

4. A process as claimed in either of claims 1 and 2 wherein the water-miscible lower aliphatic alcohol used in either of steps (a) and (b) is ethanol.

5. A process as claimed in any one of claims 1 to 4 wherein the water-miscible lower aliphatic ketone used in either of steps (a) and (b) is acetone.

6. A process as claimed in any one of claims 1 to 5 wherein potassium cyanide or sodium cyanide or potassium metabisulfite or sodium metabisulfite is used as the compound capable of providing cyanide or sulfide ions respectively.

7. A process as claimed in any one of claims 1 to 6 wherein a non-ionic, non-polar or slightly polar macroreticular adsorption resin is used as a polymeric adsorbent having a large specific surface.

8. A process as claimed in claim 7 wherein said macroreticular adsorption resin has a pore size of 10-8 to 10-7m and a specific surface of at least 200m2/g.

9. A process substantially as herein described with reference to the Examples.

10. A process substantially as herein described.