CA2012927C - Removal of thiamine monophosphate from a solution of thiamine phosphates - Google Patents
Removal of thiamine monophosphate from a solution of thiamine phosphates Download PDFInfo
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- CA2012927C CA2012927C CA 2012927 CA2012927A CA2012927C CA 2012927 C CA2012927 C CA 2012927C CA 2012927 CA2012927 CA 2012927 CA 2012927 A CA2012927 A CA 2012927A CA 2012927 C CA2012927 C CA 2012927C
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- thiamine
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- monophosphate
- cocarboxylase
- thiamine monophosphate
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Abstract
Thiamine monophosphate is~
separated from a solution of thiamine phosphates containing thiamine monophosphate and cocarboxylase and having a pH of 2-7 by using a cation exchanger resin having a pK a of 1.0-4.5.
separated from a solution of thiamine phosphates containing thiamine monophosphate and cocarboxylase and having a pH of 2-7 by using a cation exchanger resin having a pK a of 1.0-4.5.
Description
201292' O.Z. 0050/40611 Removal of thiamine monophosphate from a solution of thiamine phosphates The present invention relates to a process for removing thiamine monophosphate from a solution of thia mine phosphates which contains thiamine monophosphate and cocarboxylase as useful products by using a cation exchanger and eluting the thiamine monophosphate with an acid.
The phosphorylation of thiamine generally pro-duces a mixture of the following composition: about 70~
of thiamine monophosphate, about Z0~ of cocarboxylase (thiamine diphosphate) and about 5~ of thiamine triphos-phate and thiamine tetraphosphate. As described in DE-A-1 085 527, the mixture is customarily separated by first removing the phosphoric: acid and then passing the mixture over a weakly basic ion exchanger. This elimi-nates residual amounts of phosphoric acid. The deacidi-fied mixture is then passed over a strongly acidic ion exchanger, eg. Amberlite~ IR-1.20. Thiamine monophosphoric acid adheres, while the cocarboxylase and the higher phosphates, essentially thiamine tri- and tetraphosphate, are eluted. This procedure gives the cocarboxylase in the form of a very dilute solution, causing not inconsider-able evaporation costs. Furthermore, some of the cocar-boxylase is hydrolysed by the strongly acid ion exchanger to thiamine monophosphate, which reduces the total yield of cocarboxylase. Instead of using a strongly acidic ion exchanger resin use is also made of a weakly acidic ion exchanger resin, eg. Amber:lite~ IRC-50. This again represents a selective ion exchange process which like-wise produces the eluted cocarboxylase in a highly dilute solution.
It is an object of the present invention to develop a process of the type mentioned at the beginning in such a way as to give significantly more efficient removal of thiamine monophosphate. Another object is that the solution of cocarboxylase obtained should not be very 201292' - 2 - O.Z. 0050/40611 dilute.
We have found that these objects are achieved by a process of the type mentioned at the beginning, wherein a solution which contains the thiamine monophosphate and the cocarboxylase has a pH of 2-7 and the cation ex-changer used has a pKa of 1.0--4.5.
The pRa of a cation exchanger is the apparent pK, which can be determined by the method of F. Helfferich, Ionenaustauscher, vol. 1, p.84 (1959), Verlag Chemie GmbH, Weinheim, BergstraBe.
In a preferred embodiment of the present inven-tion, the cation exchanger resin used possesses imino-diacetic acid residues or aminoalkylene- and/or iminodialkylene-phosphoric acid residues as functional groups. It is particularly advantageous to use a cation exchanger resin possessing aminomethylene- and/or iminodimethylene-phosphoric acid residues as functional groups. It is similarly particularly advantageous to use a solution with a pH of 4.5-5..5. A particularly suitable eluent is aqueous 1-30~ strength hydrochloric acid.
The cation exchanger resins with aminoalkylene-and/or iminoalkylene-phosphon,ic acid groups are poly-styrene/divinyl polymers which preferably contain an aminomethylene- and/or iminomethylene-phosphoric acid group as functional group. Cation exchanger resins with aminoalkylene- and/or iminoalkylene-, in particular aminomethylene- and/or iminomethylene-phosphoric acid groups are known (cf. for example J. Appl. Chem. 8, (1958), 458; US-A-4 002 564; CA 93, 168909x) and are commercially available. It is particularly advantageous to use the cation exchanger resin with aminoalkylene-and/or iminoalkylene-phosphon.ic acid groups available from Bayer AG under the tradename Lewatit~ OC 1060.
The cation exchanger resins with aminoalkylene and/or i.minoalkylene-phosphoric acid groups show par ticularly high selectivity and a surprisingly high capacity for thiamine monophosphate. If a solution which 201292' - 3 - O.Z. 0050/40611 has the above composition and a pH of 2-7, preferably 4.5-5.5, is passed over these resins, the thiamine monophosphate is selectively adsorbed. The cocarboxylase and the higher phosphates are eluted in an eluate which in the course of the elution surprisingly becomes more and more concentrated. The process in question here is thus not a conventional exchange process but, surprising-ly, a displacement chromatography process. The conse-quence is that the concentration of cocarboxylase in the eluate is equal to the concentration in the applied solution. The cocarboxylase can be isolated from the eluate in a conventional manner, for example by evapora tion. The thiamine triphosphate and tetraphosphate are separated from the cocarboxylase by a conventional method.
Once the capacity of the ion exchanger is ex-hausted, it is rinsed with water and then with 1-30~
strength aqueous HC1, preferably 7.5~ strength HC1, as eluent . The thiamine monophosphate is eluted as a satura-ted solution in the form of the hydrochloride. After rinsing with water until neutral, the ion exchanger is ready again for use. This permits a rapid sequence of separating cycles in an indusitrial process. The capacity of Lewatit~ OC-1060 is limited only by the proportion of thiamine monophc~phate. For optimal separation, this capacity must not be exceeded.. The capacity was found to be about 140 g of thiamine monophosphate/1 of resin.
According to the present invention it is thus possible to separate a solution of thiamine, thiamine monophosphate, cocarboxylase and higher phosphates in any desired composition by the above-described method without dilu-ting the cocarboxylase yet selectively removing the thiaminemonophosphoric acid up to an amount of 140 g/1 of Lewatit~ OC-1060. The method has been successfully used to separate a mixture in which the cocarboxylase had already been concentrated to about 37~ (see Example below).
201292' O.Z. 0050/40611 The process according to the present invention has many advantages . For instance, the product concentra-tion remains constant or even increases in the course of the purification step. In addition, the hydrochloric acid used as displaces restores the ion exchanger resin to its initial state, obviating the need for a separate re-generation step. The procesa according to the present invention also permits the industrially extremely impor-tant rapid sequence of separating cycles . A particular advantage is the fact that t:he resin capacity is high, being for example up to 140 g of thiamine monophosphate/1 of resin in the case of Lewat:it~ OC-1060.
The present invention is further illustrated by the following Example:
EXAMIPLE
2,000 g of a solution of the following compo- -sition, and pH 6 is applied to a separating column measuring 330 * 65 mm:
Composition:
2 0 Amount % share of concentration in in g dry solution substance Thiamine monophosphate 150 (57%) 7.5% strength Cocarboxylase 95 (36%) 4.8% strength Remainder (thiamine tri- and tetraphosphate) 18 (7%) 0.9% strength Dry substance 263 (:100%) 13.2% strength solution =
2,000 g of solution (about 2,000 ml) The following volumes were applied:
SOlutiori 1.8 BV (.L BV = 1,100 ml; BV = bed volume) Water 1.0 BV
HC1 7.5~ 0.5 BV
Water 2.2 BV
Total applied volume 5.5 BV
The following fractions were obtained:
Prerun 0.9 BV
Cocarboxylase 1.8 BV 4.8% strength Monophosphate 1.0 BV 13.5% strength Afterrun 1.8 BV
Total volume of fractions 5.5 BV
The result obtained is shown as a graph in Figure 1. The run was carried out with the Lewatit~ OC-1060 at about 100% capacity for thiamine monophosphate. Figure 1 shows the elution diagram of the separation of thiamine monophosphate and carboxylase at about 100% capacity for thiamine monophosphate. It can be seen that the concen-tration of cocarboxylase in the eluate rises continuously as the mixture is being applied. The monophosphate starts to break through toward the end of the cocarboxylase elution peak. Following a short intermediate rinse, the application of HC1 elutes the mono-phosphate in high purity and concentration. The elution peak is sharp; there is no tailing. Following a rinse phasE~ of water, the next product mixture can be applied.
Figure 2 :is likewise an elution diagram of the separation of thiamine monophosphate and cocarboxylase at 50% capacity for thiamine monophosphate. The same resin Lewatit~ OC-1060 was used. However, it was loaded to only 50% of the capacity for thiamine monophosphate and with a solution of about 20% of cocarboxylase and 75% of thiamine monophosphate. Between the application of the solution and the eluating with HCl, the resin was rinsed with plenty of _y, 5a water. It was found in this connection that the monophosphate remained in a atable state on the resin despite the long rinse. ThE: subsequent elution with hydrochloric acid takes place abruptly. The intermediate rinse with water makes it possible to purify the thiamine monophosphate thoroughly, and the eluate obtained is pure.
The phosphorylation of thiamine generally pro-duces a mixture of the following composition: about 70~
of thiamine monophosphate, about Z0~ of cocarboxylase (thiamine diphosphate) and about 5~ of thiamine triphos-phate and thiamine tetraphosphate. As described in DE-A-1 085 527, the mixture is customarily separated by first removing the phosphoric: acid and then passing the mixture over a weakly basic ion exchanger. This elimi-nates residual amounts of phosphoric acid. The deacidi-fied mixture is then passed over a strongly acidic ion exchanger, eg. Amberlite~ IR-1.20. Thiamine monophosphoric acid adheres, while the cocarboxylase and the higher phosphates, essentially thiamine tri- and tetraphosphate, are eluted. This procedure gives the cocarboxylase in the form of a very dilute solution, causing not inconsider-able evaporation costs. Furthermore, some of the cocar-boxylase is hydrolysed by the strongly acid ion exchanger to thiamine monophosphate, which reduces the total yield of cocarboxylase. Instead of using a strongly acidic ion exchanger resin use is also made of a weakly acidic ion exchanger resin, eg. Amber:lite~ IRC-50. This again represents a selective ion exchange process which like-wise produces the eluted cocarboxylase in a highly dilute solution.
It is an object of the present invention to develop a process of the type mentioned at the beginning in such a way as to give significantly more efficient removal of thiamine monophosphate. Another object is that the solution of cocarboxylase obtained should not be very 201292' - 2 - O.Z. 0050/40611 dilute.
We have found that these objects are achieved by a process of the type mentioned at the beginning, wherein a solution which contains the thiamine monophosphate and the cocarboxylase has a pH of 2-7 and the cation ex-changer used has a pKa of 1.0--4.5.
The pRa of a cation exchanger is the apparent pK, which can be determined by the method of F. Helfferich, Ionenaustauscher, vol. 1, p.84 (1959), Verlag Chemie GmbH, Weinheim, BergstraBe.
In a preferred embodiment of the present inven-tion, the cation exchanger resin used possesses imino-diacetic acid residues or aminoalkylene- and/or iminodialkylene-phosphoric acid residues as functional groups. It is particularly advantageous to use a cation exchanger resin possessing aminomethylene- and/or iminodimethylene-phosphoric acid residues as functional groups. It is similarly particularly advantageous to use a solution with a pH of 4.5-5..5. A particularly suitable eluent is aqueous 1-30~ strength hydrochloric acid.
The cation exchanger resins with aminoalkylene-and/or iminoalkylene-phosphon,ic acid groups are poly-styrene/divinyl polymers which preferably contain an aminomethylene- and/or iminomethylene-phosphoric acid group as functional group. Cation exchanger resins with aminoalkylene- and/or iminoalkylene-, in particular aminomethylene- and/or iminomethylene-phosphoric acid groups are known (cf. for example J. Appl. Chem. 8, (1958), 458; US-A-4 002 564; CA 93, 168909x) and are commercially available. It is particularly advantageous to use the cation exchanger resin with aminoalkylene-and/or iminoalkylene-phosphon.ic acid groups available from Bayer AG under the tradename Lewatit~ OC 1060.
The cation exchanger resins with aminoalkylene and/or i.minoalkylene-phosphoric acid groups show par ticularly high selectivity and a surprisingly high capacity for thiamine monophosphate. If a solution which 201292' - 3 - O.Z. 0050/40611 has the above composition and a pH of 2-7, preferably 4.5-5.5, is passed over these resins, the thiamine monophosphate is selectively adsorbed. The cocarboxylase and the higher phosphates are eluted in an eluate which in the course of the elution surprisingly becomes more and more concentrated. The process in question here is thus not a conventional exchange process but, surprising-ly, a displacement chromatography process. The conse-quence is that the concentration of cocarboxylase in the eluate is equal to the concentration in the applied solution. The cocarboxylase can be isolated from the eluate in a conventional manner, for example by evapora tion. The thiamine triphosphate and tetraphosphate are separated from the cocarboxylase by a conventional method.
Once the capacity of the ion exchanger is ex-hausted, it is rinsed with water and then with 1-30~
strength aqueous HC1, preferably 7.5~ strength HC1, as eluent . The thiamine monophosphate is eluted as a satura-ted solution in the form of the hydrochloride. After rinsing with water until neutral, the ion exchanger is ready again for use. This permits a rapid sequence of separating cycles in an indusitrial process. The capacity of Lewatit~ OC-1060 is limited only by the proportion of thiamine monophc~phate. For optimal separation, this capacity must not be exceeded.. The capacity was found to be about 140 g of thiamine monophosphate/1 of resin.
According to the present invention it is thus possible to separate a solution of thiamine, thiamine monophosphate, cocarboxylase and higher phosphates in any desired composition by the above-described method without dilu-ting the cocarboxylase yet selectively removing the thiaminemonophosphoric acid up to an amount of 140 g/1 of Lewatit~ OC-1060. The method has been successfully used to separate a mixture in which the cocarboxylase had already been concentrated to about 37~ (see Example below).
201292' O.Z. 0050/40611 The process according to the present invention has many advantages . For instance, the product concentra-tion remains constant or even increases in the course of the purification step. In addition, the hydrochloric acid used as displaces restores the ion exchanger resin to its initial state, obviating the need for a separate re-generation step. The procesa according to the present invention also permits the industrially extremely impor-tant rapid sequence of separating cycles . A particular advantage is the fact that t:he resin capacity is high, being for example up to 140 g of thiamine monophosphate/1 of resin in the case of Lewat:it~ OC-1060.
The present invention is further illustrated by the following Example:
EXAMIPLE
2,000 g of a solution of the following compo- -sition, and pH 6 is applied to a separating column measuring 330 * 65 mm:
Composition:
2 0 Amount % share of concentration in in g dry solution substance Thiamine monophosphate 150 (57%) 7.5% strength Cocarboxylase 95 (36%) 4.8% strength Remainder (thiamine tri- and tetraphosphate) 18 (7%) 0.9% strength Dry substance 263 (:100%) 13.2% strength solution =
2,000 g of solution (about 2,000 ml) The following volumes were applied:
SOlutiori 1.8 BV (.L BV = 1,100 ml; BV = bed volume) Water 1.0 BV
HC1 7.5~ 0.5 BV
Water 2.2 BV
Total applied volume 5.5 BV
The following fractions were obtained:
Prerun 0.9 BV
Cocarboxylase 1.8 BV 4.8% strength Monophosphate 1.0 BV 13.5% strength Afterrun 1.8 BV
Total volume of fractions 5.5 BV
The result obtained is shown as a graph in Figure 1. The run was carried out with the Lewatit~ OC-1060 at about 100% capacity for thiamine monophosphate. Figure 1 shows the elution diagram of the separation of thiamine monophosphate and carboxylase at about 100% capacity for thiamine monophosphate. It can be seen that the concen-tration of cocarboxylase in the eluate rises continuously as the mixture is being applied. The monophosphate starts to break through toward the end of the cocarboxylase elution peak. Following a short intermediate rinse, the application of HC1 elutes the mono-phosphate in high purity and concentration. The elution peak is sharp; there is no tailing. Following a rinse phasE~ of water, the next product mixture can be applied.
Figure 2 :is likewise an elution diagram of the separation of thiamine monophosphate and cocarboxylase at 50% capacity for thiamine monophosphate. The same resin Lewatit~ OC-1060 was used. However, it was loaded to only 50% of the capacity for thiamine monophosphate and with a solution of about 20% of cocarboxylase and 75% of thiamine monophosphate. Between the application of the solution and the eluating with HCl, the resin was rinsed with plenty of _y, 5a water. It was found in this connection that the monophosphate remained in a atable state on the resin despite the long rinse. ThE: subsequent elution with hydrochloric acid takes place abruptly. The intermediate rinse with water makes it possible to purify the thiamine monophosphate thoroughly, and the eluate obtained is pure.
Claims (6)
1. A process for removing thiamine monophosphate from a solution of thiamine phosphates which contains thiamine monophosphate and cocarboxylase using a cation exchanger resin and eluting the thiamine monophosphate with an acid, wherein said solution has a pH of 2-7 and the cation exchanger resin used has a pK a of 1.0-4.5.
2. A process as claimed in claim 1, wherein the cation exchanger resin used contains iminodiacetic acid residues or aminoalkylene- and/or iminodialkylene-phos-phonic acid residues as functional groups.
3. A process as claimed in claim 1, wherein the cation exchanger resin used contains aminomethylene-and/or imindiomethylene-phosphonic acid residues as functional groups.
4. A process as claimed in claim 1, wherein the solution has a pH of 4.5-5.5
5. A process as claimed in claim 1, wherein the acid used for eluting the thiamine monophosphate is aqueous 1-30% strength hydrochloric acid.
6. A process for removing thiamine monophosphate from a solution of thiamine phosphates which contain thiamine monophosphate and cocarboxylase using a cation exchanger resin and eluting the thiamine monophosphate with aqueous hydrochloric acid, which comprises using a solution of pH 2-7 and a cat.ion exchanger resin which possesses iminodiacetic acid residues or aminoalkylene-and/or iminodialkylene-phosphonic acid residues as functional groups.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2012927 CA2012927C (en) | 1990-03-23 | 1990-03-23 | Removal of thiamine monophosphate from a solution of thiamine phosphates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2012927 CA2012927C (en) | 1990-03-23 | 1990-03-23 | Removal of thiamine monophosphate from a solution of thiamine phosphates |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2012927A1 CA2012927A1 (en) | 1991-09-23 |
| CA2012927C true CA2012927C (en) | 2000-08-29 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2012927 Expired - Lifetime CA2012927C (en) | 1990-03-23 | 1990-03-23 | Removal of thiamine monophosphate from a solution of thiamine phosphates |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2012927C (en) |
-
1990
- 1990-03-23 CA CA 2012927 patent/CA2012927C/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| CA2012927A1 (en) | 1991-09-23 |
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| EEER | Examination request | ||
| MKEX | Expiry |