JP2006292394A - Method of regenerating silica gel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem that a conventional method of column chromatography using a silica gel as an adsorbent performed as a refining method of coenzyme Q is insufficient for the regeneration of the silica gel from an aspect of the separability of coenzyme Q and impurities in column chromatography and is difficult to reuse the silica gel, and that the column chromatography using the silica gel has a problem as an industrial refining method from an aspect of workability and the use amount of the silica gel. <P>SOLUTION: In order to reutilize the silica gel which is used in order to refine coenzyme Q by the column chromatography using the silica gel as the adsorbent, an organic solvent which forms an azeotropic mixture along with water is heated to treat the silica gel so as to adjust the moisture contained in the silica gel. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for regenerating silica gel, and more specifically, provides a method for regenerating silica gel used for purification of coenzyme Q.

  Coenzyme Q is used as a therapeutic agent for diseases such as heart failure. This coenzyme Q is produced by methods such as synthesis, fermentation and extraction from natural products.

As a purification method of coenzyme Q, column chromatography using silica gel as an adsorbent is performed. From the standpoint of separability between coenzyme Q and impurities in column chromatography, the regeneration of silica gel is inadequate with conventional methods, and it is difficult to reuse.

Further, column chromatography using silica gel has a problem as an industrial purification method in terms of workability and the amount of silica gel used.

Factors that determine the adsorption capacity of organic compounds on silica gel used for chromatography include silica gel particle size, surface area, apparent specific gravity, water content, and the like. In particular, the water content of silica gel is a factor that greatly affects the adsorption capacity (adsorption capacity) in which the silica gel holds the organic compound and the efficiency (recovery rate) in which the organic compound held in the silica gel can be recovered.
That is, if the amount of water is small, the adsorption capacity increases, but the recovery rate decreases. Moreover, when there is much moisture content, adsorption capacity will fall. Therefore, when the silica gel is recycled, it is necessary to return the water content to the state before the regeneration.

The water molecules embedded in the silica gel can be divided into first layer water molecules (adsorbed on the silica gel surface) and second layer water molecules (covalently bonded to silica gel). When the used silica gel containing sufficient water molecules is regenerated, the water molecules in the first layer can be desorbed from the pores of the silica gel at a relatively low temperature. On the other hand, hot air at 150 ° C. or higher is generally required for desorption of the second layer water molecules having a large adsorption interaction between the silica gel surface and water molecules.
In order to perform hot air regeneration, the silica gel must be taken out from the column, and cumbersome operations such as hot air regeneration and refilling of the column must be performed.

Further, as a method of regenerating silica gel, a method of regenerating using a nonpolar solvent having a boiling point of 40 ° C. or lower (JP-B-2-53032) is known, but the second layer of water molecules adsorbed on the silica gel is used. It cannot be removed. For this reason, since the adsorption capacity of regenerated silica gel is low, it is not put to practical use.

Japanese Patent Publication 2-53032

  The present invention relates to a method for regenerating silica gel, and more specifically, provides a method for regenerating silica gel used for purification of coenzyme Q in order to solve the problems associated with the above-described method.

As a result of various examinations of silica gel regeneration methods in silica gel column chromatography, coenzyme Q was adsorbed on silica gel and purified with elution organic solvent to purify coenzyme Q, and then silica gel was treated with organic solvent for washing. In the regeneration method, the silica gel regeneration method is characterized by using an organic solvent that forms an azeotrope with water.

By treating with an organic solvent that forms an azeotrope with water, the water molecules in the first layer adsorbed on the silica gel can be removed, and the adsorption capacity of the silica gel can be recovered. The column chromatography used allows good separation of coenzyme Q and impurities, allowing repeated use of silica gel.

Further, since this regeneration method is applied to silica gel as it is packed in a column container, it has been found that operations such as loading and unloading of silica gel can be omitted, and the present invention has been completed.

By using the method of the present invention, coenzyme Q used as a pharmaceutical or the like can be efficiently produced at low cost.

The starting material to be purified in the present invention is a crude coenzyme Q containing impurities such as various lipids mixed when producing coenzyme Q by a method such as synthesis, fermentation and extraction from natural products. is there.
Coenzyme Q in the present invention may be any of coenzymes Q1 to Q12, or a mixture of two or more thereof.

The silica gel used in the present invention is not particularly limited, but practically, for example, silica gel (trade name Wakosil C-300, manufactured by Wako Pure Chemical Industries), silica gel (trade name silica gel 60N, manufactured by Kanto Chemical), silica gel (trade name) Commercially available products such as silica gel 60 (manufactured by Nacalai Tesque) and silica gel (trade name BW60, manufactured by Fuji Silysia) are preferably used.

Adsorption and elution with silica gel are carried out by conventional methods.
The elution organic solvents used in the elution treatment are nonpolar solvents such as benzene, toluene, n-hexane, n-pentane, isooctane, cyclohexane, benzene and petroleum ether, chloroform, ethyl ether, isopropyl ether, butyl ether, methyl ethyl ketone, A mixed solvent obtained by mixing a polar solvent such as ethyl acetate, acetone, methanol and ethanol is used.

  In order to remove organic impurities adsorbed on the silica gel after elution and adjust the water content, the silica gel is washed and regenerated. In the present invention, there is no particular limitation as long as it is an organic solvent that forms an azeotrope with water as a regeneration solvent for silica gel, but chloroform, ethyl ether, isopropyl ether, butyl ether, and methyl ethyl ketone have low water solubility and azeotrope with water. Since the mixture is separated into two layers of water and an organic solvent at room temperature, water can be easily removed out of the system and is particularly suitable as a silica gel regenerating solvent.

Polar solvents that form an azeotrope with water, such as chloroform, ethyl ether, isopropyl ether, butyl ether, methyl ethyl ketone, or a mixture thereof, or a mixture thereof, or n-hexane, n-pentane, isooctane, cyclohexane, benzene, petroleum ether It is carried out using a mixture with a nonpolar solvent.

The amount of solvent used for washing and regeneration is not particularly limited, but may be an amount of solvent necessary for desorbing impurities and water adsorbed on silica gel. In general, a solvent having a volume of 3 to 10 times the silica gel volume is used.

  The treatment temperature at the time of washing and regeneration in the present invention is from 20 ° C. to the boiling point of the solvent used. The treatment temperature is preferably higher because the free energy of the water molecules adsorbed on the silica gel is increased, but if the temperature is higher than the boiling point of the solvent used, a part of the solvent is gasified at normal pressure, and therefore the column. The solvent flow is not smooth and the silica gel is not regenerated or becomes insufficient.

Distillation for removing water may be batch-type or continuous. The latter is preferable for practical use.

The silica gel used for the purification of coenzyme Q has heretofore been inadequately regenerated and cannot be reused. However, according to the present invention, the adsorption separation ability of the silica gel can be recovered and reused. The present invention can be made unnecessary and the amount of silica gel used can be reduced. Therefore, the cost and the operation are improved, and the present invention is industrially significant.
Hereinafter, the present invention will be described more specifically with reference to examples.

(1) Preparation method of crude coenzyme Q10 2.3-Dimethoxy-5-methyl-benzohydroquinone-1 / 4 (17.6 g) and 5.8 g of boric acid are added to 80 ml of toluene and heated. After removing the azeotrope, the toluene is distilled off. Add 10 ml of toluene to the residue, heat at 50-55 ° C., add 23 g of isodecaprenol (purity 87.6%) with stirring, and continue stirring under the same conditions for 5 hours.
After completion of the reaction, the reaction mixture is extracted with 400 ml of ethyl ether, and the ether extract is washed with water, washed with alkaline water and then dried with sodium sulfate.
8 g of silver oxide is added to this ether extract and left overnight at room temperature with stirring.
The reaction mixture was filtered, and 24.5 g of an oily residue obtained by distilling off the solvent from the filtrate was dissolved in acetone and cooled to obtain 12.4 g of crude coenzyme Q10 crystals (purity: 87.6%).

(2) Purification of coenzyme Q10 200 g (water content: 6.7%) of silica gel (trade name BW60, manufactured by Fuji Silysia) suspended in n-hexane was packed in a column container having a diameter of 30 mm and a length of 500 mm, 10 g of the crude crystals dissolved in n-hexane are charged and adsorbed on the top of the column, and then washed with 1000 ml of n-hexane. When a mixture of isopropyl ether having an isopropyl ether content of 5 Vol% and n-hexane was allowed to flow down at a flow rate of 800 ml / hr, an effluent containing coenzyme Q10 started to elute from the column about 50 minutes after the start of the flow. As a fraction containing pure coenzyme Q10, 4200 ml was collected, and the solvent was removed therefrom to obtain 3.80 g (recovery rate: 86.8%) of pure coenzyme Q10. The product was identified as a standard product by the ultraviolet absorption spectrum measurement value, the infrared absorption spectrum measurement value, the nuclear magnetic resonance spectrum measurement value, and the mass spectrum measurement value.

(3) Purification of Coenzyme Q10 with Regenerated Silica Gel Next, experimental examples of silica gel regeneration and purification of coenzyme Q10 using regenerated silica gel according to the present invention are shown.
After the operation (2) was performed, 2000 ml of isopropyl ether was passed through the silica gel column (water content: 7.2%) while the column was heated from the outside and maintained at 55 ° C. Thereby, impurities and water adsorbed on the silica gel were removed from the column. Thereafter, 1000 ml of n-hexane was flowed to make the silica gel reusable.
In a column packed with regenerated silica gel (water content: 6.9%), 5 g of the crude crystals of (1) dissolved in n-hexane were charged and adsorbed on the top of the column, followed by isopropyl ether content. A mixture of 5 Vol% isopropyl ether and n-hexane was allowed to flow down at a flow rate of 800 ml / hr. As a fraction containing pure coenzyme Q10, 4200 ml was collected, and the solvent was removed therefrom to obtain 3.68 g (recovery rate: 84.0%) of pure coenzyme Q10. This corresponds to about 86% of coenzyme Q10 in the crude crystals.

Comparative Example 1
Next, an experimental example for purification of coenzyme Q10 on silica gel that has been washed and regenerated at room temperature is shown.
After performing the operation of (2), n-hexane was carried out in the same manner as in Example 1 except that 2000 ml of isopropyl ether was passed through this silica gel column (water content: 7.2%) at room temperature. After charging 10 g of crude crystals of crude coenzyme Q10 dissolved in the column and adsorbing it on the top of the column, 200 ml of n-hexane was allowed to flow, and Q10 was distilled off.

Reference Example Next, an experimental example for purification of coenzyme Q10 on silica gel performed by washing at normal temperature and regenerating treatment is shown.
After performing the operation of (2) above, 2000 ml of isopropyl ether was allowed to flow through this silica gel column (water content: 7.2%) at room temperature without heating the column, and the column was heated from the outside and maintained at 55 ° C. -1000 ml of hexane was allowed to flow. Thereafter, 10 g of crude crystals of crude coenzyme Q10 dissolved in n-hexane were charged and adsorbed on the top of the column, and when 10 ml of n-hexane was passed, distillation of Q10 was observed, and separation and purification from impurities could not be performed. It was.

(1) Preparation Method of Crude Coenzyme Q10 After 1 kg of fresh beef heart was saponified, extracted with hexane and concentrated by a conventional method, the obtained oily substance was dissolved in ethanol. The ethanol solution was cooled to 0 to 3 ° C. to precipitate crystals to obtain 621 mg of crude crystals (purity: 73.7%) containing coenzyme Q10.
This operation was repeated three times for the following examination.

(2) Purification of coenzyme Q10 50 g (water content: 4.7%) of silica gel (trade name Silica Gel 60 manufactured by Nacalai Tesque) suspended in n-toluene was packed in a column container having a diameter of 8 mm and a length of 500 mm. After 200 mg of the crude crystals dissolved in n-hexane were charged and adsorbed on the top of the column, a mixture of methyl ethyl ketone and n-hexane having a methyl ethyl ketone content of 3 Vol% was flowed down at a flow rate of 200 ml / hr. About 1 hour after the start, an effluent containing coenzyme Q10 began to flow out of the column.
As a fraction containing pure coenzyme Q10, 1000 ml was collected, and the solvent was removed therefrom to obtain 131 mg (recovery rate: 88.9%) of pure coenzyme Q10.

(3) Purification of Coenzyme Q10 with Regenerated Silica Gel Next, experimental examples for regenerating silica gel and purifying coenzyme Q10 using regenerated silica gel according to the present invention are shown.
After the operation of (2) above, this silica gel column (moisture content: 7.4%) is heated from the outside to flow 500 ml of methyl ethyl ketone, and then 250 ml of n-hexane is flowed to re-run the silica gel column. It was made available.
A column packed with regenerated silica gel (water content: 6.4%) was charged with 200 mg of the crude crystals of (1) dissolved in n-hexane on the top of the column and adsorbed, and then the methyl ethyl ketone content A mixed solution of 3 Vol% methyl ethyl ketone and n-hexane was allowed to flow down at a flow rate of 200 ml / hr.
As a fraction containing pure coenzyme Q10, 1000 ml was collected, and the solvent was removed therefrom to obtain 125 mg (recovery rate: 84.8%) of pure coenzyme Q10.

Claims (3)

In a method of refining coenzyme Q by adsorbing coenzyme Q to silica gel by column chromatography using silica gel as an adsorbent and eluting it with an elution organic solvent, and then washing the silica gel with an organic solvent for washing, A method for regenerating silica gel, comprising adjusting water contained in silica gel with an organic solvent that forms an azeotrope with water. When adjusting the water content in silica gel with an organic solvent that forms an azeotrope with water, the water is removed from the system by distillation from the equilibrium mixture of water and organic solvent by distillation. The method for regenerating silica gel according to claim 1. The method for regenerating silica gel according to claim 1, wherein the organic solvent that forms an azeotropic mixture with water is chloroform, ethyl ether, isopropyl ether, butyl ether, or methyl ethyl ketone.