JPH0253032B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for regenerating silica gel, and more particularly to a method for regenerating silica gel used for purification of coenzyme Q. Coenzyme Q is involved in the electron transport system in vivo, and 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, but as it is a pharmaceutical, it is required to be highly pure. Column chromatography using silica gel as an adsorbent is used as a purification method for this purpose, but due to the separation of coenzyme Q and impurities in column chromatography, the regeneration of silica gel is insufficient. Therefore, it was difficult to reuse it. For these reasons, column chromatography using silica gel has been problematic as an industrial purification method in terms of workability and the amount of silica gel used.

The present inventors investigated various methods for regenerating silica gel in silica gel column chromatography, and found that by contacting silica gel after adsorption, elution, and washing with a nonpolar solvent at a temperature of 40°C to below the boiling point. It is possible to remove water adsorbed by silica gel and the solvent used for washing, and column chromatography using regenerated silica gel obtained by this method can effectively separate coenzyme Q from impurities. Silica gel can be used repeatedly, and the amount of silica gel used can be reduced. Furthermore, it has been found that this regeneration method is applied to the silica gel that is still filled in the column container, so that operations such as filling and taking out the silica gel can be omitted, and the present invention has been completed.

That is, the present invention purifies coenzyme Q by adsorbing coenzyme Q to silica gel using column chromatography using silica gel as an adsorbent and eluting it with an organic solvent for elution. In a method for regenerating silica gel in a column in which the silica gel is washed with a cleaning organic solvent and then brought into contact with a nonpolar solvent, the silica gel is heated at 40°C.
This is a method for regenerating silica gel characterized by using a nonpolar solvent at a temperature between or below the boiling point.

The starting materials to be purified in the present invention are synthesized,
This is crude coenzyme Q that contains impurities such as various lipids that are mixed in when coenzyme Q is produced by methods such as fermentation and extraction from natural products. Coenzyme Q in the present invention may be any of coenzymes _Q1 to _Q12 , or may be a mixture of two or more thereof.

The silica gel used in the present invention is not particularly limited, but for practical purposes, for example, silica gel (trade name Wako Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd.), silica gel (trade name Wako Gel C-300, manufactured by Wako Pure Chemical Industries, Ltd.), silica gel (product name Art 7734, manufactured by Merck & Co.), and silica gel (product name Art 9385,
Merck), silica gel (product name KT-3063,
Commercial products such as Fujigel (manufactured by Fujigel) and silica gel (trade name 4B, manufactured by Fujigel) are preferably used.

Adsorption with silica gel, elution and washing are performed by conventional methods.

In practice, the elution organic solvent used in the elution treatment is usually a single solvent such as benzene and toluene, or n-hexane, n-pentane,
A mixed solvent whose polarity is adjusted by mixing a nonpolar solvent such as isooctane, cyclohexane, benzene, and petroleum ether with a polar solvent such as chloroform, ethyl ether, isopropyl ether, ethyl acetate, acetone, methanol, and ethanol is used.

To remove impurities adsorbed on the silica gel after elution, wash the silica gel with chloroform, ethyl ether, isopropyl ether, ethyl acetate, acetone, methanol, ethanol, etc. alone or in a mixture thereof. and n-hexane, n-pentane, isooctane, cyclohexane, benzene,
It is carried out using a mixture with a non-polar solvent such as stone-free ether. Note that the cleaning solvent used for cleaning must have greater polarity than the elution organic solvent used for elution. The amount of solvent used for washing is not particularly limited and may be any amount necessary to remove impurities adsorbed on the silica gel, but is generally preferably about 2 to 15 times the amount per 1 kg of silica gel.

Non-polar organic solvents to be brought into contact with silica gel during regeneration in the present invention include n-hexane, n-heptane, n-pentane, isooctane, cyclohexane, petroleum ether, benzene, and toluene.

The silica gel is brought into contact with these nonpolar solvents to regenerate the silica gel, and this regeneration is performed using a nonpolar solvent heated to a temperature of 40° C. or below the boiling point. If this temperature is higher than the boiling point of the solvent used, at normal pressure some of the solvent will gasify and therefore the flow of the solvent in the column will not be smooth and the regeneration of the silica gel will not take place or will be insufficient. Become. Furthermore, if this temperature is lower than 40°C, the regeneration of the silica gel will be insufficient or impossible. As a heating method, a heated solvent may be injected into the column, or the entire column may be heated, but the latter is preferred in practice.

In the regeneration of the present invention, it is preferable to apply pressure from the top of the column in order to increase the flow rate of the solvent.

There is no particular limit to the amount of nonpolar solvent that is brought into contact with the silica gel during regeneration, but it is usually about 5 to 30, preferably 15 to 25, per kg of silica gel.

In this way, through the regeneration in the present invention, at least the water adsorbed on the silica gel, as well as the cleaning solvent used in the cleaning, are removed by being discharged from the column together with the regeneration solvent used in the regeneration. The silica gel is recycled. The silica gel used to purify coenzyme Q was previously thought to be unable to be reused due to insufficient regeneration, but the present invention restores the adsorption separation ability of silica gel and makes it possible to reuse it. Eliminates the need for replacement,
Furthermore, the amount of silica gel used can be reduced, resulting in improvements in terms of cost and operation, and the present invention has great industrial significance.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (1) Preparation method of crude coenzyme Q ₁₀ Protaminobacter ruber, a methanol-assimilating bacterium, was grown in a synthetic medium containing methanol.
NCIB 2879 was cultured at 28°C with aeration of air and mechanical agitation. After culturing, the bacteria were collected using a centrifuge and dried using a spray dryer to obtain 18 kg of dried bacterial cells.

Add 18kg of dried bacterial cells to 60% acetone at 30°C.
After stirring and extraction for 1 hour (first extraction), the mixture was filtered to obtain an extract and extracted residual bacterial cells. After adding 60% acetone to the extracted residual bacteria obtained by filtration after this first extraction and stirring and extracting at 30°C for 1 hour (second extraction),
The mixture was filtered to obtain an extract and residual bacterial cells. Furthermore, 60% acetone was newly added to the extracted residual bacteria obtained by filtration after this second extraction, and the mixture was stirred and extracted at 30°C for 1 hour (third extraction).
The mixture was filtered to obtain an extract and residual bacterial cells. The above three extracts were combined and concentrated under reduced pressure to dryness, and 1000 ml of n-hexane was added to dissolve the solution to remove insoluble impurities. Add 200ml of methanol solution containing 5% of 28wt% ammonia water to this,
After stirring for 20 minutes, the mixture was stopped, the n-hexane layer and the ammonia/methanol layer were separated, and the ammonia/methanol layer was removed. Then, add 200 ml of methanol solution containing 5 vol% water to the hexane layer.
After stirring for 20 minutes, the mixture was allowed to stand to separate the n-hexane layer and the water/methanol layer, and the water/methanol layer was removed. The n-hexane layer was concentrated under reduced pressure and dried, and dissolved in 150 ml of acetone to remove insoluble materials. Add 150 ml of methanol to this liquid, place it in a deep freezer at -10°C, and let it stand overnight to precipitate crystals, which are filtered out.
20 g of crude crystals (purity 81.0%) containing coenzyme Q ₁₀ were obtained.

(2) Purification of coenzyme Q ₁₀ A column container with a diameter of 26 mm and a length of 300 mm was filled with 50 g of silica gel suspended in n-hexane (trade name: Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd.).
- After charging 5 g of the above crude crystals dissolved in hexane to the top of the column and adsorbing them, a mixture of acetone and n-hexane with an acetone content of 1 vol% was flowed down at a flow rate of 200 ml/hr. After 10 minutes, an effluent containing coenzyme Q ₁₀ began to flow out of the column. Pure coenzyme Q ₁₀
A 500 ml portion was taken as a containing fraction, and the solvent was removed from it to obtain 3.63 g of pure coenzyme Q ₁₀ . This corresponds to about 90% of the coenzyme Q ₁₀ in the crude crystals.

(3) Purification of coenzyme Q ₁₀ using regenerated silica gel Next, an experimental example of regenerating silica gel and purifying coenzyme Q ₁₀ using the regenerated silica gel according to the present invention will be shown.

After performing the above operation (2), wash the silica gel by pouring 300 ml of a 1:1 mixed solvent (vol) of acetone:n-hexane into the silica gel column to remove impurities, and then heat the column from the outside. 1000% n-hexane while keeping at 55℃
ml was flowed to regenerate the column.

The crude crystals 5 of the above (1) dissolved in n-hexane were placed in a column packed with regenerated silica gel.
After charging the column to the top of the column and adsorbing it, a mixture of acetone and n-hexane containing 1 vol % of acetone was allowed to flow down at a flow rate of 200 ml/hr. A 500 ml portion was taken as a fraction containing pure coenzyme Q ₁₀ , and the solvent was removed from it to obtain 3.50 g of pure coenzyme Q ₁₀ . This corresponds to about 86% of the coenzyme Q ₁₀ in the crude crystals.

Comparative Example 1 The same procedure as Example 1 was carried out except that the silica gel was brought into contact with n-hexane at room temperature to regenerate _it .
250 ml was obtained from which the solvent was removed to obtain 1.6 g of pure coenzyme Q ₁₀ . This is a coenzyme in the crude crystals
This corresponds to approximately 40% of _Q10 .

Example 2 (1) Preparation method of crude coenzyme Q ₁₀ Isodecarenol and 2,3-dimethoxy-5
- Add 2500 ml of acetonitrile as an extractant to 50 g of the reaction solution containing coenzyme Q ₁₀ obtained by reacting with methylhydroquinone, perform extraction at 30°C for 10 minutes to obtain an extract, and filter this extract. did. This operation was repeated five times, and the resulting extracts were combined and concentrated under reduced pressure to dryness to obtain a solid. Add 200ml of methyl ethyl ketone to this solid and
Dissolve with stirring at ℃, filter, and cool the filtrate to -10℃.
After placing the liquid in a deep freezer for 2 hours and confirming that the temperature has reached -10℃, the purity is 99.4.
% of powdered coenzyme Q ₁₀ was added thereto, and the mixture was allowed to stand for an additional 5 days to precipitate crystals. When the precipitated crystals were filtered and dried under vacuum, the coenzyme
20 g of crude crystals (purity 73.7%) containing _Q10 were obtained.

(2) Purification of coenzyme Q ₁₀ A column container with a diameter of 26 mm and a length of 300 mm was filled with 50 g of silica gel (trade name: Art 7734, manufactured by Merck & Co., Ltd.) suspended in n-heptane.
After charging and adsorbing 5 g of the above crude crystals dissolved in n-heptane onto the top of the column, a mixture of ethyl acetate and n-heptane containing 5 vol% ethyl acetate was flowed down at a flow rate of 180 ml/hr. After about 2 hours, an effluent containing coenzyme Q ₁₀ began to flow out of the column. 400 ml was collected as a fraction containing pure coenzyme Q ₁₀ , and the solvent was removed from it to obtain 3.25 g of pure coenzyme Q ₁₀ . This corresponds to about 89% of coenzyme Q ₁₀ in the crude crystals.

(3) Purification of coenzyme Q ₁₀ using regenerated silica gel Next, an experimental example of regenerating silica gel and purifying coenzyme Q ₁₀ using the regenerated silica gel according to the present invention will be shown.

After performing the above operation (2), wash the silica gel column with 300 ml of ethyl acetate to remove impurities, and then heat the column from the outside.
Flow 600ml of n-heptane while keeping the temperature at 75℃.
The column was regenerated.

Crude crystals 5 of the above (1) dissolved in n-heptane in a column packed with regenerated silica gel.
After charging the column to the top of the column and adsorbing it, a mixed solution of ethyl acetate and n-heptane containing 5 vol % of ethyl acetate was allowed to flow down at a flow rate of 180 ml/hr.

Collect 400 ml as a pure coenzyme Q ₁₀ -containing section,
Remove the solvent from this and get 3.10g of pure coenzyme Q ₁₀
I got it. This is about 85% of the coenzyme _Q10 in the crude crystals.
corresponds to

Comparative Example 2 The same procedure as in Example 2 was carried out except that the silica gel was brought into contact with n-heptane at room temperature to regenerate the silica gel. 300 ml was collected as a fraction containing pure coenzyme Q ₁₀ , and the solvent was removed from it. and 2.5
g of pure coenzyme Q ₁₀ was obtained. This corresponds to about 68% of coenzyme Q ₁₀ in the crude crystals.

Claims (1)

[Claims] 1 Purify coenzyme Q by adsorbing coenzyme Q onto silica gel using column chromatography using silica gel as an adsorbent, eluating it with an organic solvent for elution, and then using an organic solvent for washing that is more polar than the organic solvent for elution. A method for regenerating silica gel in a column in which the silica gel is washed with water and then the silica gel is brought into contact with a nonpolar solvent, the method comprising using a nonpolar solvent at a temperature of 40° C. or below the boiling point.