JPH0578538B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for purifying coenzyme Q, and more specifically, it relates to a method for purifying coenzyme Q, and more specifically, by using a plurality of adsorption column chromatographs connected in series to each other, a large amount of coenzyme Q can be purified. A novel method for purifying crude coenzyme Q is provided.

Coenzyme Q is involved in the electron transport system of the terminal respiratory system in vivo, and is a useful substance that exhibits excellent pharmacological effects on various diseases such as myasthenia gravis and emphysema.

[Problems to be solved by conventional technology and invention]

Coenzyme Q can be obtained by synthesis or extraction from microbial cells or natural products, but the crude coenzyme Q obtained by these methods is
It also contains many impurities and has extremely low purity. When coenzyme Q is used as a medicine, it is required that this coenzyme Q has extremely high purity, which requires an industrially excellent purification method.

Here, an industrially superior purification method means that purified coenzyme Q (hereinafter referred to as purified coenzyme Q) has high purity and that the amount of packing material used in adsorption column chromatography is used per unit of purified coenzyme Q. (g-filler/g
-purified coenzyme Q) and amount of solvent used (-solvent/g
- Purification method with less purified coenzyme Q).

Many methods are known for obtaining purified coenzyme Q from crude coenzyme Q by adsorption column chromatography, as shown below. That is, there is a method of using an inorganic adsorption carrier as a filler, and methods of using silica gel as a carrier include, for example, JP-A-54-52790, JP-A-57-18988, JP-A-59-
No. 33354, Special Publication No. 58-53917, Special Publication No. 59-26273
The methods described in Japanese Patent Publication No. 57-21309 and Japanese Patent Application Laid-Open No. 56-30941 are known. On the other hand, the method using Florisil as a carrier includes
For example, JP-A-54-110389, JP-A-58-
There are methods described in JP-A No. 12266 and JP-A-54-122795, and methods using alumina as a carrier include, for example, JP-A-59-122795.
There are a large number of methods, including the method described in No. 45894.

All purification methods using these inorganic adsorption carriers are chromatography using a single column.

Furthermore, when trying to obtain highly purified coenzyme Q using an inorganic adsorption carrier, the recovery rate (purified coenzyme Q/crude coenzyme Q) is extremely low;
Generally, large amounts of filler and solvent must be used per unit of purified coenzyme Q. Therefore, it is necessary to make the purification equipment large-scale for purification on an industrial scale, but when making it large-scale, there are problems with local uneven flow of the solvent, mixing, and diffusion of crude coenzyme Q within the column. Due to the nature of adsorption column chromatography, it is extremely difficult to perform it on a large scale.

There is also a method of using porous synthetic resin as a filler. In other words, as an adsorption column chromatography system that uses a non-polar porous synthetic resin and a polar solvent as a solvent, for example, JP-A No. 55-39701
No., JP-A-56-121490, JP-A-57-2686, JP-A-57-6910, JP-A-59-50648, etc.
In addition, adsorption column chromatography using a polar porous synthetic resin and a nonpolar solvent is known, for example, in Japanese Patent Publication No. 11302/1983, but it is not suitable for industrial use due to the same reasons as when using an inorganic adsorbent alone. It is difficult to obtain highly purified coenzyme Q on a large scale, and furthermore, due to the nature of adsorption column chromatography using porous synthetic resins, it is difficult to obtain purified coenzyme Q with a high recovery rate. For this purpose, purification such as adsorption column chromatography followed by adsorption column chromatography using an inorganic adsorption carrier and recrystallization is required.

The purpose of the present invention is to understand that purification of crude coenzyme Q by conventional adsorption column chromatography is unsuitable for large-scale production, and that this accuracy alone usually does not produce coenzyme Q of sufficiently high purity. The object of the present invention is to provide a method for purifying coenzyme Q that can solve the problem that coenzyme Q cannot be purified.

[Means and actions for solving problems]

The present inventors have conducted extensive research in order to obtain coenzyme Q of sufficiently high purity even though it is a method using adsorption column chromatography, which can be purified on an industrial scale and without further post-treatment. As a result of repeated efforts, we have arrived at the present invention.

That is, the present invention uses a plurality of columns connected in series to purify crude coenzyme Q by adsorption column chromatography, and sequentially passes a crude coenzyme Q solution through the columns to purify the crude coenzyme Q. This is a method for purifying coenzyme Q, which is characterized by sequentially reducing the impurity content in the Q solution and recovering a fraction whose main component is coenzyme Q from the final column.

The crude coenzyme Q of the present invention refers to a coenzyme Q-containing product itself containing at least one of coenzymes Q ₄ to _{Q 12} and a large amount of one or more types of impurities, or a coenzyme Q-containing product that has been extracted, concentrated and/or crystallized in advance. It has undergone a purification process known per se, such as chemical conversion. There are no particular restrictions on the manufacturing method or origin of coenzyme Q-containing materials, and examples include extracts from living organisms such as microbial cells, animal organs, and mixed blood, and reaction products obtained by synthesis. or its concentrate.

In the present invention, the content of impurities in the crude coenzyme Q is sequentially reduced by sequentially passing the crude coenzyme Q through a plurality of columns connected in series as a solution. There are means for sequentially discharging impurities from the bottom of each column and sequentially retaining other impurities in each column, and (b) means for sequentially retaining impurities in each column. The former (a) is preferable.

The present invention will be specifically explained with reference to the drawings. It is assumed that the crude coenzyme Q used in this explanation has an elution pattern as shown in FIG. That is, the main component of this crude coenzyme Q is coenzyme Q, and the impurities x ₁ , x 2 and x 3 are eluted sequentially over time earlier than coenzyme Q, and impurities x 1 , x ₂ and x ₃ are eluted later than coenzyme Q. Contains impurities x ₄ , x ₅ and x ₆ that are sequentially eluted over time. However, the curves of both coenzyme Q and each impurity have long tails, and each component is not clearly classified and always contains small to trace amounts of other components.

An example of a flow sheet for a purification apparatus used for the present invention is shown in FIG. That is, this device has a first column 1, a second column 2, and a third column 3.
It has three columns. At the top of each column there is a first top pipe 4, a second top pipe 5 and a third top pipe 6, respectively. These tower top pipes are connected to each other by a tower top communication pipe 7 branched from the first tower top pipe 4. In addition, these tower top pipes are provided with a first tower top valve 8, a second tower top valve 9, and a third tower top valve 1, respectively.
0 is set. Further, at the bottom of each column, there are a first bottom pipe 11, a second bottom pipe 12, and a third bottom pipe 13, respectively. Each bottom pipe is provided with a first bottom valve 14, a second bottom valve 15, and a third bottom valve 16, respectively. Further, each bottom pipe is provided with a first detector 17, a second detector 18, and a third detector 19, respectively, between the bottom of each column and each bottom valve. The first of the first bottom pipe 11
The part between the bottom valve 14 and the first detector 17 and the part between the top of the second column 2 of the second top pipe 5 and the second isotop valve 9 are the first communication pipe. I was contacted at 20,
Thus, the first column 1 and the second column 2 are connected in series. Similarly, the second column 2 and the third column 3 are connected to each other in series through a second communication pipe 21. Each communication pipe is provided with a first communication valve 22 and a second communication valve 23, respectively. A supply pipe 24 is provided at the top of the first column 1 . Note that as the detector at the bottom of each column, for example, an ultraviolet absorption measuring device (UV monitor) and an infrared absorption measuring device can be used. Moreover, instead of using these measuring instruments, the valves can also be opened and closed according to a time schedule determined experimentally in advance.

First, the operation (a) for removing impurities in crude coenzyme Q by sequentially discharging them from the bottom of each column while leaving other impurities remaining in each column using this apparatus will be explained. That is, a solution in which crude coenzyme Q is dissolved in a solvent is supplied to the first column 1 through the supply pipe 24, and each component is developed and adsorbed onto the packing material in the first column 1. Next, while discharging the liquid from the first isobottom valve 14 to the outside of the system, the solution is poured from the first column top pipe 4, and the fraction whose main component is impurity x ₁ is transferred from the first column 1 to the first column bottom. It is discharged outside the system through the valve 14.

When coenzyme Q in this discharged liquid from the first column 1 is detected by the first detector 17, the first tower bottom valve 14 is immediately closed and the first communication valve 22 is opened to remove the coenzyme Q from the first column 1. The drained liquid is passed through the first communication pipe 20 to the second
Move to column 2. At this time, the first detector 17
Immediately when impurity x ₆ begins to be detected in
Before closing the communication valve 22 and moving to the second column 2, the classification whose main component is impurity x ₆ is transferred to the first column 1.
to remain. In the second column 2, each component is developed and adsorbed in the same manner as in the first column 1, and then the second column top valve 9 is opened and the solvent is poured from the second column top pipe 5 to remove the main components. The fraction containing impurities x ₂ is discharged from the system through the second bottom valve 15.

When the second detector 18 detects coenzyme Q in the effluent from the second column 2, the effluent is transferred to the third column 3 as described above, while the main component is impurity x ₅ . A portion is left in the second column 2. In the third column 3, as in the first column 1 and the second column 2, the development of each component,
Separation and elution are performed, and impurity x 3 , coenzyme _Q and impurity x ₄ are removed from the third bottom valve 16 of the third column 3.
Each category having each of them as a main component is sequentially discharged. Coenzyme Q in this discharged liquid is detected by the third detector 19, and a fraction whose main component is coenzyme Q is collected. The fraction whose main components are impurities remaining in each column is washed out with a solvent, the packing material in each column is regenerated, and each column is again subjected to adsorption column chromatography. The time for the development, adsorption, and elution of each component in each column and the washing out of impurities are appropriately combined. 3. By washing out a small amount of residual impurities in column 3, the idle time of each column can be reduced and each column can be used effectively.

Next, use this device to perform an operation to remove impurities in the crude coenzyme Q while leaving them in each column.
(b) will be explained. That is, a solution in which crude coenzyme Q is dissolved in a solvent is supplied to the first column 1 from the supply pipe 24. Next, a solvent is supplied from the first tower top pipe 4, and the first tower top valve 8, first communication valve 22, second communication valve 23, and third tower bottom valve 13 are opened, and the other valves are closed, and the solvent is removed. is sequentially passed through the first column 1, the first connecting tube 20, the second column 2, the second connecting tube 21, and the third column 3, and along with this, each component of the crude coenzyme Q also moves, and each component is It is developed and adsorbed in each column. Almost the entire amount of coenzyme Q in the effluent from the first column 1
When almost no coenzyme Q is detected in the effluent from the first column 1 by the first detector 17, and impurity x ₆ begins to be detected, the first communication valve 22 is closed and the A fraction whose main component is impurity x ₆ remains in the column 1, while the second column top valve 9 is opened and the solvent is poured from the second column top pipe 5.
This solvent is transferred to the third column 3 along with the components of the crude coenzyme Q. When the second detector 18 detects almost no coenzyme Q in the liquid discharged from the second column 2 and when impurity x ₅ begins to be detected, the second communication valve 23 is closed and the second column 2 is The main component is impurity 5.
On the other hand, the third tower top valve 10 is opened to pour the solvent from the third tower top pipe 6, and the third tower bottom valve 16 is opened to allow the discharged liquid from the third column 3 to flow out of the system. This discharged liquid is sequentially discharged in this order into sections whose main components are impurities x ₁ , x ₂ , x ₃ , coenzyme Q, and impurity x ₄ , and the third detector 19 detects that the main components are coenzyme Q. Detect and collect the classification. A section whose main components are impurities x ₆ and ₅ remaining in the first column 1 and second column 2, respectively, and a small amount of impurities x ₄ remaining in the third column 3.
The main component is removed by washing the packing material by flowing a solvent down each column, the packing material in each column is regenerated, and each column is subjected to adsorption column chromatography again. Further, as in the above operation (a), it is possible to effectively utilize each column without leaving it idle by appropriately combining the operations of each column.

There are no particular limitations on the filler used in the present invention, and fillers commonly used in the past can be used, but representative examples are as follows.

That is, examples of the inorganic filler include silica gel, alumina, florisil, activated carbon, and hydroxy abatanite. Examples of commercially available silica gel products include Wako Gel C-200 and Wako Gel C-300 (both manufactured by Wako Pure Chemical Industries), Silica Gel Art (Art) 9385, and Silica Gel Art 7734.
(all manufactured by Merck), and silica gel KT
-3063 and Silica Gel 4B (both manufactured by Fujigel).

In addition, porous synthetic resins are generally used as organic fillers, and representative examples of non-polar synthetic resins include Amberlite XAD-2 (manufactured by Rohm and Haas) and Amberlite.
Examples of polar synthetic resins include styrene-divinyl copolymers such as XAD-4 (manufactured by Rohm and Haas) and High Porous Polymer HP (manufactured by Mitsubishi Chemical Corporation). Manufactured by Rohm and Haas)
and polyacrylic esters such as Amberlite XAD-8 (manufactured by Rohm and Haas),
Sulfoxides such as Amberlite XAD-9 (Rohm & Haas) and Amberlite XAD-11 (Rohm & Haas)
Examples include polar synthetic resins such as.

The packing materials packed in each column may be the same or different. Preferably they are the same. Particular preference is given to inorganic fillers of the same type.

In the present invention, the solvent for dissolving the crude coenzyme Q and the solvent added to each column may be any solvent that can dissolve the crude coenzyme, and either a single solvent or a mixed solvent may be used.

Generally, when using an inorganic filler, a non-polar solvent is preferred as the sole solvent, such as hydrocarbons such as n-hexane, n-pentane, n-heptane, isooctane, cyclohexane and mixtures thereof, petroleum ether, etc. is particularly preferred for practical purposes. The mixed solvent is particularly preferably a mixed solvent in which an ether type such as ethyl ether, an ester type such as ethyl acetate, butyl acetate, or a ketone type organic solvent such as acetone or methyl ethyl ketone is added to the above-mentioned nonpolar solvent. In addition, when using a porous synthetic resin as a filler, methanol, ethanol, isopropanol, n-propanol, acetone, methyl ethyl ketone, isopropyl ether, tetrahydrofura, dioxane, methyl cellosolve, dimethyl formamide,
Polar solvents such as acetonitrile and water can be used alone or in mixtures with each other; mixed solvents are preferred. Mixed solvents such as methanol and water, methanol and acetone, methanol and n-hexane, and acetone and water are particularly preferably used.
On the other hand, when using polar porous resin, benzene, toluene, n-hexane, petroleum ether,
Non-polar solvents such as petroleum benzene, isopentane, carbon tetrachloride and chloroform can be used alone or in mixtures with each other. Also,
A mixed solvent obtained by mixing each of these solvents with acetone, ether, and ethyl acetate may also be used. Mixed solvents are generally preferred. A mixed solvent of n-hexane and each of acetone, ether and ethyl acetate is particularly preferred.

The solvent injected from the top of each column depends on the type of packing material packed in each column, crude coenzyme Q
Generally, a solvent suitable for each column condition is used depending on the type, number, and amount of impurities contained in the column, as well as the processing temperature.
It is also possible to use a common solvent for each column.
Further, the solvent supplied from the top of each column may be changed to another solvent during the supply.

Note that when each column is filled with an inorganic filler or a polar porous resin, it is preferable to increase the polarity of the solvent in the later columns. On the other hand, when each column is filled with a non-polar porous resin, it is preferable to reduce the polarity of the solvent in later columns. Incidentally, in order to adjust the polarity of the solvent, a plurality of solvents having different polarities may be mixed as in a conventional method.

For example, hexane/acetone (volume ratio)
If they are mixed at a ratio of 100/0, 98/2, and 95/5, the polarity will increase in this order. Also, methanol/acetone (volume ratio) is 7/3, 5/5, 3/
If mixed at 7, the polarity will decrease in this order.

The temperature of the column, the temperature of the crude coenzyme Q solution supplied to the column, the supply rate, the amount of solvent poured into each column, the supply rate, etc. are determined by the type of packing material used and the impurities in the crude coenzyme Q. The number, type, and amount of solvents, the type of solvent, etc., and cannot be determined unambiguously. These conditions are for each column:
Although conditions suitable for each column may be selected, it is also possible to use the same conditions for each column. However, in practice, the following conditions are usually adopted.
That is, for example, the temperature of the crude coenzyme Q solution supplied to the column and the temperature of the column are such that the packing material does not lose its adsorption ability, the coenzyme Q in the crude coenzyme Q is dissolved in the solvent, and the boiling point of the solvent is maintained. The temperature may be lower, usually from 15 to 50°C, preferably from room temperature to normal temperature, and heating and cooling are not particularly necessary, but heating and cooling are not prohibited. Note that it is preferable that the temperature of the columns be higher for later columns.

When an inorganic packing material is used, the supply amount of the crude coenzyme Q solution is usually 50% by weight or less of the total weight (g) of the packing material used in each column,
Practically speaking, the content is preferably 30% by weight or less.

In addition, when porous synthetic resin is used as a packing material, it is usually less than 50% by weight (g - crude coenzyme Q/ml - total volume of packing material) of the total volume (ml) of packing material used in each column. In practice,
It is preferably 35% by weight or less. Note that the total filler volume (ml) here is not an apparent volume but an actual volume.

The amount of solvent for dissolving crude coenzyme Q is not particularly limited, but it is usually 0.5 to 1 g of crude coenzyme Q.
The ratio is 5 ml, preferably 1.3 to 2.5 ml.

There is no particular restriction on the ratio of the weight (g) of the packing material between each column used in the present invention, but the ratio of the maximum packing amount (weight) to the minimum packing amount (weight) is usually 0.1 to 0.1 for practical purposes. 1.

In the present invention, the apparent column cylinder velocity of the solvent poured into each column [solvent supply amount per unit time]
ml/sec/column cross-sectional area (cm ² ) = cm/sec] is not particularly limited, but in practice it is usually 0.001 to 0.3 cm/sec,
Preferably it is 0.01-0.1 cm/sec. Furthermore, the pressure at the top of the column may be normal pressure, increased pressure, or reduced pressure as long as the apparent cylinder velocity of the supplied solvent, which is preferable for separation, is maintained.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples. However, the present invention is not limited to these examples.

Example 1 Two columns (corresponding to the first column 1 and the second column 2 connected in series with each other in FIG. 2 - therefore, the third column 3 and the second connecting pipe 21 are not used) (diameter 22 mm) , a cylinder with a length of 450 mm),
Filler Wakogel C- suspended in n-hexane
300 140g was divided into two equal parts and 70g each was packed into two columns.

Add 27 g of crude coenzyme Q ₁₀ obtained from microbial cells to 54 ml of n-hexane at 25°C (coenzyme Q ₁₀ content 18.9
A solution in which g) was dissolved was supplied to the upper part of the first column 1.

Next, while extracting the liquid from the bottom of the first column 1, a solvent (acetone content 0.5% by volume) was added from the top of the column.
A mixed solvent of acetone and hexane (same below)
was poured out. The solvent supply rate at this time is 684
ml/hr, apparent column cavity velocity 0.05cm/sec
The temperature of the first column 1 was maintained at 25°C.

By this operation, the first bottom valve 14 of the first column 1
When 260 ml of the impurity solution was taken out of the system, coenzyme Q ₁₀ was detected by the first detector 17 (UV monitor wavelength 254 nm or less). At this time, the first tower bottom valve 14 is closed, and the first communication valve 22 and the second
The bottom valve 15 was opened and the fraction containing coenzyme Q ₁₀ was transferred to the second column 2. At the same time, the second tower top valve 9 was opened, and the solvent (acetone content 8% by volume) was
A mixed solvent of acetone and n-hexane (the same applies hereafter) was poured into the solution. The solvent supply rate at this time is
It was set to 80ml/hr.

At this time, the amount of solvent supplied to the first column 1 was changed from 684 ml/hr to 604 ml/hr. Second
The column temperature of column 2 was 35°C. The top pressures of the first column 1 and the second column 2 were 0.3 Kg/cm ² G and 0.1 Kg/cm ² -G, respectively.

Here, the apparent cavity velocity of the solvent in the first column 1 and the second column 2 was 0.044 cm/sec and 0.05 cm/sec, respectively.

From the start of adsorption column chromatography, when the total amount of eluate reached 2000 ml (260 ml from the bottom of the first column 1, 1740 ml from the bottom of the second column 2), the first
The detection of coenzyme Q ₁₀ was completed by the detector 17, and the first tower top valve 8, first tower bottom valve 14, and first communication valve 22 were closed. At this time, in the second column 2, the solvent (acetone with an acetone content of 1.0% by volume and n
- mixed solvent with hexane (same below) and the supply rate was set at 684 ml/hr. Here, the apparent cavity velocity of the solvent in the second column 2 is 0.05 cm/
sec. The temperature in the second column 2 remained at 35°C. The eluate is separated from the second bottom valve 15 of the second column 2 into an elution section containing only impurities, impurities and coenzyme.
The samples were separated into four categories in the order of elution: a category containing coenzyme Q ₁₀ , a category mainly containing only coenzyme Q ₁₀ , and a category containing impurities and coenzyme Q ₁₀ . The respective eluate volumes are 1520ml, 365ml, 1335ml and 125ml.
It was hot.

The total amount of solvent used was poured from the top of each column.
It was 3605ml. Removing the solvent from the 1335 ml section containing mainly only coenzyme Q ₁₀ yielded 15.12 g of coenzyme Q _10.
I got it. The melting point of this coenzyme Q ₁₀ is 48℃, and its purity is determined by reverse phase and normal phase high performance liquid chromatography, mass spectrum, and infrared absorption spectrum.
Confirmed to be 100% coenzyme _Q10 .

The obtained purified coenzyme _Q10 recovery rate corresponds to 80%. The total amount of solvent used and the total amount of filler used per 1 g of obtained purified coenzyme Q ₁₀ were 239 g and 9.3 g, respectively.

Note that a large amount of impurities remained in the first column 1.

Comparative Example 1 The solvent was poured only from the top of the first column 1 without pouring the solvent from the top of the second column 2,
The procedure of Example 1 was followed except that the drained liquid was not extracted from the bottom of the first column 1 to the outside of the system.

That is, the solvent injected into the first column 1 is
It is a mixed solvent of acetone and n-hexane with an acetone content of 0.5% by volume, and the supply rate is 684ml/hr.
The apparent velocity of the cylinder was 0.05 cm/sec, and the total amount supplied was 2000 ml. Also, set the temperature of each column to 25℃.
I kept it.

Next, this solvent was changed to a mixed solvent of acetone and n-hexane with an acetone content of 1% by volume, and the feed rate and apparent cylinder velocity remained unchanged. Note that the total amount of this solvent supplied was 1670 ml.

Additionally, the temperature of each column was changed to 35°C. The top pressure of each column is 0.3Kg/cm ² -G and
It was 0.1Kg/cm ² -G.

The eluate from the second bottom valve 15 is divided into elution sections containing only impurities, sections containing impurities and coenzyme Q ₁₀ , sections mainly containing only coenzyme Q ₁₀ , and sections containing impurities and coenzyme Q ₁₀ . It was divided into four sections in the order of elution. Eluate volume is 1580ml, 775ml, respectively.
The total volume was 3670ml with 920ml and 395ml.

The solvent was removed from a 920 ml portion containing mainly only coenzyme Q ₁₀ to obtain 10.5 g of coenzyme Q ₁₀ with a purity of 99%. The recovery rate of the obtained purified coenzyme Q ₁₀ was equivalent to 55%, and the total amount of solvent used and total amount of filler used per 1 g of obtained purified coenzyme Q ₁₀ (purity 99%) were 353 ml and 13.3 g, respectively. .

The recovery rate obtained here was significantly inferior to that in Example 1, and the purity of the purified coenzyme Q ₁₀ obtained was also inferior to that in Example 1. Furthermore, the total amount of solvent used and the total amount of filler used per 1 g of purified coenzyme Q ₁₀ were extremely large compared to Example 1.

Example 2 54 g of crudely purified coenzyme (coenzyme Q ₁₀ content: 37.8 g) obtained from microbial cells was purified using the apparatus shown in FIG. 70 g of 210 g of Wakogel C-300 suspended in n-hexane was packed into each cylinder of three columns (all 22 mm in diameter and 450 mm in length).

A solution of 54 g of the crude coenzyme dissolved in 108 ml of n-hexane at 25° C. was supplied to the upper part of the first column 1.

Then, while the liquid was drawn out of the system from the bottom of the first column 1, the solvent was poured from the top of the column. The column temperature was maintained at 25° C. with a solvent supply rate of 684 ml/hr and an apparent column cavity velocity of 0.05 cm/sec.

By this operation, the first bottom valve 14 of the first column 1
When 170 ml of the impurity solution was taken out of the system, coenzyme Q was detected by the first detector 17 (UV monitor). At this time, the first column bottom valve 14 was closed, the first communication valve 22 and the second column bottom valve 15 were opened, and the fraction containing coenzyme Q ₁₀ 1 was transferred to the second column 2. At the same time, the second tower top valve 9 was opened and the solvent was poured. At this time, the solvent supply rate was set to 80ml/
hr.

At this time, the supply rate of the solvent supplied to column 1 was changed from 684 ml/hr to 604 ml/hr.

The temperature of the second column 2 was also 25°C. Also, the first
The top pressures of column 1 and second column 2 are 0.3 Kg/cm ² -G and 0.1 Kg/cm ² -G, respectively.
It was hot.

Here, the apparent cavity velocity of the solvent in the first column 1 and the second column 2 was 0.044 cm/sec and 0.05 cm/sec, respectively.

The eluate from the second bottom valve 15 of the second column 2 is detected by the second detector 18 and eluted as two sections: a 570 ml elution section containing only impurities and a 200 ml section containing impurities and coenzyme Q ₁₀ . The samples were separated one by one and taken out of the system.

Next, the second column bottom valve 15 was closed, the second communication valve 23 and the third column bottom valve 16 were opened, and the fraction containing a large amount of coenzyme Q ₁₀ was transferred to the third column 3. At the same time, open the third tower valve 10 and pump out the solvent at 80ml/hr.
It was poured at a feed rate of .

The apparent cavity velocity of the solvent in the third column 3 is
It became 0.056cm/sec.

The temperatures of the first column 1 and the second column 2 were not changed, and the temperature of the third column 3 was set at 35°C.

The top pressure of each column is 0.4Kg/cm ² -G,
They were 0.2Kg/cm ² -G and 0.1Kg/cm ² -G.

A total of 2050 ml of eluate from the start of adsorption column chromatography (170 ml from the bottom of the first column, 170 ml from the bottom of the second column,
770ml from the bottom of column 2, from the bottom of column 3
1110ml), the first detector 17 detects the coenzyme.
Q ₁₀ is almost no longer detected, and the first tower top valve 8
Then, the first communication valve 22 was closed, and the transfer of the section containing coenzyme Q ₁₀ from the first column 1 to the second column 2 and the pouring of the solvent into the first column 1 were completed.

In addition, in the third column 3, the transfer of the section containing coenzyme Q ₁₀ from the second column 2 to the third column 3 and the pouring of the solvent are continuing.

At this time, the solvent poured into the second column 2 was changed to the solvent, and the supply rate was changed from 80 ml/hr to 604 ml/hr.
Changed to ml/hr.

Here, the apparent cylinder velocity of the solvent in the second column 2 was 0.044 ml/hr.

The top pressures of the second column 2 and third column 3 are 0.3Kg/cm ² -G and 0.1Kg/cm ^{2 -G} , respectively.
It was G.

From the start of adsorption column chromatography, the total amount of eluate was 4010 ml (from the bottom of the first column, 170 ml,
770 ml from the bottom of the second column and 3070 ml from the bottom of the third column 3), almost no coenzyme Q ₁₀ is detected by the second detector 18, and the second communication valve 23 and the second top valve 9 was closed, the solvent was poured into the second column 2, and the transfer of the section containing coenzyme Q ₁₀ from the second column 2 to the third column 3 was completed. Note that impurity elution in the third column 3 is continuing.

Also, at this time, the solvent poured into the third column 3 was used as a solvent (a mixed medium of acetone and n-hexane with an acetone content of 1.5% by volume).
In addition, the feed rate was changed from 80 ml/hr to 684 ml/hr.

Here, the apparent cylinder velocity in the third column 3 is
It becomes 0.05cm/sec.

Next, the eluate from the third bottom valve 16 of the third column 3 is divided into an elution section containing only impurities, a section containing impurities and coenzyme Q ₁₀ , a section mainly containing only coenzyme Q ₁₀ , and an elution section containing impurities and coenzyme Q. The _samples were separated into four sections in the order of elution. These eluate volumes are 2400ml, 600ml, 1800ml and
It was 400ml. In addition, the total amount of solvent used from the start of adsorption column chromatography was 6140 ml (170 ml from the bottom of the first column 1, 770 ml from the bottom of the second column 2).
ml, 5200 ml from the bottom of the third column 3).

The solvent was removed from the 1800 ml portion containing primarily Coenzyme Q ₁₀ to yield 28.35 g of Coenzyme Q ₁₀ . The melting point of this coenzyme Q ₁₀ is 48°C, and it has been analyzed by reverse-phase and normal-phase high performance liquid chromatography and mass spectrometry.
100% pure coenzyme by infrared absorption spectrum
It was confirmed that _Q10 .

The recovery rate of the obtained purified coenzyme Q ₁₀ was 75%, and the total amount of solvent and filler used per 1 g of the obtained purified coenzyme Q ₁₀ were 217 ml and 7.4 g, respectively.

Note that a large amount of impurities remained in the first column 1 and the second column 2.

Comparative Example 2 The solvent was poured only into the first column 1 without pouring the solvent into the second column 2 and the third column 3.
The procedure of Example 2 was followed except that the liquid was not discharged from the bottom of each of column 1 and second column 2 to the outside of the system. i.e. n-hexane at 25°C
A solvent in which 54 g of the same crude coenzyme Q ₁₀ as in Example 2 was dissolved in 108 ml was supplied to the first column 1. The third column bottom valve 13 was opened, and the crude coenzyme Q ₁₀ was allowed to pass through each column in sequence to develop and adsorb each component.

The solvent is supplied from the top of the first column 1, and the feeding rate is
684 ml/hr, apparent empty cylinder velocity was 0.05 cm/sec, and 3000 ml was poured.

The temperature of each column was maintained at 25°C.

The solvent was then changed from one to another, and 3170 ml of the solvent was poured without changing the feed rate.

In addition, the temperature of each column was changed to 35°C. The top pressures of the first column 1, second column 2 and third column 3 are 0.4Kg/cm ² -G and 0.2, respectively.
Kg/cm ² -G and 0.1 Kg/cm ² -G.

The eluate from the third bottom valve 16 is divided into elution sections containing only impurities, sections containing impurities and coenzyme Q ₁₀ , sections mainly containing only coenzyme Q ₁₀ , and sections containing impurities and coenzyme Q ₁₀ . The samples were separated into four sections in the order of elution. The volume of each eluate is
They were 3140ml, 1200ml, 1080ml and 750ml.
The solvent was removed from the 1080 ml portion containing mainly only coenzyme Q ₁₀ to obtain 17.01 g of coenzyme Q ₁₀ with a purity of 99%.

The recovery rate of the purified coenzyme Q ₁₀ obtained was 45%, and the total amount of solvent and filler used per 1 g of purified coenzyme Q ₁₀ (purity 99%) was 363 ml.
and 12.3g. The recovery rate obtained in Comparative Example 2 was significantly inferior to that in Example 2, and the purity of purified coenzyme Q ₁₀ was inferior to that in Example 2.

Furthermore, the total amount of solvent used and the total amount of filling used per 1 g of purified coenzyme were both extremely large compared to Example 2.

Example 3 Crude coenzyme obtained in the same manner as Example 1
10g of Q ₁₀ (coenzyme Q ₁₀ content 7.0g) was divided into two equal parts of 60ml each using 120ml of high porous polymer powder HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.) as a filler, packed into a column, and mixed with acetone/methanol (by volume). ratio 4:6
Purification was carried out in accordance with Example 1, except that the mixture was filled with (the same applies hereinafter).

That is, acetone/methanol (4:
A solution prepared by dissolving 10 g of the crude coenzyme Q ₁₀ in the mixed solvent of 6) was supplied to the upper part of the first column.

Next, while extracting the liquid from the bottom of the first column 1, the solvent (acetone:methanol=
A mixed solvent of 4:6 (the same applies hereafter) was poured.

At this time, the solvent supply rate was 684 ml/hr, the apparent cavity velocity was 0.05 cm/sec, and the first column 1
The temperature was maintained at 25℃. The top pressure of the first column 1 was normal pressure.

Next, when the amount of solvent supplied to the first column 1 reached 600 ml, the solvent was changed to a solvent (acetone:methanol=5:5 or below).

By this operation, the first isobasic valve 14 of the first column 1
When 720 ml of a solution containing impurities was taken out of the system, coenzyme Q ₁₀ was detected by the first detector 17. At this time, the first column bottom valve 14 was closed, the first communication valve 22 and the second column bottom valve 15 were opened, and the fraction containing coenzyme Q ₁₀ was transferred to the second column 2. At the same time, open the second column top valve 9 and use the solvent (acetone: methanol = 9:1 mixed solvent).
was poured out.

The solvent supply rate at this time was 80 ml/hr.

The column temperatures in the first column 1 and the second column 2 were 25°C and 35°C, respectively, and the top pressure was normal pressure. Here, the apparent cavity velocity of the solvent in the second column 2 is 0.056 cm/sec.

From the start of adsorption column chromatography, the total amount of eluate was 1020 ml (720 ml from the bottom of the first column, 720 ml from the bottom of the second column,
300 ml from the bottom of column 2), coenzyme Q ₁₀ was no longer detected by the first detector 17, and the first top valve 8, first bottom valve 14, and first communication valve 22 were closed.

At this time, in the second column 2, the solvent was changed to a solvent (acetone:methanol=6:4 and the same applies hereafter), and the feeding rate was also changed to 684 ml/hr.
Here, the apparent cavity velocity of the solvent in the second column 2 is 0.05 cm/sec.

The temperature in the second column 2 remained at 35°C.

The eluate from the second bottom valve 15 of the second column 2 is separated into an elution section containing only impurities, an elution section containing only impurities, and an elution section containing only impurities and coenzymes.
Category 4 that includes coenzyme Q ₁₀ , category that mainly contains only coenzyme Q ₁₀ , and category 4 that includes impurities and coenzyme Q ₁₀
The samples were divided into two sections in the order of elution. The respective eluate volumes were 120 ml, 50 ml, 545 ml and 50 ml.

The total amount of solvent used was poured from the top of each column.
It was 1485ml. By removing the solvent from the 545 ml section containing only coenzyme Q ₁₀ , 5.9 g of coenzyme Q ₁₀ was obtained.
I got it. This coenzyme Q ₁₀ has a purity of 99.1% as determined by reverse phase and normal phase high performance liquid chromatography, mass spectrometry and infrared absorption spectroscopy.
It was confirmed that _Q10 .

The obtained purified coenzyme Q ₁₀ recovery rate corresponds to 85%.

The total amount of solvent and filler used per 1g of purified coenzyme _Q10 was 252ml and 252ml, respectively.
It was 20.3ml.

Note that a large amount of impurities remained in the first column 1.

Comparative Example 3 The solvent was poured only from the top of the first column 1 without pouring the solvent from the top of the second column,
The same procedure as in Example 3 was carried out except that the effluent was not drawn out from the bottom of the first column 1.

That is, 650 ml of solvent, 300 ml of solvent, and 420 ml of solvent were poured into the first column 1 in sequence.
ml was used. All solvents have a feed rate of 684ml/
hr, and the apparent velocity of the cylinder was 0.05 cm/sec. Additionally, the temperature of each column was maintained at 25°C. The top pressure of each column was normal pressure.

The eluate from the second bottom valve 15 is divided into elution sections containing only impurities, sections containing impurities and coenzyme Q ₁₀ , sections mainly containing only coenzyme Q ₁₀ , and sections containing impurities and coenzyme Q ₁₀ . The sample was divided into four sections in the order of elution. The volumes of each eluate were 720 ml, 200 ml, 400 ml, and 50 mm, respectively.

The solvent was removed from a 400 ml portion containing mainly only coenzyme Q ₁₀ to obtain 4.3 g of coenzyme Q ₁₀ with a purity of 99%. The recovery rate of the purified coenzyme Q ₁₀ obtained was 55%, and the total amount of solvent used and filler used per 1 g of purified coenzyme Q ₁₀ (purity 99%) were respectively
They were 319ml and 22.6ml.

The recovery rate obtained here was significantly inferior to that in Example 3, and the purity of purified coenzyme Q ₁₀ was also inferior to that in Example 3. Furthermore, the total amount of solvent used and the total amount of filler used per 1 g of purified coenzyme Q ₁₀ were extremely large compared to Example 3.

Example 4 54 g of crude coenzyme (coenzyme Q ₁₀ content: 37.8 g) obtained from microbial cells was purified using the apparatus shown in FIG. 3 columns (all diameter 22mm, length
70 g of Wakogel C-300 suspended in n-hexane was packed into each column (450 mm cylinder). A solution of 54 g of the crude coenzyme dissolved in 108 ml of n-hexane at 25° C. was supplied to the upper part of the first column 1. Next, the first communication valve 22 and the second communication valve 23 were opened, and the solvent was poured from the top of the first column 1 while drawing out the liquid from the bottom of the third column 3. The solvent supply rate was 684 ml/hr, the apparent velocity of each column was 0.05 cm/sec, and the temperature of each column was maintained at 25°C. The top pressure of each column is 0.9Kg/cm ² G, 0.4Kg/cm ² G and 0.1Kg/cm ² respectively.
It was G.

When the total amount of eluate reaches 2200 mm from the start of adsorption chromatography, coenzyme Q ₁₀ is almost no longer detected in the first detector 17, the first column top valve 8 and the first communication valve 22 are closed, and the first Injection of the solvent into column 1 was completed. At the same time, the second tower top valve 9 was opened and the solvent was poured from the second tower top pipe 5.
The temperature of the second column 2 and the third column 3 was maintained at 35° C. with a solvent supply rate of 684 ml/hr and an apparent cavity velocity of 0.05 cm/sec. The top pressures of the second column 2 and the third column 3 are respectively
They were 0.4Kg/cm ² G and 0.1Kg/cm ² G.

When the total volume of eluate reaches 4500 ml from the start of adsorption column chromatography, the coenzyme is added to the second outlet 18.
_Q10 is almost no longer detected, and the second communication valve 2
3 and the second column top valve 9 were closed, and the pouring of the solvent into the second column 2 was completed. At the same time, the third tower top valve 10 was opened and the solvent was poured.

The supply rate of the solvent to the third column 3 was set to 684 ml/
hr, the apparent cavity velocity was 0.05 cm/sec, and the temperature of each column was maintained at 35°C. The top pressure of the third column 3 was 0.1 Kg/cm ² G.

Next, the eluate from the third bottom valve 16 of the third column 3 is divided into an elution section containing only impurities, a section containing impurities and coenzyme Q ₁₀ , a section mainly containing only coenzyme Q ₁₀ , and an elution section containing only impurities and coenzyme. The samples were separated into four sections in the order of elution, including _Q10 . The volumes of these eluates were 3520 ml, 650 ml, 1800 ml and 830 ml, respectively. Furthermore, the total amount of solvent used from the start of adsorption column chromatography was 6800 ml. Removal of solvent from 1800 ml section containing mainly only Coenzyme Q ₁₀ yields 99.5% pure Coenzyme Q ₁₀ 22.45
I got g.

The recovery rate of the purified coenzyme Q ₁₀ obtained was 59.4%, and the total amount of solvent and filler used per 1 g of purified coenzyme Q ₁₀ was 303 ml and 303 ml, respectively.
It was 9.4g.

Note that a large amount of impurities remained in the first column 1 and the second column 2.

〔Effect of the invention〕

In the present invention, the recovery rate of coenzyme Q is significantly improved, and the amount of filler and solvent used per unit weight of purified coenzyme is greatly reduced. Therefore,
Although the present invention is a method using adsorption column chromatography, it is possible to purify coenzyme Q on an industrial scale. Moreover, the purity of coenzyme Q obtained by the present invention is high.

[Brief explanation of the drawing]

FIG. 1 shows an example of the elution pattern of crude coenzyme Q, and FIG. 2 shows an example of the flow sheet of the purification apparatus used for the present invention. In the drawings, 1...first column, 2...second column, 3...
Third column, 4...First tower top pipe, 5...Second tower top pipe, 6...Third tower top pipe, 7...Tower top connection pipe, 8...
...First tower top valve, 9...Second tower top valve, 10...Third
Tower top valve, 11...first tower bottom pipe, 12...second tower bottom pipe, 13...third tower bottom pipe, 14...first tower bottom valve,
15...Second tower bottom valve, 16...Third tower bottom valve, 17
...First detector, 18...Second detector, 19...
Third detector, 20...first communication pipe, 21...second
Communication pipe, 22...first communication valve, 23...second communication valve, and 24...supply pipe.

Claims (1)

[Claims] 1. When purifying crude coenzyme Q by adsorption column chromatography, multiple columns connected in series are used, and the crude coenzyme Q solution is sequentially passed through the columns to remove impurities in the crude coenzyme Q solution. A method for purifying coenzyme Q, which comprises sequentially reducing the content and recovering a fraction whose main component is coenzyme Q from the final column.