JPS63154702A - Separation of cyclodextrins from each other - Google Patents

Abstract

PURPOSE:To separate cyclodextrins from each other efficiently without the use of an organic solvent, by contacting a cyclodextrin mixture with a chemically modified silica carrier thereby adsorbing the mixture onto the carrier, and then eluting it fractionally with warm water having specific temperatures. CONSTITUTION:Silica gel is reacted with an alkylchlorosilane, and then, if desired, further reacted with trimethylchlorosilane, to produce a chemically modified silica carrier (B) having a structure in which the silanol group of the silica gel is substituted by an 8-18C linear alkylsilyl group. A solution (A) of a cyclodextrin mixture containing alpha-, beta- and gamma-cyclodextrins and, if desired, glucose, an oligosaccharide, an enzyme, etc., in various ratios is contacted with and adsorbed onto component (B) and, if desired, washed with cold water to remove the glucose and the oligosaccharide, and then eluted at a liquid velocity (SV) of 3-30 with warm water (C) in a volume scores of times as much as the volume of component (B) of which temperature is raised continuously or stepwise in the range of 20-80 deg.C. The eluats are separately collected in fraction collectors in order of the elution, concentrated and, if desired, dried.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for separating cyclodextrins, and more specifically, to adsorbing cyclodextrins onto a chemically modified silica carrier, and then separating the adsorbed cyclodextrins. This invention relates to a method for mutually separating cyclodextrins, which comprises fractionating and eluting cyclodextrins using warm water.

[Prior art j Cyclodextrins are produced by adding Bacillus macerans to starch or starch decomposition products.
It is a decomposition product obtained by the action of a cyclodextrin-producing enzyme produced by microorganisms such as P. eerans), and is expected to have a wide range of applications in the fields of food, medicine, cosmetics, etc. by utilizing its inclusion effect. .

Recently, efforts have been made in various fields to develop industrial production methods for such cyclodextrins. Therefore, the raw J&K contains not only various cyclodextrins but also a large amount of linear or branched oligosaccharides. Therefore, in order to obtain highly pure cyclodextrin, it is necessary to separate and collect only specific cyclodextrins from these sugar solutions, but conventional methods do not allow such highly purified cyclodextrin products to be produced at low cost. have not been successful in manufacturing.

Typical methods for purifying cyclodextrins known so far are as follows.

■ Method of precipitating cyclodextrin by adding 77 tons of organic solvent to sugar solution (Japanese Patent Publication No. 52-8385
issue),.

■ A method of purification using anion exchange membrane (fat) (Japanese Patent Publication No. 9223/1983).

(2) A method using a hydrophobic synthetic adsorption resin made of a porous polymer (Japanese Unexamined Patent Publication No. 56-805).

■ A method of fractionation using an alkali metal salt of a strongly acidic ion exchange resin (Japanese Patent Application Laid-open No. 30702/1983).

However, although these methods are effective to some extent in separating cyclodextrins from other oligosaccharides, dextrins, etc., they have a drawback in that they can hardly be used to separate cyclodextrins from each other.

[Problems to be Solved by the Invention] Taking these circumstances into consideration, the present inventors have conducted extensive research to find an efficient and practical method for separating cyclodextrin, and as a result, have developed a chemical solution. They discovered that a decorated silica carrier selectively adsorbs only cyclodextrins, and using this, they succeeded in separating oligosaccharides and cyclodextrins. A patent application has been filed.

However, since this method usually uses hot water of 80°C or higher to elute cyclodextrins, various cyclodextrins are eluted almost simultaneously.
Although it is possible to separate cyclodextrins from other saccharides, it has the disadvantage that it is virtually impossible to separate cyclodextrins from each other.

As a result of a detailed study of the elution pattern of cyclodextrins by varying the temperature of this hot water, the present inventors found that
When the temperature of hot water is below room temperature, only glucose and oligosaccharides are eluted and cyclodextrins are hardly eluted, but when the temperature of hot water is raised to above about 20°C, elution occurs gradually, and the elution rate varies considerably depending on the type of cyclodextrin. I discovered that there is a difference.

[Means for Solving the Problems 1] The present invention was completed based on the above new findings, and involves bringing a cyclodextrin mixture into contact with a chemically modified silica carrier to adsorb the cyclodextrins onto the silica carrier. This is a method for mutually separating cyclodextrins, which consists of then fractionating and eluting the adsorbed cyclodextrins using hot water.

The chemically modified silica carrier used in the method of the present invention has a structure in which the silica 7-l group of the silica sol is substituted with a CB C10 linear alkylsilyl group. Particularly preferred is a silica support in which C1l is substituted with a linear alkylsilyl group such that the carbon content is 7-20%. In addition, the remaining silica gel (silica gel substituted with an octadecylsilyl group) may be further substituted with a trimethylsilyl group (endcapping), or further substituted with an octylsilyl group instead of the octadecylsilyl group. It is also possible to use structures.

These chemically modified silica carriers can be produced by reacting silica sol with furkylchlorosilane and, if desired, reacting the reaction product with trimethylchlorosilane. Already has Preparative Boot 〇18 (Waters Company product), YMC-ODS-ALL%YMC-
Since they are commercially available under trade names such as GEL-C8 (all products manufactured by Yamamura Kagaku Kenkyusho), it is convenient to purchase and use them as appropriate.

According to the invention, the contacting of the cyclodextrin mixture with the chemically modified silica support can take place in various ways.

The most preferred method is to flow a solution of cyclodextrins through a column filled with a chemically decorated silica carrier, but it is also possible to use a method in which a silica carrier is added to the solution and mixed.

In the solution of the cyclodextrin mixture that is brought into contact with the chemically modified silica support, α-cyclodextrin, β-
Cyclodextrin and γ-cyclodextrin may be contained in any ratio, and glucose, oligosaccharides, etc. may also be mixed. The content of cyclodextrins in the solution may be as low as 1% or less, and enzymes and the like may be included in the solution as long as they do not inhibit the adsorption of cyclodextrins. In any of the above methods, the concentration of the solution brought into contact with the chemically modified silica support ranges from 1% or less (good quality) to 6.
It can be used in a very wide range up to a high quality of 0% or higher.

According to the present invention, cyclodextrins adsorbed on a chemically modified silica carrier are eluted as follows.

First, if necessary, the chemically modified silica carrier on which the cyclodextrins have been adsorbed is washed with a predetermined amount (2-3 times the volume of the carrier) of cold water to remove coexisting glucose and oligosaccharides, and then the temperature of the eluate is The temperature is raised continuously from about 20°C to 80°C, usually from about 30°C to 60°C, and then the liquid is passed through the column to elute the cyclodextrins.

According to the present invention, the cyclodextrins adsorbed on the chemically modified silica carrier become γ as the temperature of the eluate increases.
-cyclodextrin is eluted first, then β-
Cyclodextrin is eluted, followed by α-cyclodextrin.

The rate of elution of cyclodextrins from a chemically modified silica support varies somewhat depending on the type of silica support used, so the temperature of the eluate may be adjusted to around 10-20°C depending on the chemistry used (l) Therefore, in the present invention, while taking the above points into consideration, the temperature of the eluate (hot water) is appropriately adjusted according to the cyclodextrin composition in the raw material solution, and the temperature is preferably It is necessary to obtain an elution pattern.

In addition, in the present invention, as described above, the eluent (warm water)
In addition to the so-called gradient elution method, in which elution is performed by continuously increasing the temperature of the eluate (hot water) between approximately 20 and 80°C, a method in which elution is performed by increasing the temperature of the eluate (warm water) in stages is used. It is possible.

The amount of eluent used for elution is usually several tens of times the volume of the chemically decorated silica carrier.

In the present invention, the flow rate of the eluate has almost no effect on the separation efficiency and can be carried out at any speed, but the range of 5V = 3-30 is usually selected in consideration of work efficiency etc. .

The eluate from the column is fractionated into a 7 lac collector. In the elution of the present invention, as described above, γ-cyclodextrin is eluted first, followed by β-cyclodextrin and a-cyclodextrin, so the eluate collected in each 7-cyclodextrin collector is Confirm the cyclodextrin composition by HPLC etc., collect the same components, and use WkML.
-1 If necessary, it can be further dried into a powder. In addition, in the case of large-scale processing, similar fractionation/separation can be performed using a fractionator using a peak fraction using an RI detector or the amount of eluted a.

Further, by repeating the above-described adsorption/separation/elution operations on the cyclodextrins thus separated, it is possible to obtain cyclodextrins with even higher purity.

[Effects of the Invention] According to the present invention, it is possible to efficiently separate α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin from each other without using any growth solvent, and moreover, it is possible to efficiently separate α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin with extremely high purity. It is characterized by being able to obtain cyclodextrins. Therefore, the present invention is extremely useful for industrially producing high-purity cyclodextrins.

[Example] Next, an example will be shown to explain the present invention in more detail and concretely.

Example 1 Dissolution of a cyclodextrin mixture obtained by treating 100 g of commercially available cyclodextrin powdered candy (Dixie Pearl KM-50, manufactured by Shisui Minato Seito KK) with glucoamylase.
e, (sugar composition: a-cyclodextrin 27.8%,
β-cyclodextrin 14.3%, γ-cyclodextrin 3.9%, sugars such as glucose 54.0%)
is adjusted to a concentration of 21.0%.

Next, convert 8 g of this sugar solution into octadecylsilyl (ODS).
column (diameter 2.0 ctaXl6, Ocn, carrier amount 8 g), and the cyclodextrins are adsorbed onto the carrier. Next, water 40a+I is passed through the column at 5V=10 to elute and remove coexisting sugars such as glucose. Next, while passing the liquid at the same rate, the temperature of the eluate in the column was gradually raised to around 60'(:'), and the
The cyclodextrins deposited on the lJi are fractionated and eluted (the eluate is fractionated into 2.7 ml portions using a 72 ml collector).

The sugar composition of each 7-lactone fractionated above was
The elution pattern shown in Figure 1 was obtained by confirming (measurement) with C (in the figure, the solid line ■ represents α-cyclodextrin;
The dotted line ■ represents β-cyclodextrin, the chain line ■ represents γ-cyclodextrin, and the solid line ■ represents the temperature at which the eluate passes through the column. ).

Next, each of these 7-lactones was divided according to its cyclodextrin composition, concentrated and dried to obtain five types of powder. The yield and composition of each of these powders are f5
It is shown in Table 1. In addition, 111.6 mg of β-cyclodextrin (purity 98
．． 5%).

Table 1 (Note: CD in the table is an abbreviation for cyclodextrin.)
(Margin below)

[Brief explanation of the drawing]

FIG. 1 is a curve diagram showing the elution pattern of the cyclodextrins obtained in Example 1. Patent applicant Nikken Chemical Co., Ltd.

Claims (1)

[Claims] Cyclodextrin interaction characterized by bringing a cyclodextrin mixture into contact with a chemically modified silica carrier, adsorbing the cyclodextrins in the liquid onto the silica carrier, and then separately eluting the adsorbed cyclodextrins with hot water. Separation method.