JP3142065B2 - Cellulose gel filtration filler - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a cellulosic gel filtration filler. More specifically, the present invention relates to a gel filtration filler for separation and preparative separation of a water-soluble low-molecular substance mixture, which has a characteristic that a distribution coefficient changes rapidly in a specific molecular weight range.

[0002]

2. Description of the Related Art Granules of cellulose or various derivatives thereof have recently been used as chromatography materials.

[0003] The particle size, pore size inside the carrier and the like of the cellulose carrier used as a packing material for chromatography are designed according to the purpose of separation.

The Chemical Society of Japan, 1984, No. 5, 722
On page 727, there is a report on the production of porous cellulose spherical particles from cellulose organic acid esters and on the properties of the particles.

Japanese Patent Application Laid-Open No. 57-38801 similarly discloses a method for producing porous cellulose spherical particles from a cellulose organic acid ester.

[0006] These cellulose supports for chromatography have been shown to be useful for the separation of proteins having a relatively large molecular weight.

[0007] However, the porous cellulose particles produced by these methods have a problem that the pore size inside the cellulose carrier is reduced and the amount of the pores is reduced, thereby lowering the separation accuracy.

In recent years, ion-exchange resins have been used to separate and fractionate products having a specific molecular weight range from a mixture of water-soluble low-molecular substances (molecular weight of several thousand or less), for example, a mixture of oligosaccharides and a mixture of amino acids and peptides. Gel filtration agents and the like are used. For example, it is difficult to fractionate a substance having a molecular weight of 500 to 1000, such as oligosaccharide trisaccharide or more, with good separation performance by gel filtration.

The Chemical Society of Japan 1981 No 12, 188
On pages 3 to 1889, there are reports on the production of cellulose spherical gel and its performance on gel chromatography.

In this report, the crosslinking effect on cellulose is examined. It is reported that the cross-linking reaction of spherical cellulose with epichlorohydrin does not reduce the pore size of uncrosslinked spherical cellulose. The report argues that this phenomenon is due to the fact that the hydrogen bond is broken by the crosslinking reaction, and instead the network structure of cellulose is enlarged and the pore size is increased. That is, it has been conventionally understood that cellulose particles having a small pore size having a molecular weight of several thousands or less are difficult to obtain even by crosslinking.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide a cellulosic gel filtration filler suitable for high-speed separation of a mixture of water-soluble low-molecular substances.

It is another object of the present invention to provide a cellulosic gel filtration filler for high-speed preparative separation, which has a characteristic that the distribution coefficient changes abruptly in a specific relatively low molecular weight range.

[0013] Still other objects and advantages of the present invention will become apparent from the following description.

[0014]

According to the present invention, the above objects and advantages of the present invention are substantially achieved by (a) a type II cellulose crystalline phase and a cellulose amorphous phase, and (b) a cellulose amorphous phase. Has cross-links between the cellulose molecular chains, (c) is substantially composed of spherical particles having an average particle diameter of less than 50 μm, (d) the distribution coefficient of maltohexaose is 0.25 or less, maltose The difference between the partition coefficient of maltohexaose and that of maltohexaose is greater than or equal to 0.25, and the difference between the partition coefficient of maltose and that of glucose is less than or equal to 0.10, and (e) maltotriose and maltotetraose. And a cellulosic gel filtration filler characterized by having a degree of separation of 0.40 or more.

The cellulose-based gel filtration filler of the present invention comprises:
First, it is composed of a type II cellulose crystal phase and a cellulose amorphous phase. Therefore, natural cellulose or I
It is completely different from cellulose fine particles composed of type cellulose.

As is well known, type II cellulose and type I cellulose are distinguished by X-ray diffraction. In the X-ray diffraction diagram of the type II cellulose, there is substantially no diffraction peak at a diffraction angle (2θ) of 17 ° clearly present in the type I cellulose.

The type II cellulose refers to cellulose obtained by converting cellulose into a single cellulose derivative and then regenerating the cellulose by hydrolysis or the like.

Second, in the amorphous phase of cellulose, crosslinks exist between cellulose molecular chains. Type II cellulose (10
X-ray diffraction angles (2θ) derived from 1) plane and (002) plane
It is characterized in that a diffraction peak around 20 ° is clearly present.

The crosslinking between the cellulose molecules present in the amorphous phase of the cellulose bridges the hydroxyl groups of the cellulose molecules via the crosslinking agent molecules.

For example, when epichlorohydrin is used as a cross-linking agent, the degree of cross-linking can be specified by the absorbance of an absorption peak attributed to CH stretching vibration of alkylene in an infrared absorption spectrum by a KBr tablet method. The cellulosic gel filtration filler of the present invention is 0.065.
Preferably has an absorbance of ~0.156mg ^-1 .cm ^-2, and more preferably has an absorbance of 0.094~0.140mg ^-1 .cm ^-2.

Third, the particles substantially consist of spherical particles having an average particle diameter of less than 50 μm. When the average particle diameter is 50 μm or more, the separation peak becomes broad, and the degree of separation between the target substances decreases. In order to increase the degree of separation, it is necessary to increase the length of the column, which undesirably increases the separation time. On the other hand, when the average particle size is 50 μm or less, the pressure when the eluent is passed through the column increases, and it is not preferable because separation cannot be performed at a high flow rate.

In order to increase the degree of separation, it is preferable that the average particle size is small. In addition, if the particle size distribution is large, there are many problems such as an increase in column pressure and a decrease in degree of separation as described above. Those having a particle size distribution in which 90% of the total number of particles are present within a particle size range of ± 20% are particularly preferably used.

Fourth, when a water-soluble polymer mixture is separated, it is characterized in that the distribution coefficient changes abruptly in a specific molecular weight range. For example, when a mixture of maltooligosaccharides is separated into each peak, it has the following characteristics. That is, the distribution coefficient of maltohexaose is 0.1.
25 or less, the difference between the partition coefficient of maltose and the partition coefficient of maltohexose is 0.25 or more, and the difference between the partition coefficient of maltose and the partition coefficient of glucose is 0.1.
0 or less, and the degree of separation between maltotriose and maltotetraose is 0.40 or more.

The distribution coefficient of maltohexaose is 0.25
When the molecular weight is higher, it is difficult to exclude relatively high molecular weight substances (thousands or more). For example, from a mixture having a wide molecular weight range of several hundreds to tens of thousands, a specific molecular weight of several hundred to several thousand When trying to separate substances, the required gel bed layer becomes too long, which is undesirable. In other words, when applied as a preparative column, there are disadvantages such as an increase in the column length, and when performing repetitive preparative separation, the time per cycle becomes longer and the amount of preparative per unit time decreases. There is not preferred.

When the difference between the distribution coefficient of maltose and the distribution coefficient of maltohexaose is less than 0.25, the distance between the separation peaks of hundreds to thousands of substances having a specific molecular weight range becomes narrow, and the separation becomes difficult. It is not preferable because it becomes worse.

Further, if the difference between the distribution coefficient of maltose and the distribution coefficient of glucose exceeds 0.10, the required gel bed layer in the low molecular weight region other than the specific molecular weight range of several hundred to several thousand becomes long, This also increases the column length,
When repetitive fractionation is performed, it is not preferable because the time per cycle becomes long and the fractionated quantity per unit time decreases.

The cellulose-based gel filtration filler of the present invention can be produced, for example, by the following production method. The manufacturing method will be described.

The cellulose-based gel filtration filler of the present invention comprises:
The exclusion limit molecular weight of polyethylene glycol is about 1,
The fine non-crosslinked cellulose particles in the range of 000 to about 10,000 are swelled in an aqueous medium containing a basic compound, washed with water, dried, and then in a hydrophilic aprotic organic solvent in the presence of the basic compound. , By a cross-linking reaction.

The exclusion limit molecular weight is about 1,000 to about 1
As the fine non-crosslinked cellulose particles in the range of 000, those obtained by the production method described in Japanese Patent Application No. 2-47149 can be used, for example.

According to the above method, (1) viscose and a water-soluble anionic polymer compound are mixed in the presence of calcium hydroxide to form a viscose fine particle dispersion, and (2) ( i) heating the dispersion or mixing the dispersion with a coagulation liquid to coagulate the viscose in the dispersion and then neutralizing with an acid to produce cellulose fine particles; or (ii) The dispersion is coagulated and neutralized with an acid to form cellulose fine particles, and then (3) the cellulose fine particles are separated from the mother liquor and, if necessary, desulfurized, pickled, washed or dried to obtain a sharp particle size distribution. Can be obtained.

Such fine non-crosslinked cellulose particles are
For example, it is subjected to a swelling treatment in an aqueous medium containing a basic compound such as an aqueous solution of caustic soda. By this swelling treatment, the strain generated during particle formation inside the non-crosslinked cellulose particles can be removed, and the subsequent crosslinking reaction can be smoothly and easily controlled. As described above, it has heretofore been considered that such fine non-crosslinked cellulose particles do not reduce the pore size expressed as the exclusion limit molecular weight by crosslinking, but rather increase the network structure.

As described above, it is possible to prevent the network structure of the cellulose particles from being enlarged by performing the swelling treatment before the crosslinking reaction to adjust the swellability of the cellulose particles during the crosslinking reaction. Some inventors of the present invention have clarified this.

The crosslinking reaction to be carried out next is carried out by a hydrophilic aprotic organic solvent such as dimethyl sulfoxide,
The reaction is carried out by reacting a cross-linking agent such as epichlorohydrin with the swelled non-cross-linked cellulose particles in dimethylformamide in the presence of a basic compound such as caustic soda and caustic potash.

As described above, the thus obtained cellulose gel filtration material of the present invention has a small average particle size, a small exclusion limit molecular weight, and a distribution coefficient in a range of several hundreds to several thousands of specific molecular weights. Because of the rapid change (that is, the difference in distribution coefficient is large), it shows good performance for high-speed separation and fractionation of a mixture of water-soluble low-molecular substances such as oligosaccharides.

[0035]

The present invention will be described below in detail with reference to examples. Prior to that, methods for measuring various characteristic values in this specification will be described.

Particle Size Measurement Method About 0.1 g of a sample is taken, put into 25 ml of pure water, dispersed by stirring, and measured with a light transmission type particle size distribution analyzer. The average particle diameter was calculated on a volume basis.

Absorbance of CH stretching absorption band: 1 to 3 mg of fine cellulose particles were precisely weighed, and potassium bromide powder (abbreviated as KBr) for infrared absorption spectrum measurement was about 200 μm.
mg is precisely weighed, and both are well kneaded and ground in an agate mortar.
Next, the KBr powder kneaded and kneaded with the cellulose particles is scraped and collected, and a KBr tablet for measuring an infrared absorption spectrum is measured by a tableting machine, and a transmission infrared absorption spectrum is measured. In the obtained infrared spectrum, 2800-30
The absorbance of the CH stretching absorption band having a peak at 00 cm ^-1 is determined and converted to the absorbance per 1 mg of cellulose.

That is, w: collection amount of microcellulose particles (mg), M: collection amount of KBr powder (mg), m: weight of KBr tablet (mg), A: absorbance of CH stretching absorption band, T (p): CH stretching Transmittance (%) at the absorption band peak top, T (b); Baseline transmittance (%) at the CH stretching absorption band peak wavenumber. However, the baseline is 2000-4
In the range of 000 cm ⁻¹ , the peaks of both the OH stretching absorption band and the CH stretching absorption band are scooped at the contact point. Ao: Absorbance of CH stretching absorption band per 1 mg of fine cellulose particles,

[0039]

(Equation 1) Is represented by

A gel is packed into a resin column with a distribution coefficient of 16 mmφ × 35 cm at a flow rate of 3 ml / min for 60 minutes using water as a filling liquid. After the temperature in the column was maintained at 60 ° C. and the column was stabilized, each elution volume was determined using a maltooligosaccharide having a known molecular weight, and a partition coefficient Kn defined by the following equation was calculated.

[0041]

(Equation 2)

Vn: elution volume (ml) of maltooligosaccharide, Vo: elution volume (ml) of blue dextran (molecular weight 2,000,000), Vt: column internal volume (ml).

The analysis conditions are as follows.

1 Sample size 100 mg / ml, 2 μ
1, 2 eluent pure water, 3 temperature 60 ° C, 4 flow rate 0.5 ml / min, 5 detector RI detector, 6 pump LC manufactured by Shimadzu Corporation
-6A type.

Degree of Separation The degree of separation R between maltotriose and maltotetraose was calculated from the following equation under the same conditions as in the method for determining the distribution coefficient.

[0046]

(Equation 3)

V ₃ and V ₄ represent the elution volumes from the time of sample injection until the maximum value of the peak is obtained. V ₃ : elution volume of maltotriose (ml), V ₄ : elution volume of maltotetraose (ml), and W ₃ and W ₄ indicate the widths of the peaks on the baseline. W ₃ : maltotriose Elution volume of peak width (ml), W ₄ : maltotetraose Elution volume of peak width (m
l).

Embodiment 1 (1). 500 g of softwood pulp at 20 ° C, 18
Immersed in 20 liters of a ca.
Squeezed twice. The mixture was pulverized for 1 hour while raising the temperature from 25 ° C. to 50 ° C., aged, and then added with 35% by weight of carbon disulfide (175 g) based on cellulose, and sulfurized at 25 ° C. for 1 hour to obtain cellulose xanthate. . The xanthate was dissolved in an aqueous solution of caustic soda to obtain viscose. The viscose had a cellulose concentration of 9.3% by weight, a caustic soda concentration of 5.9% by weight, and a viscosity of 6,200 centipoise.

(2). 10.8% aqueous solution of sodium polyacrylate (molecular weight 100,000) 3200 in a 5 liter plastic container
g, calcium carbonate 130 g and calcium hydroxide 40
g and stirred with a homomixer. After dispersion, liquid temperature 25
At 800C, 800 g of the above viscose liquid was added, and the mixture was stirred at 500 rpm for 10 minutes with a lab stirrer. After generating the viscose fine particles, the liquid temperature was raised from 25 ° C. to 75 ° C. in 25 minutes while continuously stirring,
The viscose fine particles were solidified by maintaining at 15 ° C. for 15 minutes. The coagulated viscose particles were separated from the mother liquor by a G4 type glass filter, neutralized with 5% by weight hydrochloric acid to obtain cellulose particles, washed with a large excess of water, and dried with a ventilation dryer at 105 ° C. for 5 hours. The obtained cellulose particles have an exclusion limit molecular weight of 6 by polyethylene glycol.
000.

(3). Next, the cellulose particles 200
g was swelled in a 10% by weight aqueous solution of caustic soda at 20 ° C. and then separated from the mother liquor by a glass filter.
Washed and dried.

(4). Next, the cellulose particles 100
g, dimethyl sulfoxide 1000 g and epichlorohydrin 200 g were placed in a 2 liter flask, and the cellulose particles were sufficiently swollen under stirring. Then, 315 g of an aqueous solution of caustic soda in which 15 g of caustic soda was dissolved was gradually added. The addition of the aqueous sodium hydroxide solution was performed over 1 hour while adjusting the liquid temperature to 20 ° C.

Next, the liquid temperature was raised to 50 ° C. in one hour, and thereafter the crosslinking reaction was completed while adjusting the temperature to 50 ° C. The resulting finely crosslinked cellulose particles are separated from the mother liquor by a glass filter and then neutralized with 1% by weight of hydrochloric acid,
Washed with a large excess of water.

The obtained crosslinked cellulose particles had an average particle size of 30 μm, and the absorbance of the absorption peak attributable to the CH stretching vibration of alkylene in the infrared absorption spectrum obtained by the KBr tablet method was 0.009 mg ⁻¹ .cm ⁻² .

The crosslinked cellulose particles were packed in a column having an inner diameter of 16 mm and a length of 35 cm to separate maltooligosaccharides.

Table 1 shows the distribution coefficient of maltooligosaccharides, the difference between the distribution coefficients, and the degree of separation between maltotriose and maltotetraose.

[0056]

[Table 1]

The above-mentioned crosslinked cellulose particles were packed in a column having an inner diameter of 22 mm and a length of 60 cm.
As a result of separating the laminarioligosaccharide mixture under the l / min condition, laminaritriose and laminaripentaose could be completely separated into two peaks.

Example 2 Crosslinked cellulose particles were obtained under the same conditions as in Example 1 except that the average particle diameter was changed to 17 μm.

Table 2 shows the distribution coefficient of maltooligosaccharides in the crosslinked cellulose particles, the difference in distribution coefficient, and the degree of separation between maltotriose and maltotetraose.

[0060]

[Table 2]

The above-mentioned crosslinked cellulose particles were packed in a column having an inner diameter of 22 mm and a length of 60 cm.
As a result of separating the dextran hydrolyzate under the condition of 1 / min, the separation of the oligosaccharide component having a molecular weight of 1,000 or less was good.

Comparative Example 1 In Example 1, crosslinked cellulose particles having an average particle diameter of 32 μm obtained by performing the crosslinking treatment in the step (4) without performing the swelling treatment in the aqueous sodium hydroxide solution in the step (3) Are shown in Table 3.

[0063]

[Table 3]

As in Example 1, the inner diameter was 22 mm and the length was 60 cm.
As a result of separating the laminari-oligosaccharide mixture on the column,
The peak width of each component became broad, and good separation was not possible.

Comparative Example 2 Crosslinked cellulose particles were obtained under the same conditions except that the average particle diameter was changed to 120 μm in Example 1.

The distribution characteristics of maltooligosaccharides of the crosslinked cellulose particles are shown in Table 4.

[0067]

[Table 4]

As in Example 1, the inner diameter was 22 mm and the length was 60 cm.
As a result of separating the laminari-oligosaccharide mixture on the column,
The peak width of each component became broad, and good separation was not possible.

[0069]

EFFECT OF THE INVENTION Since the partition coefficient of the cellulose gel filtration agent of the present invention rapidly changes in a specific molecular weight range, oligosaccharides, which are water-soluble substances having a molecular weight of several thousand or less, are separated by gel filtration. When used for preparative applications, high performance separations are achieved. Further, the gel filtration agent of the present invention is excellent in safety and chemical stability, so that it can be widely used as a separating material for preparative separation in the food and pharmaceutical fields.

Claims (1)

(57) [Claims] Claims: 1. An amorphous cellulose phase comprising (a) a type II cellulose crystalline phase and a cellulose amorphous phase, (b) a cellulose amorphous phase having crosslinks between cellulose molecular chains, and (c) an average particle diameter of Substantially consisting of spherical particles smaller than 50 μm, (d)
The distribution coefficient of maltohexaose is 0.25 or less,
The difference between the partition coefficient of maltose and the partition coefficient of maltohexaose is 0.25 or more, and the difference between the partition coefficient of maltose and the partition coefficient of glucose is 0.10 or less;
(E) A cellulose gel filtration filler characterized in that the degree of separation between maltotriose and maltotetraose is 0.40 or more.