JPH04273058A - Cellulosic gel filtering filler - Google Patents

Abstract

PURPOSE:To be adaptable for quickly decomposing water-soluble low molecular weight material mixture by using II type selulose crystal phases and selulose non-crystal phases to provide property that distribution coefficient is abruptly changed in a range of specified relatively low molecular weight. CONSTITUTION:II type selulose has the existance of cross linkages between selulose molecular chains in selulose non-crystal phases, apart from selulose particles formed of I type selulose. It contains spherical particles with an average size of 50mum or smaller. When water-soluble high molecular weight material mixture is decomposed, distribution coefficient is abruptly changed in a range of specified molecular weight, for example, such that the distribution coefficient of maltohexaose is 0.25 or less, a difference in distribution coefficient between maltose and maltohexaose or glucose is 0.25 or more and 0.10 or less, respectively, and a separation rate between maltotriose and maltotetraose is 0.4 or more. Such property and a small average particle size are adequate for quickly decomposing and taking up water-soluble low molecular weight material mixture.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cellulose-based gel filtration filler. More specifically, the present invention relates to a cellulose-based gel filtration packing material for separating and fractionating water-soluble low-molecular substance mixtures, which has a property that its partition coefficient changes rapidly within a specific molecular weight range.

0002

BACKGROUND OF THE INVENTION In recent years, granular materials of cellulose or its various derivatives have come to be used as chromatography materials.

[0003] Cellulose carriers used as packing materials for chromatography are designed in terms of particle size, pore size inside the carrier, etc. depending on the purpose of separation.

[0004] Journal of the Chemical Society of Japan 1984 No. 5, 72
On pages 2 to 727, there is a research report on the production of porous cellulose spherical particles from cellulose organic acid esters and the properties of the particles.

[0005] Furthermore, JP-A-57-38801 similarly discloses a method for producing porous cellulose spherical particles from cellulose organic acid ester.

These cellulose carriers for chromatography have been shown to be useful in separating proteins with relatively large molecular weights.

However, the porous cellulose particles produced by these methods have a problem in that the pore size inside the cellulose carrier becomes smaller and the amount of pores also becomes smaller, resulting in lower separation accuracy.

In recent years, ion exchange resins, Gel filtration agents and the like are used, but it is difficult to fractionate substances with a molecular weight in the range of 500 to 1000, such as trisaccharides or more of oligosaccharides, by gel filtration with good separation performance.

[0009] Journal of the Chemical Society of Japan 1981 No. 12, 1
A research report on the production of cellulose spherical gel and its performance in gel chromatography is published on pages 883-1889.

[0010] This report examines the crosslinking effect on cellulose, and reports as a fact that when spherical cellulose is subjected to a crosslinking reaction using epichlorohydrin, the pore size does not become smaller than that of uncrosslinked spherical cellulose. The same report considers that this phenomenon is caused by the destruction of hydrogen bonds due to the crosslinking reaction, which actually enlarges the network structure of cellulose and increases the pore size. That is, it was conventionally understood that cellulose particles with a molecular weight of several thousand or less and a small pore size were difficult to obtain even by crosslinking.

[0011]

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

Another object of the present invention is to provide a cellulose-based gel filtration packing material for high-speed preparative separation, which has the characteristic that the partition coefficient changes rapidly in a specific relatively low molecular weight range.

Further objects and advantages of the invention will become apparent from the following description.

[0014]

According to the present invention, the above objects and advantages of the present invention are achieved by: (a) consisting essentially of a type II cellulose crystalline phase and a cellulose amorphous phase; There are crosslinks between cellulose molecular chains, (
c) it consists essentially of spherical particles with an average particle diameter of less than 50 μm, and (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, and (e) the degree of separation between maltotriose and maltotetraose is 0.40 or more.

[0015] The cellulose gel filtration filler of the present invention is
First, it is composed of a type II cellulose crystalline phase and a cellulose amorphous phase. Therefore, natural cellulose i.e. I
It is completely different from cellulose fine particles made of type cellulose.

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

[0017] The type II cellulose is cellulose obtained by converting cellulose into a single-layer cellulose derivative and then regenerating the cellulose by hydrolysis or the like.

Secondly, crosslinks exist between cellulose molecular chains in the cellulose amorphous phase. Type II cellulose (101
) plane and (002) plane X-ray diffraction angle (2θ)2
It is characterized by the presence of a clear diffraction peak near 0°.

Furthermore, the crosslinking between cellulose molecules existing in the amorphous phase of cellulose involves crosslinking the hydroxyl groups of cellulose molecules with each other via crosslinking agent molecules.

For example, when epichlorohydrin is used as a crosslinking agent, the degree of crosslinking can be determined by the absorbance of an absorption peak attributable to the CH stretching vibration of alkylene in an infrared absorption spectrum measured by the KBr tablet method. The cellulose gel filtration filler of the present invention is from 0.065 to
0.156mg-1. It is preferable to have an absorbance of 0.094 to 0.140 mg-1.cm-2. cm-2
More preferably, it has an absorbance of .

Thirdly, it consists essentially 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 target substances becomes small. In order to increase the degree of separation, it becomes necessary to increase the column length, which increases the separation time, which is undesirable. Moreover, if the average particle diameter is less than 50 μm, the pressure when the eluent is passed through the column becomes high, making it impossible to separate at a high flow rate, which is not preferable.

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

Fourthly, when separating a mixture of water-soluble polymeric substances, the separation coefficient is characterized in that the distribution coefficient changes rapidly within 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.
25 or less, the difference between the partition coefficient of maltose and the partition coefficient of maltohekiose 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 furthermore, the degree of separation between maltotriose and maltotetraose is 0.40 or more.

[0024] The distribution coefficient of maltohexaose is 0.25.
For example, from a mixture with a wide range of molecular weights ranging from several hundred to tens of thousands, it becomes difficult to exclude substances with relatively high molecular weights (several thousand or more). When attempting to separate substances, the required gel bed layer becomes too long, which is undesirable. In other words, when applied as a preparative column, the column length becomes large, and when performing repeated preparative separations, the time per cycle becomes longer and the amount of preparative separation per unit time decreases. Yes, it's not good.

Furthermore, when the difference between the distribution coefficient of maltose and the distribution coefficient of maltohexaose becomes less than 0.25, the distance between the separated peaks of several hundred to several thousand substances in a specific molecular weight range becomes narrow, and the separation becomes difficult. This is not desirable because it makes things worse.

Furthermore, if the difference between the partition coefficient of maltose and the partition coefficient of glucose exceeds 0.10, the required gel bed layer in the low molecular weight range other than the specific molecular weight range of several hundred to several thousand becomes longer. This also increases the column length,
When fractionation is performed repeatedly, the time per cycle becomes longer and the amount of fractionation per unit time decreases, which is not preferable.

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

[0028] The cellulose gel filtration filler of the present invention is
The exclusion limit molecular weight for polyethylene glycol is approximately 1,
000 to about 10,000 are swollen in an aqueous medium containing a basic compound, washed with water, dried, and then swelled in a hydrophilic aprotic organic solvent in the presence of a basic compound. , obtained by crosslinking reaction.

[0029] The exclusion limit molecular weight is about 1,000 to about 1
As fine non-crosslinked cellulose particles in the range of 0,000, for example, those obtained by the manufacturing method described in Japanese Patent Application No. 047149/1998 can be used.

According to the above method, (1) viscose and a water-soluble anionic polymer compound are mixed in the presence of calcium hydroxide to produce a fine particle dispersion of viscose, and (2) ( i) coagulating the viscose in the dispersion by heating said dispersion or mixing said dispersion with a coagulating liquid and then neutralizing with acid to produce fine particles of cellulose; or (ii) as described above. The dispersion is coagulated and neutralized with acid to produce fine particles of cellulose, and then (3) the fine particles of cellulose are separated from the mother liquor and optionally desulfurized, pickled, washed with water, or dried to obtain a sharp particle size distribution. It is possible to obtain non-crosslinked cellulose particles having the following properties.

[0031] 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. This swelling treatment makes it possible to remove the distortion generated inside the non-crosslinked cellulose particles during particle formation, making it possible to make the subsequent crosslinking reaction smooth and easy to control. As mentioned above, conventionally, such microscopic non-crosslinked cellulose particles are
It was believed that crosslinking did not reduce the pore size, expressed as the exclusion limit molecular weight, but rather increased the network structure.

As mentioned above, it is possible to prevent the network structure of cellulose particles from increasing by performing a swelling treatment before carrying out the crosslinking reaction to adjust the swelling property of the cellulose particles during the crosslinking reaction. Some of the inventors of the present invention have disclosed the following.

The crosslinking reaction carried out next can be carried out using a hydrophilic aprotic organic solvent such as dimethyl sulfoxide,
This is carried out by reacting a crosslinking agent such as epichlorohydrin with swelling-treated non-crosslinked cellulose particles in dimethylformamide in the presence of a basic compound such as caustic soda or caustic potassium.

As mentioned above, the cellulose-based gel filtration material of the present invention thus obtained has a small average particle diameter and a small exclusion limit molecular weight, and has a distribution coefficient within a specific molecular weight range of several hundred to several thousand. , changes rapidly (that is, the difference in partition coefficients is large), so it shows good performance for high-speed separation and fractionation of mixtures of water-soluble low-molecular substances such as oligosaccharides.

[0035]

[Examples] The present invention will be explained in detail with reference to Examples below. Before that, methods for measuring various characteristic values in this specification will be described.

Particle Size Measurement Method Approximately 0.1 g of a sample is collected, poured into 25 ml of pure water, stirred and dispersed, and measured using 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 accurately weighed, and about 20 mg of potassium bromide powder (abbreviated as KBr) for infrared absorption spectrum measurement was prepared.
Weigh out exactly 0 mg, and thoroughly knead and crush both in an agate mortar. Next, the cellulose particles and the kneaded and pulverized KBr powder are collected and used in a tablet molding machine to form a KBr tablet for infrared absorption spectrum measurement, and the transmission method 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; the amount of microcellulose particles collected (mg);
M; Amount of KBr powder collected (mg), m
;KBr tablet weight (mg), A ;C
Absorbance of the H stretching absorption band, T(p); transmittance (%) at the peak top of the CH stretching absorption band, T(b): transmittance (%) of the baseline at the peak wave number of the CH stretching absorption band. However, the baseline is 2000-4
In the range of 000 cm-1, the peak bases of both the OH stretching absorption band and the CH stretching absorption band are drawn so as to be scooped at the contact point. Ao: absorbance of CH stretching absorption band per 1 mg of microcellulose particles, when

[0039]

[Formula 1] It is expressed as

A jacketed resin column with a distribution coefficient of 16 mmφ x 35 cm was filled with water at a flow rate of 3 ml/min for 60 minutes.
Fill with gel. After the column temperature was maintained at 60°C and the column was stabilized, each elution volume was determined using maltooligosaccharides with known molecular weights, and the partition coefficient Kn defined by the following formula was calculated.

[0041]

[Formula 2]

[0042] Vn: Elution volume of maltooligosaccharide (ml)
, Vo: elution volume (ml) of blue dextran (molecular weight 2 million), Vt: column internal volume (ml).

The analysis conditions are as follows.

1 Sample size 100mg
/ml, 2 μl, 2 eluent pure water, 3 temperature
60℃, 4 flow rate
0.5 ml/min, 5 detectors
RI detector, 6
Pump: LC-6A type manufactured by Shimadzu Corporation.

Separation degree The separation degree R of maltotriose and maltotetraose was calculated from the following formula under the same conditions as the method used to calculate the partition coefficient above.

[0046]

[Formula 3]

V3 and V4 represent the elution volumes from the time of sample injection until the maximum peak value is obtained. V3: Elution volume of maltotriose (ml), V4: Elution volume of maltotetraose (ml), W3 and W4 indicate the width of the peak on the baseline, W
3: Elution volume of maltotriose peak width (ml)
, W4: Elution volume of maltotetraose peak width (
ml).

Example 1 (1). 500g of pulp made from coniferous wood was heated at 20℃, 18
Immersed in 20 liters of wt% caustic soda solution for 1 hour, 2.8
Squeezed twice. The mixture was pulverized for 1 hour while increasing the temperature from 25°C to 50°C for aging, and then 35% by weight of carbon disulfide (175 g) was added to the 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 6200 centipoise.

(2). 3200g of 10.8% sodium polyacrylate aqueous solution (molecular weight 100,000) in a 5L plastic container
, 130 g of calcium carbonate and 40 g of calcium hydroxide
g and stirred with a homomixer. After dispersion, the liquid temperature is 25
800 g of the above viscose liquid was added at 0.degree. C., and stirred at 500 rpm for 10 minutes using a lab stirrer. After producing viscose fine particles, the liquid temperature was raised from 25°C to 75°C over 25 minutes while stirring, and the mixture was heated to 75°C.
The temperature was maintained at 5° C. for 15 minutes to solidify the viscose microparticles. The coagulated viscose particles were separated from the mother liquor using a G4 type glass filter, and then neutralized with 5% by weight hydrochloric acid to obtain cellulose particles, which were washed with a large excess of water and then dried in a ventilation dryer at 105° C. for 5 hours. The obtained cellulose particles have an exclusion limit molecular weight of 6 by polyethylene glycol.
It was 000.

(3). Next, the cellulose particles 200
After swelling g in a 10% by weight aqueous sodium hydroxide solution at 20°C and separating it from the mother liquor through a glass filter,
Washed with water and dried.

(4). Next, the cellulose particles 100
g, 1000 g of dimethyl sulfoxide and 200 g of epichlorohydrin were placed in a 2 liter flask, and after stirring the cellulose particles sufficiently, 15 g of caustic soda was added.
315 g of an aqueous solution of caustic soda dissolved therein was gradually added little by little. Addition of the caustic soda aqueous solution was carried out over 1 hour while adjusting the liquid temperature to 20°C.

[0052] Next, the liquid temperature was raised to 50°C in 1 hour, and thereafter the crosslinking reaction was completed while being controlled at 50°C. The produced fine crosslinked cellulose particles were separated from the mother liquor by a glass filter, and then neutralized with 1% by weight hydrochloric acid.
Washed with large excess of water.

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

[0054] The crosslinked cellulose particles had an inner diameter of 16 mm,
It was packed into a column with a length of 35 cm to separate maltooligosaccharides.

Table 1 shows the partition coefficients of malto-oligosaccharides, the difference in partition coefficients, and the degree of separation between maltotriose and maltotetraose.

[0056]

[Table 1]

[0057] The crosslinked cellulose particles had an inner diameter of 22 mm,
Packed into a column with a length of 60 cm, at room temperature, with 0 pure water eluent.
．． As a result of separating the laminarioligosaccharide mixture under conditions of 5 ml/min, 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 malto-oligosaccharides, the difference in the distribution coefficients, and the degree of separation between maltotriose and maltotetraose of the crosslinked cellulose particles.

[0060]

[Table 2]

[0061] The crosslinked cellulose particles had an inner diameter of 22 mm,
Packed into a column with a length of 60 cm, at room temperature, with 0 pure water eluent.
．． As a result of separating the dextran hydrolyzate under conditions of 3 ml/min, oligosaccharide components having a molecular weight of 1000 or less were successfully separated.

Comparative Example 1 Crosslinked cellulose particles with an average particle diameter of 32 μm obtained in Example 1 by performing the crosslinking treatment in step (4) without undergoing the swelling treatment in a caustic soda aqueous solution in step (3). The characteristics are shown in Table 3.

[0063]

[Table 3]

[0064] As in Example 1, the inner diameter is 22 mm and the length is 60 mm.
As a result of separating the laminarioligosaccharide mixture using a cm column, the peak width of each component became broad, and good separation could not be achieved.

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

Table 4 shows the maltooligosaccharide distribution characteristics of the crosslinked cellulose particles.

[0067]

[Table 4]

[0068] As in Example 1, the inner diameter was 22 mm and the length was 60 mm.
As a result of separating the laminarioligosaccharide mixture using a cm column, the peak width of each component became broad, and good separation could not be achieved.

[0069]

Effects of the Invention The cellulose-based gel filtration agent of the present invention has a partition coefficient that changes rapidly within a specific molecular weight range. When used in preparative applications, it achieves high performance separation. Furthermore, since the gel filtration agent of the present invention has excellent safety and chemical stability, it can be widely used as a preparative separation material in the food and pharmaceutical fields.

Claims (1)

[Claims] Claim 1: (a) consists essentially of a type II cellulose crystalline phase and a cellulose amorphous phase, (b) the cellulose amorphous phase has crosslinks between cellulose molecular chains, and (c) the average particle size is consists essentially of spherical particles smaller than 50 μm, (d) the distribution coefficient of maltohexaose is 0.25 or less, and the difference between the distribution coefficient of maltose and the distribution coefficient of maltohexaose is 0.25 or more; and (e) a cellulosic system characterized by having a difference between the partition coefficient of maltose and the partition coefficient of glucose of 0.10 or less, and (e) a degree of separation between maltotriose and maltotetraose of 0.40 or more. Gel filtration filler.