JPH088984B2 - Cellulose gel filtration filler - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cellulosic gel filtration packing material. More specifically, it relates to a gel filtration filler for separation / separation of a mixture of water-soluble low-molecular substances, and a cellulose-based gel filtration filler having a characteristic that the distribution coefficient rapidly changes 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.

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

Journal of the Chemical Society of Japan 1984 No5, 722-
On page 727, a research report on the production of porous cellulose spherical particles from a cellulose organic acid ester and the properties of the particles is given.

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

These cellulose carriers for chromatography have been shown to be useful for separating proteins having a relatively large molecular weight.

However, the porous cellulose particles produced by these methods have a problem that the pore size inside the cellulose carrier becomes small and the amount of the pores also becomes small, so that the separation accuracy decreases.

In recent years, in order to separate / separate a product having a specific molecular weight range from a mixture of water-soluble low-molecular substances (molecular weight of several thousand or less) such as a mixture of oligosaccharides and a mixture of amino acids / peptides, an ion exchange resin, Gel filtration materials and the like are used. For example, it is difficult to fractionate gel filtration with good separation performance for substances having a molecular weight of 500 to 1000 such as oligosaccharide trisaccharides or more.

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

In this report, the cross-linking effect on cellulose was examined, and it was reported as a fact that the cross-linking reaction of spherical cellulose with epichlorohydrin did not result in a pore size smaller than that of uncross-linked spherical cellulose. In the same report, it is considered that this phenomenon is because the hydrogen bond is broken by the cross-linking reaction, and the network structure of cellulose is rather increased and the pore size is increased. That is, it was conventionally understood that cellulose particles having a molecular weight of several thousand or less and a small pore size 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 packing material suitable for industrially separating a mixture of water-soluble low molecular weight substances.

[0012] Another object of the present invention is to provide a cellulosic gel filtration packing material for industrial fractionation, which has a characteristic that the partition coefficient changes rapidly in a specific relatively low molecular weight range. .

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

[0014]

According to the present invention, the above objects and advantages of the present invention consist essentially of (a) a type II cellulose crystalline phase and a cellulose amorphous phase, and (b) a cellulose amorphous phase. Has crosslinks between cellulose molecular chains, (c) is substantially composed of spherical particles having an average particle size of 50 to 300 μm, and (d) the distribution coefficient of maltohexaose is 0.25 or less, and maltose is The difference between the distribution coefficient of malthexaose and that of maltohexaose is 0.25 or more, and the difference between the distribution coefficient of maltose and that of glucose is 0.10 or less, and (e) maltotriose and maltotetra This is achieved by a cellulosic gel filtration filler characterized by having a degree of separation from aus of 0.35 or more.

The cellulosic gel filtration filler of the present invention comprises
First, it is composed of a type II cellulose crystalline 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, the diffraction peak with a diffraction angle (2θ) of 17 ° clearly present in the type I cellulose is substantially absent.

The type II cellulose is cellulose obtained by converting cellulose to a cellulose-bearing derivative and then regenerating the cellulose by hydrolysis or the like.

Secondly, the cellulose amorphous phase has crosslinks between cellulose molecular chains. Type II cellulose (10
X-ray diffraction angle (2θ) derived from 1) plane and (002) plane
It is characterized in that there is a clear diffraction peak near 20 °.

Crosslinking between the cellulose molecules existing in the amorphous phase of cellulose bridges the hydroxyl groups of the cellulose molecules through the crosslinking agent molecules.

The degree of crosslinking can be specified by the absorbance of the absorption peak attributed to CH stretching vibration of alkylene in the infrared absorption spectrum by the KBr tablet method, when epichlorohydrin is used as a crosslinking agent. 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.

Thirdly, it consists essentially of spherical particles having an average particle size of 50 to 300 μm. If the average molecular diameter exceeds 300 μm, the separation peak becomes broad and the degree of separation between the target substances becomes small, which is not preferable. 50 μm
When used in an industrial preparative column, for example, a simulated moving bed type column, since a large amount of liquid is circulated, the fluid loss pressure increases, which not only increases equipment costs, but also increases the concentration due to each separation layer. Since it changes, the pressure loss also greatly differs in each layer, and it becomes difficult to set operating conditions such as the flow rate of each part. When used as an industrial preparative column, there are restrictions on using such a small particle size column, and a column having a particle size of 100 μm or more is more preferable.

However, as a matter of course, when the particle size becomes large, the separation peak becomes broad and the specific molecular weight of 2
In order to increase the degree of separation between two substances, it is necessary to increase the distance between the separation peaks, and therefore a particle having a too large particle size is not preferable. The particle size is preferably 200 μm or less.

Fourthly, when the water-soluble polymer substance mixture is separated, the partition 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.
25 or less, the difference between the maltose distribution coefficient and the maltohexose distribution coefficient is 0.25 or more, and the difference between the maltose distribution coefficient and the glucose distribution coefficient is 0.1.
It has a characteristic that the separation degree between maltotriose and maltotetraose is 0.35 or more.

The distribution coefficient of maltohexaose is 0.25.
If it exceeds, it is difficult to exclude substances having a relatively high molecular weight (thousands or more), and therefore, for example, from a wide range of mixtures having molecular weights of hundreds to tens of thousands, a specific molecular weight of hundreds to thousands of When trying to separate substances, the gel bed layer required is too long, which is undesirable. In other words, when applied as an industrial preparative column, the column length becomes long and the apparatus becomes large, and in the case of repeated preparative columns, the time per cycle becomes long and the productivity decreases. Is not preferred.

If 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 several hundreds to several thousands of substances in a specific molecular weight range becomes narrow, and the separation occurs. It is not preferable because it gets worse.

Further, when the difference between the distribution coefficient of maltose and the distribution coefficient of glucose exceeds 0.10, the gel bed layer required in a low molecular weight region other than the specific molecular weight range of several hundreds to several thousands becomes long, This also increases the column length,
In the case of repeating preparative collection, the time per cycle becomes long and the productivity is lowered, which is not preferable.

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

The cellulosic gel filtration filler of the present invention comprises
The exclusion limit molecular weight of polyethylene glycol is about 1,
000 to about 10,000 fine non-crosslinked cellulose particles are swollen 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. It can be obtained by crosslinking reaction.

The exclusion limit molecular weight is about 1,000 to about 1.
As the fine non-crosslinked cellulose particles in the range of 0000, for example, those obtained by the production methods described in JP-A-63-90501 and JP-A-57-38801 can be used.

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 the 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 been conventionally said that such fine non-crosslinked cellulose particles do not have a small pore size, which is expressed as an exclusion limit molecular weight by crosslinking, but have a large network structure.

As described above, it is possible to prevent enlargement of the network structure of the cellulose particles by carrying out a swelling treatment before carrying out the crosslinking reaction to control the swelling property of the cellulose particles during the crosslinking reaction. It was made clear by some inventors of the present invention.

Further, the crosslinking reaction carried out next is carried out by using a hydrophilic aprotic organic solvent such as dimethyl sulfoxide,
It is carried out by reacting a swelling-treated non-crosslinked cellulose particle with a crosslinking agent such as epichlorohydrin in the presence of a basic compound such as caustic soda and potassium hydroxide in dimethylformamide.

As described above, the cellulosic gel filtration material of the present invention thus obtained has a small exclusion limit molecular weight, and the partition coefficient rapidly changes within a specific molecular weight range of several hundreds to several thousands ( That is, since the difference in partition coefficient is large), high performance is shown for separation / preparation of a mixture of water-soluble low-molecular substances such as oligosaccharides.

[0035]

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

Particle size measuring method Approximately 0.1 g of a sample is sampled, put into 25 ml of pure water, stirred and dispersed, and measured by a light transmission type particle size distribution measuring device. The average particle diameter was calculated on a volume basis.

Absorbance of CH stretchable absorption band 1-3 mg of fine cellulose particles are precisely weighed and potassium bromide powder (abbreviated as KBr) for infrared absorption spectrum measurement is about 200.
Precisely weigh mg and knead and crush both in an agate mortar.
Then, KBr powder kneaded and pulverized with the cellulose particles is scraped and collected into a KBr tablet for infrared absorption spectrum measurement by a tablet molding machine, and a transmission infrared absorption spectrum is measured. 2800 to 30 in the obtained infrared spectrum
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; microcellulose particle collection amount (mg), M; KBr powder collection amount (mg), m; KBr tablet weight (mg), A; absorbance of CH stretching absorption band, T (p); CH stretching Transmittance (%) at peak of absorption band, T (b); Transmittance (%) of baseline at peak wavenumber of CH stretching absorption band. However, the baseline is 2000-4
In the range of 000 cm ^-1 , squeeze the peak ridges of both the OH stretch absorption band and the CH stretch absorption band at the contact points. Ao; absorbance of CH stretching absorption band per 1 mg of microcellulose particles,

[0039]

(Equation 1)

It is represented by

Partition coefficient A gel column is packed in a resin column with a jacket of 16 mmφ × 35 cm at a flow rate of 3 ml / min for 60 minutes using water as a packing liquid. After maintaining the column temperature at 60 ° C. and stabilizing the column, each elution volume was obtained using maltooligosaccharide having a known molecular weight, and the partition coefficient Kn defined by the following formula was calculated.

[0042]

[Formula 2]

Vn: Elution volume of maltooligosaccharide (ml), Vo: Elution volume of blue dextran (molecular weight of 2,000,000) (ml), Vt: Internal volume of the column (ml).

The analysis conditions are as follows.

1 sample size 100 mg / ml, 2 μl, 2 eluent pure water, 3 temperature 60 ° C., 4 flow rate 0.5 ml / min, 5 detector RI detector, 6 pump Shimadzu LC-6A
Type.

Degree of Separation The degree of separation R of maltotriose and maltotetraose was calculated from the following formula under the same conditions as in the method of obtaining the partition coefficient.

[0047]

(Equation 3)

V ₃ and V ₄ represent the elution volume 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 width of the peak on the baseline. W ₃ : Maltotriose Elution volume of peak width (ml), W ₄ : Maltotetraose Elution volume of peak width (m
l).

Example 1 (1). 500g of softwood pulp at 20 ℃, 18
Immerse in 20 liters of caustic soda solution of 1% by weight for 1 hour, and then 2.8
Squeezed twice. Grinding for 1 hour while raising the temperature from 25 ° C to 50 ° C, aging, and then adding 35% by weight of carbon disulfide (175 g) to cellulose and sulfiding 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). 3200 g of a 10.8% aqueous solution of sodium polyacrylate (molecular weight 50,000) and 40 g of calcium carbonate were placed in a 5 l poly container and stirred with a homomixer. After dispersion, 800 g of the above viscose solution was added at a liquid temperature of 25 ° C., and stirring was carried out at 250 rpm for 10 minutes with a lab stirrer. After the fine particles of viscose were generated, the liquid temperature was raised from 25 ° C. to 75 ° C. in 25 minutes with continuous stirring and maintained at 75 ° C. for 15 minutes to solidify the viscose fine particles. The coagulated viscose particles were separated from the mother liquor with a G4 type glass filter, neutralized with 5% by weight hydrochloric acid to give cellulose particles, which were washed with a large excess of water and then dried for 2 hours in a ventilation dryer at 105 ° C. The exclusion limit molecular weight of the obtained cellulose particles by polyethylene glycol was 8200.

(3). Then, the cellulose particles 200
After swelling g in a 10% by weight caustic soda aqueous solution at 15 ° C. and separating from the mother liquor with a glass filter,
It was washed with water and dried.

(4). Then, the cellulose particles 100
g, 1000 g of dimethyl sulfoxide and 200 g of epichlorohydrin were placed in a 2 l flask, and after sufficiently swelling the cellulose particles under stirring, 315 g of a caustic soda aqueous solution in which 15 g of caustic soda was dissolved was gradually added little by little. The caustic soda aqueous solution was added over 1 hour while controlling the liquid temperature at 20 ° C.

Then, the liquid temperature was raised to 50 ° C. for 1 hour and then the crosslinking reaction was completed while adjusting the temperature to 50 ° C. The micro-crosslinked cellulose particles produced are separated from the mother liquor by a glass filter and then neutralized with 1% by weight hydrochloric acid,
It was washed with a large excess of water.

The obtained crosslinked cellulose particles had an average particle size of 130 μm, and the absorbance of the absorption peak attributed to CH stretching vibration of alkylene in the infrared absorption spectrum by the KBr tablet method was 0.012 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 partition coefficient of maltooligosaccharides, the difference in partition coefficient, and the degree of separation between maltotriose and maltotetraose.

[0057]

[Table 1]

The crosslinked cellulose particles were packed in 6 columns (in series connected) having an inner diameter of 16 mm and a length of 95 cm, and the tetrasaccharide component in the maltooligosaccharide was fractionated under the following condition A.
The recovery rate of the product having a purity of 70% was 61%.

When the linear velocity and pressure loss were measured in a column having an inner diameter of 20 mm and a length of 60 cm, the linear velocities of 20, 40 and 8 were obtained.
0.02, 0.05, 0.12 kg for 0 ml / hr.cm ² respectively
The pressure loss was / cm ² .

Condition A Maltooligosaccharide composition (%): As shown in Table 2.

[0061]

[Table 2]

Filling volume 1145.7 cm ³ (1.6 cmφ
X 95 cm x 6), temperature 60 ° C, flow rate 2.0 ml / min, eluent pure water, raw material concentration 610 mg / ml, raw material load 45 ml.

Comparative Example 1 By changing the crosslinking reaction conditions in Example 1, the difference between the distribution coefficient of maltose and the distribution coefficient of maltohexaose was 0.27,
Crosslinked cellulose particles having a degree of separation of maltotriose and maltotetraose of 0.28 were obtained.

Using the particles, the tetrasaccharide component in the maltooligosaccharide was fractionated under the condition A of Example 1 to find that the purity was 70%.
The recovery rate of the product was 33%.

Comparative Example 2 The distribution coefficient of maltohexaose was 0.05, the difference between the distribution coefficients of maltose and glucose was 0.07, and the difference between the distribution coefficient of maltose and that of maltohexaose was 0.2.
0, using a commercially available gel filter medium having a degree of separation of maltotriose and maltotetraose of 0.24, Example 1 Condition A
As a result of fractionating the tetrasaccharide component in the maltooligosaccharide, the recovery rate of the product having a purity of 70% was 26%.

Examples 2 and 3, Comparative Example 3 Maltose, maltohexaose and exclusion were obtained using a commercially available gel filter medium having a distribution coefficient of maltohexaose of 0.39 and crosslinked cellulose particles having a small distribution coefficient of the present invention. A mixture of dextran showing molecular weights, inner diameter 16 mm,
Column length of 35 cm, temperature 60 ℃, flow rate 0.5 ml / m
The elution times of the three peaks were determined under the in condition. The results are shown in Table 3.

[0067]

[Table 3]

As is clear from the results, in the case of fractionation on an industrial scale, when the distribution coefficient of maltohexaose was large (Comparative Example 3), the time per cycle was long, the productivity was lowered and the column length was further increased. It can be seen that this also leads to an increase in equipment costs.

Comparative Example 4 Crosslinked cellulose particles having an average particle size of 20 μm and 40 μm were obtained in the same manner as in Example 1 except that the particle forming conditions were changed.
This particle was treated in the same manner as in Example 1 to have an inner diameter of 22 mm and a length of 6
When packed in a 0 cm column and examined for pressure loss, 4
A particle diameter of 0 μm exceeds a pressure loss of 1 kg / cm ² at a linear velocity of 80 ml / hr.cm ² , and a linear velocity of 40 m for a particle of 20 μm.
The pressure loss exceeded 1 kg / cm ² at l / hr.cm ² .

It is needless to say that it is necessary to select a particle size having a low pressure loss depending on various factors such as column diameter and length depending on the method of the industrial fractionation apparatus, for example, application to simulated moving bed. Nor.

[0071]

EFFECTS OF THE INVENTION Since the cellulosic gel filtration material of the present invention has a sharp distribution coefficient change in a specific molecular weight range, oligosaccharides, which are water-soluble substances having a molecular weight of several thousand or less, are separated by a gel filtration method. Achieves high performance separation when used for preparative applications. Further, since the gel filtration material of the present invention is excellent in safety and chemical stability, it can be widely used as a preparative separation material in the fields of food and medicine.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hayami Ito 1-1 Kawasaki-cho Akashi-shi, Hyogo Kawasaki Heavy Industries Ltd. Akashi Technical Research Institute (72) Masao Hayashiya 1-1 Kawasaki-cho Akashi-shi, Hyogo Kawasaki Heavy Industry Co., Ltd. Akashi Technical Research Institute (72) Inventor, Takashi Kajihata 1-1 Kawasaki-cho, Akashi City, Hyogo Prefecture Kawasaki Heavy Industries Ltd. Akashi Technical Research Institute

Claims (1)

[Claims] 1. (a) It consists essentially of a type II cellulose crystalline phase and a cellulose amorphous phase, (b) there is crosslinkage between cellulose molecular chains in the cellulose amorphous phase, and (c) the average particle size is Consisting essentially of spherical particles of 50-300 μm,
(d) The distribution coefficient of maltohexaose is 0.25 or less, the difference between the distribution coefficient of maltose and the distribution coefficient of maltohexaose is 0.25 or more, and the difference between the distribution coefficient of maltose and the distribution coefficient of glucose. Is 0.10 or less, and (e) the degree of separation of maltotriose and maltotetraose is 0.35 or more.