JPH03252430A - Production of cellulose particle aggregate having sharp particle size distribution - Google Patents

Abstract

PURPOSE:To obtain the subject aggregate having sharp particle size distribution and useful as a filler for chromatography column by mixing viscose with a water soluble anionic polymer compound in the presence of calcium hydroxide. CONSTITUTION:The objective aggregate is produced by mixing (A) viscose with (B) a water-soluble anionic polymer compound in the presence of Ca(OH)2 to form a dispersion of fine particles of the component A, coagulating the component A by heating the dispersion or mixing with a coagulant, forming fine particles of cellulose by neutralizing the product with an acid or coagulating and neutralizing the above dispersion with an acid, separating the cellulose particles from the mother liquor and, as necessary, subjecting the product to desulfurization, washing with acid, crosslinking and washing with water or drying.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a method for producing cellulose particle aggregates having a sharp particle size distribution.

[Prior Art J Cellulose or its various derivatives are used as packing materials for chromatography, especially when separating and purifying biological materials such as proteins and nucleic acids, from the viewpoint of safety and low non-specific adsorption with them. Its usefulness is highly evaluated. Furthermore, while fibrous or fine granular cellulose has conventionally been used in this field, cellulose formed into spherical shapes by various methods is also commercially available for the purpose of improving flow resistance.

Journal of the Chemical Society of Japan, 1981. (1,2), p, 1883~
In 1889, there was a research report titled ``Manufacture of cellulose spherical gel and its performance in gel chromatography J'', in which a cellulose gel with a particle size of 25 μm or less was packed into a column with a length of 30 c + n, and ethylene glycol was used for about 7000 plates. It is reported that the number of theoretical plates was obtained.
Publication No. 7 discloses a method for producing cellulose particles having an average particle diameter of 20 μm or less by utilizing phase separation between viscose and a water-soluble anionic polymer compound.

In addition, as shown in Proceedings of the 6th Spring Conference on Liquid Chromatography, p. 7-8, the cellulose particles obtained by the above method were classified into 8-12 μl, and then packed into a column with a length of 30 cm. 8 with ethylene glycol
The number of theoretical plates is 700.

The above method for producing cellulose particles using phase separation of viscose and a water-soluble anionic polymer compound has the advantage that relatively small particles with an average particle diameter of 20 μm or less can be easily produced and that no organic solvent is used. This is a promising method because it uses simple equipment and can be easily scaled up. However, in order to use the cellulose particles as a high-performance liquid chromatography column filler, there still remained points to be improved. In other words, in the past, it was necessary to classify cellulose particles at a complicated time before filling them into a column, but in order to eliminate the need for a classification operation, we created particles with a sharper particle size distribution. The goal is to improve the manufacturing method.

[Problems to be Solved by the Invention] An object of the present invention is to provide a method for producing cellulose particle aggregates that are abrupt and have a sharp particle size distribution from regenerated cellulose or type 1 cellulose.

Another object of the present invention is to produce sharp particles having a small average particle size of 20 μm or less and a particle size distribution in which 90% of the total number of particles in the aggregate is within a particle size range of 20% above the average particle size. The object of the present invention is to provide a method for producing a particle aggregate of type 1 cellulose having a diameter distribution.

Still another object of the present invention is the novel method described above, which comprises a step of mixing viscose and a water-soluble anionic polymer compound in the presence of a hydroxide to produce a fine particle dispersion of viscose. Our goal is to provide the following.

Still another object of the present invention is to provide a high performance liquid chromatography column that has low non-specific adsorption and is suitable for separating proteins, sugars, etc. To provide a method for producing a type 1 cellulose particle aggregate suitable as a packing material for a graphing column or a high-performance liquid chromatography column that has a high number of theoretical plates, high resolution, high pressure resistance, and enables high-speed separation. be.

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

[Means and effects for solving the problems] According to the present invention, the above objects and advantages of the present invention are as follows: (1) Viscose and a water-soluble anionic polymer compound are mixed in the presence of calcium hydroxide. (2)(i) coagulating the viscose in the dispersion by heating the dispersion or mixing the dispersion with a coagulation liquid; neutralizing with acid to produce cellulose microparticles or (u) coagulating and neutralizing the dispersion with acid to produce cellulose microparticles, and then (3) separating the cellulose microparticles from the mother liquor; This can be achieved, if necessary, by desulfurization, pickling, crosslinking, washing with water, or drying to produce a cellulose particle aggregate having a sharp particle size distribution.

As described above, according to the method of the present invention, when mixing viscose and a water-soluble anionic polymer compound to produce a fine particle dispersion of viscose, by the simple means of making calcium hydroxide present, Surprisingly, a cellulose particle aggregate having an extremely excellent and sharp particle size distribution can be obtained.

According to the method of the present invention, a viscose fine particle dispersion is produced in the first step, cellulose fine particles are produced in the second step, and the cellulose fine particles are separated from the mother liquor in the third step.

The first step of producing a fine particle dispersion of viscose is carried out by mixing viscose and a water-soluble anionic polymer compound in the presence of calcium hydroxide. At this time, calcium carbonate can also be used together.

The greatest feature of the present invention is that, in the first step, fine cellulose particles with a particle size of 20 μm or less, for example, can be obtained with a narrow particle size distribution by adding hydroxide.

The viscose used in the first step has, for example, the following properties.

The gamma value is 30-100, more preferably 35-90. The salt point is 3-20, more preferably 4-18. Cellulose concentration is 3 to 15% by weight, more preferably 5
~13% by weight. Alkali concentration is 2-15% by weight,
More preferably, it is 4 to 13% by weight. The weight ratio of alkali (as caustic soda) to cellulose in viscose is 40 to 100% by weight, more preferably 50 to 90% by weight.
Weight%. The viscosity of viscose is 5 at 20°C.
0 to 20,000 centivoise, more preferably 80 to
It is 18,000 centiboise.

The pulp source for the viscose is preferably linter pulp, and may also be softwood or hardwood. The average degree of polymerization of viscose as cellulose is usually 110 to 1,000.

The water-soluble anionic polymer compound used has, for example, a sulfonic acid group, a phosphonic acid group, or a carboxylic acid group as an anionic group. These anionic groups may be in the form of free acids or salts.

The water-soluble polymer compound having a sulfonic acid group as an anionic group may be a vinyl sulfone sickle, styrene sulfonic acid, methylstyrene sulfonic acid, allyl sulfonic acid, methallyl sulfonic acid, acrylamide metal propane sulfonic acid, or It can be derived from monomers such as these salts.

Similarly, water-soluble polymeric compounds having phosphonic acid groups as anionic groups can be derived from monomers such as styrenephosphonic acid, vinylphosphonic acid, or salts thereof.

The water-soluble polymer compound having a carboxylic acid group as an anionic group can be derived from monomers such as acrylic acid, methacrylic acid, styrene carboxylic acid, maleic acid, itaconic acid, or salts thereof.

For example, a water-soluble polymer compound having a carboxylic acid group can be produced by polymerizing sodium acrylate alone or by mixing it with other copolymerizable monomers such as methyl acrylate according to a method known per se. Supplied as a homopolymer or copolymer containing polymerized units of acid soda. Furthermore, for example, a water-soluble polymer compound having a sulfonic acid group can be produced by sulfonating a styrene homopolymer.

The same applies to the case where the sulfonic acid group is derived from a monomer other than styrene sulfonic acid, and the case where the phosphonic acid group and the carboxylic acid group are respectively derived from the above monomers.

The water-soluble anionic polymer compound preferably has at least two polymerized units of the above-mentioned monomers having an anionic group.
Contains 0 mol%. Preferred polymeric compounds include copolymers and homopolymers.

The water-soluble anionic polymer compound preferably has a number average molecular weight of at least s, ooo, more preferably 10,000 to 3,000,000.

The water-soluble anionic polymer compound in the present invention includes:
In addition to the vinyl type polymers mentioned above, other examples include carboxymethyl cellulose, sulfoethyl cellulose, and salts thereof such as Na salt.

According to the method of the present invention, viscose and a water-soluble anionic polymer compound are first mixed. For mixing, any means can be used as long as a fine particle dispersion of viscose is produced. For example, mechanical stirring using stirring blades, baffles, etc., ultrasonic stirring, or mixing using a static mixer can be carried out alone or in combination.

The water-soluble anionic polymer compound is preferably used as an aqueous solution, more preferably at a concentration of 0.5.
It is used as an aqueous solution of ~25% by weight, particularly preferably 2-22% by weight. Such an aqueous solution further has a viscosity of 3 centipoise to 50,000 centipoise at 20°C, particularly 5 centipoise.
It is preferable to have a centipoise to 30,000 centipoise.

The viscose and water-soluble anionic polymer compound are used in an amount of 0.3 to 100 parts by weight, more preferably 1 to 45 parts by weight, particularly preferably 4 to 20 parts by weight of the polymer compound per 1 part by weight of cellulose. and mixed. The mixing is advantageously carried out at a temperature below the boiling point of the carbon disulfide contained in the viscose, more preferably between 0 and 4
It is carried out in the range of 0°C.

By the first step of the method of the present invention, viscose can be finely divided into substantially spherical fine particles having an average particle size of 30 μm or less.

According to the method of the present invention, the viscose fine particle dispersion produced in the first step is then coagulated and neutralized in the second step to produce cellulose fine particles. Coagulation and neutralization may be performed simultaneously or sequentially.

If coagulation and neutralization are carried out over time, coagulation can be carried out by heating the dispersion or mixing the dispersion with a coagulant, and then neutralization is carried out by contacting with an acid. .

The above coagulation reaction produces! It is desirable to carry out the mixing operation while adding a mixing operation to the two-dispersion liquid.

Coagulation by heating can be advantageously carried out at a temperature higher than the boiling point of carbon disulfide contained in the viscose, for example at a temperature of 50° to 90°C. In the case of coagulation using a coagulant, it is not necessary to raise the temperature to such a temperature, and coagulation can be generally carried out at a temperature of 0 to 40°C.

As the coagulant, for example, lower aliphatic alcohols, alkali metal salts or alkaline earth metal salts of inorganic acids, inorganic acids, organic acids, or combinations thereof, and combinations thereof with water-soluble polymer compounds are preferably used. The lower aliphatic alcohol may be linear or branched,
For example, methanol, ethanol, 1so-propertool, n-propertool, n-butanol, etc. having 1 or more carbon atoms.
No. 4 aliphatic alcohols are preferably used. Examples of inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid, and carbonic acid. Examples of alkali metal salts of inorganic acids include NaCl2. Na.

Preferred are Na salts such as SO, and salts such as K, SO, and preferred alkaline earth metal salts include Mg salts such as Mg5O, and Ca salts such as Ca (182).Organic acids are preferred. is a carboxylic acid or a sulfonic acid, such as formic acid, acetic acid, propio/il.

The coagulant as described above is used in a proportion of, for example, about 20 to 300% by weight based on the cellulose in the viscose.

As the acid used as the neutralizing agent, strong inorganic acids such as sulfuric acid and hydrochloric acid are preferably used.

The neutralizing agent is used in an amount sufficient to neutralize the viscose;
Produces fine particles of cellulose. Furthermore, as described above, the second step of coagulation and neutralization can be carried out simultaneously. Agents effective for coagulation and neutralization are acids, preferably strong inorganic acids such as hydrochloric acid or sulfuric acid. An acid used in an amount sufficient to neutralize viscose will be an amount sufficient to coagulate and neutralize the viscose.

Simultaneous coagulation and neutralization are advantageously carried out at temperatures of, for example, 0 to 40°C.

According to the method of the present invention, the fine particles of cellulose produced in the second step are then separated from the mother liquor in the third step and, if necessary, are desulfurized, pickled, crosslinked with water, or dried. In some cases, bleaching may be performed after pickling. Separation of fine particles from the mother liquor can be performed, for example, by filtration, centrifugation, or the like. Desulfurization can be carried out using an aqueous alkali solution such as caustic soda or sodium sulfide. If necessary, to remove residual alkali, the product is then pickled with dilute hydrochloric acid, washed with water, and dried.

During the third step, the pickled cellulose particles can be subjected to a crosslinking reaction if necessary. As a crosslinking agent,
Shrimp chlorohydrin, dichlorohydrin, etc. are used. The crosslinking reaction is carried out in a liquid medium containing alkali hydroxide. When the alkali hydroxide is caustic soda, it is carried out at a concentration of 1 to 25% by weight, preferably 5 to 15% by weight.

By changing the concentration of alkali hydroxide, the pore size of the produced cellulose fine particles can be adjusted. The crosslinking agent is present in the liquid medium containing the alkali hydroxide.
The concentration is adjusted to 25% by weight. By varying the amount of crosslinking agent used, the crosslink density and strength of the resulting cellulose particles can be adjusted. As the liquid medium, water or methanol, dimethyl sulfoxide, ethanol, or acetone, which is compatible with water and is a good solvent for the crosslinking agent, is used alone or in a mixture with water.

The liquid medium is used in an amount of 10 to 30 parts by weight per 1 part by weight of cellulose. It varies depending on the liquid medium, but usually 50
The crosslinking reaction is carried out at a temperature of ~80°C.

Thus, according to the invention, for example, the individual particles (a)
'n-type cellulose, and (b) has a degree of crystallinity determined by X-ray diffraction in the range of 5 to 30%, and (c) has an average particle size of (d) sharp particles consisting of spherical or oblong particles of 20 μm or less, and having a particle size distribution in which 90% of the total number of particles in the aggregate is within a particle size range of ±20% of the average particle size; Cellulose particle aggregates having a size distribution can be advantageously produced.

The above-mentioned aggregate of microcellulose particles is the above-mentioned (a) to (c)
It is distinctive in that it has the characteristic i). These characteristics will be explained next.

The microcellulose particles first consist essentially of type II cellulose, that is, regenerated cellulose. Therefore, cellulose microparticles made of natural cellulose, ie type I cellulose, are completely different from the microparticles of the present invention. As is well known, type 1 cellulose and type I cellulose can be distinguished by X-ray diffraction.

In the X-ray diffraction diagram of type (2) cellulose, there is substantially no diffraction peak at a diffraction angle (2θ) of 15°, which clearly exists in type (2) cellulose.

Moreover, the second characteristic of these microcellulose particles is the degree of crystallinity determined by X-ray diffraction, and the degree of crystallinity is 5 to 30%. The microcellulose particles preferably have a crystallinity of 10-28%, more preferably 15-26%. Moreover, this microcellulose is not amorphous but crystalline as specified by the crystallinity described above.

Furthermore, the aggregate of these micro cellulose particles
It consists essentially of spherical or long spherical particles with an average particle size of 20 μm or less. The aggregate of microcellulose particles is thus composed of extremely small particles. The aggregate of micro cellulose particles further has an average particle size of 1-18 μm.
More preferably, it is substantially composed of spherical or long spherical particles of 1.5 to 15 μm. The term "spheroidal" as used herein is a concept that includes particles whose projected view or plan view has a shape such as an ellipse, an elongated circle, a peanut shape, or an oval shape. The individual microcellulose particles are spherical or oblong as described above, and are therefore different from particles that are rounded or irregularly shaped. When producing long spherical microcellulose particles according to the above-described method of the present invention, from the dispersion in the first step to the second step,
When proceeding to the coagulation step, if viscose and a water-soluble anionic polymer compound are coagulated while being mixed too vigorously, they are likely to form.

Fourthly, the fine cellulose particle aggregate is characterized by the fact that particles with particle sizes within a narrow range of 20% around the average particle size account for 90% by weight or more of all particles. has a very sharp particle size distribution. For example, if the average particle size of all particles is 10 μm, this means that 8 to 12
Particles with a particle size in the μm range account for 90% by weight of all particles.
This means that it occupies more than

The above-mentioned cellulose particle aggregate can be particularly advantageously used as a packing for a high performance liquid chromatography column.

The chromatography column packed with cellulose particles having the above properties obtained by the method of the present invention has less non-specific adsorption and excellent alkali resistance due to the use of cellulose as the filler material.

As packing for a high performance liquid chromatography column, the cellulose particle aggregate is preferably spherical or elongated with an average wet particle diameter of 20 μm or less. The average particle size is preferably in the range of 3 to 18 μm, more preferably 8
~12 μm. If many microparticles of 3 μm or less are mixed together, the pressure when the eluent is passed through the column becomes high, making it impossible or difficult to perform separation and analysis at a high flow rate. Furthermore, if the particle size is large, a column with high resolution cannot be obtained. Preferably, the height equivalent to a theoretical plate determined from the elution peak of ethylene glycol by the method described below is 80 μm or less.

Currently commercially available cellulose-based chromatography fillers are limited to crushed particles or relatively large particles with an average particle size of 20 μm or more, and cannot be said to have high separation performance.

By packing cellulose particles with an average particle size of 20 μm or less, a high separation performance liquid chromatography column with a height equivalent to a theoretical plate of 80 μm or less can be obtained.

Furthermore, as cellulose particle aggregates for filling high-performance liquid chromatography columns, those having a particle size distribution in which 90% of the total number of particles are within a particle size range of ±20% of the average particle size are particularly preferably used. . As mentioned above, it is possible to construct a high-performance chromatography column with a small height equivalent to a theoretical plate by reducing the average particle diameter, but when packing the column, it is necessary to As more particles are mixed in, the filling pressure increases.
It will have to be filled at a low flow rate.

A suitable method for filling a liquid chromatography column with cellulose particle aggregates is the wet slurry method described below. That is, the cellulose particles to be filled are first wetted with a slurry solvent.

As the slurry solvent, an aqueous solution of an inorganic salt such as sodium sulfate or sodium chloride, an aqueous polymer aqueous solution such as polyethylene gelcol or polyacrylic acid, or water can be used. The suitable size of the column is 4 to 10+++ m in inner diameter and 5 to 30 cm in length for analysis, and 10 to 200 m+n in inner diameter and 15 to 100 c+n in length for fractional separation. The material of the column may be either stainless steel or resin. Attach a packer to the column, pour in the slurry moistened with cellulose particles, and fill the column with the filling liquid.

As the filling liquid, use the same aqueous solution as the slurry solvent, or use a salt aqueous solution or water that is thinner than the slurry solvent. When filling is completed, the backer is removed and the inlet is closed to form a liquid chromatography column.

EXAMPLES The present invention will be explained in further detail with reference to Examples below.

Before that, the measurement method and the like in the present invention will be described below.

Particle Size Measurement Method Approximately 0.1 g of a sample is taken, poured into 25N+Q pure water, stirred and dispersed, and measured using a light transmission particle size distribution analyzer. The average particle size was calculated on a volume basis.

Measurement method of height equivalent to theoretical plate> The elution of ethylene glycol is examined using the chromatography column of the present invention (column length is 2) under the following conditions.

Flow rate: l, OmQ/min Eluent: Pure water Detector: R■ Sample (ethylene glycol) i11 degrees: 2.5
mg/mQ Sample amount: 20 pQ The height equivalent to a theoretical plate (HETP) is determined from the elution peak of ethylene glycol in Figure 1 using the following formula.

(However, ■ is the elution volume of ethylene glycol, and W is the width of the intersection of two tangent lines passing through the inflection point of the elution curve and the baseline) Example 1 500 g of pulp made of coniferous wood at 20°C 18% by weight
It was immersed in a 20°C caustic soda solution for 1 hour and squeezed 2.8 times. 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 has a cellulose concentration of 9.3% by weight and a caustic soda concentration of 5%.
9% by weight, and the viscosity was 6.200 centipoise.

In a 5Q plastic container, 3300 g of 1000% sodium polyacrylate aqueous solution (molecular weight 100,000), calcium carbonate 1:
32g of calcium hydroxide and 40g of calcium hydroxide were added thereto, and the mixture was stirred with a homomixer. After dispersion, add 868 g of the above viscose liquid at a liquid temperature of 25°C, and stir at 500 rpm using a lab stirrer.
Stir for 10 minutes. After generating viscose fine particles, the liquid temperature was raised from 25°C to 75°C in 25 minutes while stirring continuously, and maintained at 75°C for 15 minutes to solidify the viscose fine particles. Ta. The coagulated viscose particles were separated from the mother liquor using a 04 type glass filter, then neutralized with 5% by weight hydrochloric acid to obtain cellulose particles, washed with a large excess of water, and then dried in a ventilation dryer at 105°C for 5 hours. did. The results of measuring the particle size distribution of the obtained cellulose particle aggregate are shown in FIG. Average particle size is 9.5μ
m, and approximately 91% of the total particle number of the aggregate was present within the particle range of 7.6 to 11.4 μm.

Then, the cellulose particle log was 25 m12 no 0.2M
Swelled with Na, SO, inner diameter 3 mm, length 25
To the fang column, flow rate 2, 0 mQ/min, pressure 2
It was filled at 9 kg/C1n''.

The elution peak of ethylene glycol in the obtained chromatography column is shown in FIG. The number of theoretical plates is 4800
, the height equivalent to the theoretical plate is 46μα.

Example 2 Cellulose particles were prepared in the same manner as in Example 1 except that the molecular weight of sodium polyacrylate was 50,000. The average particle diameter was 17 μm.

Next, the cellulose particles were washed with a 0.5% by weight aqueous solution of caustic soda, filtered in an amount of 60g using a G4 type glass filter, and placed in an 8% by weight aqueous solution of caustic soda lQ containing 20% by weight of epichlorohydrin at 60° for 0.3 hours with stirring. Crosslinked. Subsequently, it was separated from the mother liquor using a glass filter, neutralized with 5% by weight hydrochloric acid to form crosslinked cellulose fine particles, washed with a large excess of water, further washed with methanol, and then dried in a ventilation dryer at 105°C for 5 hours. . The obtained crosslinked cellulose particles had an average particle diameter of 17 μm.

Then, 10g of the cellulose particles were added to 25m4 of 0.2M
Swelled with Na25O, inner diameter 3 mm, length 25 cm
m column at a flow rate of 1 or 2 mQ/min and a packing pressure of 9 kg/c1.

FIG. 4 shows an example in which maltooligosaccharides and glucose were separated using the obtained chromatography column.

Comparative Example A cellulose particle aggregate was prepared in the same manner as in Example 1, except that calcium hydroxide was not added when mixing viscose and sodium polyacrylate. . The results of measuring the particle size distribution of the cellulose particle aggregates obtained are shown in FIG. Average particle size is 9
．． It was 6 μm.

Approximately 60% of the total particle number of the aggregate was present within the 7.7-11.5 μm particle range. Then the cellulose particles 1
I am trying to swell 0g in 25mQ of 0.2M Na, SO4 and pack it into a column with an inner diameter of 3mm and a length of 25cm, but at a pressure of 100kg at 0.2 mal/min.
/cm2 and could not be filled.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of the elution peak of ethylene glycol for calculating the height equivalent to a theoretical plate. FIG. 2 shows the results of measuring the particle size distribution of the cellulose particle aggregate obtained in Example 1. FIG. 3 is an example of the elution peak of ethylene glycol. FIG. 4 is an example of separation of maltooligosaccharides and glucose using the chromatography column obtained in Example 2. FIG. 5 shows the measurement results of particle size distribution of cellulose particle aggregates in comparative examples. 1 Idiot Figure 2 f'/, 1 Figure 5 Figure 4 Flow 11.0.3m 1-branch pond 8: RI

Claims (1)

[Claims] 1. (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; (2) (i ) coagulating the viscose in the dispersion by heating the dispersion or mixing the dispersion with a coagulation liquid and then neutralizing with acid to form fine particles of cellulose; or (ii) 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, cross-linked with water or dried to form sharp particles. A method for producing a cellulose particle aggregate, comprising: producing a cellulose particle aggregate having a diameter distribution.