SE461149B - SET TO MANUFACTURING PARTICLES OF GEL-CREATING POLYSACCHARIDES - Google Patents

Abstract

The invention relates to a method for preparing particles of gel-forming polysaccharides which can be used as gel matrices for separating different biomolecules, such as proteins or nucleic acids. The gel particles are prepared by dissolving the polysaccharide in water at a temperature above its dissolving temperature, and emulsifying the solution in an organic solvent to form a water-in-oil emulsion, whereupon the emulsion is cooled such that the polysaccharide solidifies and is separated from the solvent. To obtain particles having a very narrow particle size distribution, the emulsification is carried out according to the invention in the presence of an inorganic hydrophobic protective colloid.

Description

461 10 15 20 25 30 35 149 '2 organic surfactants a large drop in droplet size is obtained. In order to obtain good flow properties in chromatographic columns, it is important to have a narrow size distribution of the particles. This is especially true when small gel particles are used to obtain sufficiently good chromatographic separation. Such columns give a higher back pressure and then a narrow size distribution is of the utmost importance. Otherwise, such a high pressure builds up in the column that the rather soft gel particles are deformed with the result that the flow almost completely ceases.

To obtain a sufficiently narrow size distribution, the gel particles obtained from the W / O emulsification are sieved. Screening of these soft gel particles presents considerable technical problems. Agaros is also a very expensive raw material.

Since the sieving results in a limited yield within the desired size range, the size distribution originally obtained during the emulsification will strongly affect the raw material cost in the production of agarose gel particles.

It is therefore of great economic importance to find emulsification systems that provide a narrow size distribution for the W / O emulsion of polysaccharide solution.

It is important that the emulsification system provides the opportunity to produce gel particles with different medium sizes.

Finally, it is also of great importance for the system to allow the emulsification production of polysaccharide gel particles with different dry contents and thus different pore sizes.

The present invention relates to a process for producing particles of polysaccharides using an inorganic protective colloid which largely satisfies the above-mentioned desires, as is apparent from the claims. in carrying out the process according to the invention, the gel-forming polysaccharide is dissolved in water at a temperature above its solution temperature. saccharide should be weight percent, The amount of poly is in the range from 0.5 to 20, preferably 2 - 15 weight percent. solution temperature, the polysaccharide varies with the type of compound, for agarose the temperature is about 75 ° C, and one should be at a temperature of about 80-100 ° C. In connection with the dissolution, an organic solvent with limited water solubility is added and the mixture is stirred vigorously to form a water in an oil emulsion. The organ phase: water phase ratio should be in the range from 0.5: 1 to 10: 1, preferably from 1: 1 to 5: 1. During the emulsification, depending on the type and amount of protective colloid, the amount of polysaccharide, and the stirring intensity, a droplet size is obtained in the range from 3 to 200 .mu.m, preferably from 5 to 100 .mu.m on the solution of polysaccharide.

Before the isolation of the gel particles from the organ phase, possibly after the addition of water, of the protective colloid. The dry content of those emulsions is acidified, the gel particles prepared for dissolution are in the range from 0.5 to about 50% by weight.

The protective colloid used according to the invention comprises hydrophobicized, fine-grained water-soluble inorganic compounds. Normally characterized by such water-insoluble inorganic compounds are too hydrophilic to fit in these systems. The necessary hydrophobia can be achieved by adding small amounts of co-stabilizers which are absorbed on the surface of the particles of the sparingly soluble inorganic compounds. The particles can also be made sufficiently hydrophobic by covalently bonding hydrophobic groups to the surface.

Examples of water-insoluble inorganic compounds sparingly soluble salts of phosphate polyphosphate, of sulphate, carbonate or silicate. Difficult oxides or which can be used are different or can also use fine-grained minerals. Specific examples of suitable water-insoluble inorganic compounds are calcium phosphate, calcium sulphate, calcium carbonate, iron phosphate, magnesium hydroxide, alumina, aluminum hydroxide, silica (silicic acid), iron hydroxide, bentonite, titanium dioxide, etc. The amount should be within the range. 0.1 to 50%, preferably 0.3% to 10% based on the amount of phase of polysaccharide solution. barium sulphate, zinc oxide, fine-grained glass powder, 461 149 = 10 15 20 25 30 35 4 Examples of suitable co-stabilizers for hydrophobicizing the particle surface of the soluble inorganic compounds are anionic, cationic organic compounds alcohols, amines or derivatives of ethylene oxide or propylene oxide. Specific examples are carboxylic acids, diesters of phosphoric acid, mono- or alkylsulfonic acids and quaternary ammonium compounds, etc.

As previously mentioned, however, the particles of the sparingly soluble inorganic compounds can also be hydrophobicized by binding organic compounds to the surface of the particles.

An example of such a case is the treatment of particles of silica with methylsilane to obtain hydrophobic methylsilanol groups on the particle surface. The hydrophobicity of the inorganic protective colloid is varied with the type of polysaccharide and inorganic compound, the desired particle size and especially the type of organ phase.

By simple attempts for a particular stabilizer is easily determined.

Examples of gel-forming polysaccharides are systems containing the amount of co-agar / agarose and dextran. Preferably, the invention relates to the preparation of agarose particles.

As organic phase for the production of water in the oil emulsion, various organic compounds can be used, for example paraffinic hydrocarbons, cycloalkanes, aromatics, alcohols, esters, chlorinated hydrocarbons, etc. Specific examples are C5 - C15 alkanes, toluene, xylene, C5 - C15 alcohols, carbon tetrachloride, etc.

For separation of relatively small protein molecules, agarose gel particles with higher agarose content were used, usually more than 5 because the higher content entails in the gel particles. However, it may be to produce W / O emulsions with the tightness in the size distribution that characterizes weight percent, smaller pores difficult system according to the invention if the emulsification uses polysaccharide-aqueous solution with a high polysaccharide content. Narrower size distribution is generally obtained when using agarose solutions with a lower dry content. According to a particular embodiment of this invention, gel particles can also be produced with a high dry content, usually in the range from 5 to about 30% by weight, and a narrow size distribution.

According to this variant, a W / O emulsion with a lower dry content, preferably 0.5 - 5% by weight, is first prepared, and then water is removed from the droplets of polysaccharide-aqueous solution at a temperature above which the polysaccharide solidifies into a solid gel. This concentration by water removal can be carried out in different ways: - by distillation whereby a mixture of hydrocarbon is distilled off from the W / O emulsion - by diluting the organ phase with a substance which increases the solubility of the water in the continuous phase - by adding more continuous phase if the continuous certain solubility for water, for example water and the phase has hexanol - by adding water-absorbing solid inorganic or organic character The invention is described in more detail in the following exemplary embodiments, where parts and percentages are by weight unless otherwise indicated. substances of parts by weight respectively EXAMPLE 1 Comparative example with organic surfactant 12 g of agarose and 100 g of distilled water were charged at 95 ° C. After 30 min. at 95 ° C, a solution heated to 95 ° C was charged with 15 g of nonionic surfactant (Tween 61) and 200 g of toluene. The sample was emulsified with an Ultra-turrax to a medium droplet size. The sample was then cooled to form solid gel particles of agarose-water.

The particles were washed after the particles had settled and the toluene phase had been decanted off. The experiment gave a broad particle distribution with only 40 wt * within 40 to 60 μm. Other experiments with oil-soluble surfactants, such as phosphate esters, also gave the broad distributions with a yield of about 40% in the range of 40-60 μm.

EXAMPLE 2 The procedure of Example 1 was repeated in a flask and heated with stirring to 50 μm. room temperature, by centrifugation, with the 461 10 15 20 25 30 35 149 '6 difference that the amount of Tween 61 was increased to 33 g.

In this way, particles with an average size of about 20 μm were obtained, but with a wide particle size distribution.

The yield within 15 to 20 μm was only about 30% by weight. Other experiments with oil-soluble surfactants also gave a broad particle distribution and a low yield in the range of 10-20 μm.

EXAMPLE 3 A colloid was prepared by mixing 10.2 g of MgCl 2 .6H 2 O, 8 g of 40% NaOH, 4 g of lauric acid and 20 g of toluene.

The mixture was heated with stirring so that the fatty acid melted. 3 g of agarose, 97 g of distilled water and 200 g of toluene were charged to a flask and heated with stirring to 95 ° C. After 30 min. at -95 ° C the mixture was cooled to 75 ° C and mixed with a high speed mixer, type Ultraturrax. By charging 8.5 g of the above colloid, an emulsion with a droplet size of SO 3 was obtained. The sample was cooled to room temperature to form solid gel particles of agarose water. Distilled water was added and the mixture was acidified, whereby the magnesium hydroxide was dissolved.

The aqueous phase was separated and the agarose gel particles were washed by centrifugation.

The experiment gave a narrow particle size distribution of 80% by weight within 40 to 60 microns. Further experiments showed that it was possible to regulate the size with the help of the amount of colloid invested.

If you doubled the amount of colloid, you got a narrow distribution of 65% between 15 and 20 μm.

EXAMPLE 4 The procedure of Example 3 was repeated except that stearic acid replaced lauric acid. Also in this case, a narrow particle size distribution and an average size of 50 μm were obtained.

EXAMPLE 5 The procedure of Example 3 was repeated except that the colloid consisted of 1 g of lauric acid, 11.3 g of Ca (NO 3) 2 X 4 H 2 O, 2.6 g of Na 3 PO 4 X 12 H 2 O and 20 g of toluene. 11 g of this colloid were charged, which resulted in an average size of 50 .mu.m with 70% by weight between 40 and 60 .mu.m. with the EXAMPLE 6 3 grams of agarose, 97 g of distilled water, 200 g of toluene and 0.6 g (hydrophobicized silica) of Cab-O-Sil TS-720 were mixed and heated to 95 ° C. After 30 min. the sample was emulsified to 50 μm. Then the sample was cooled to room temperature and water was added. The sample was washed by centrifugation.

In this way a narrow distribution of 80% by weight between 40 ° and 60 μm was obtained. In this case, after acidification, it was not possible to completely wash away the hydrophobicized silica particles from the surface of the agarose gel particles.

EXAMPLE 7 1.7 g of ca (No 3) 2 x 4H 2 O, 1.5 g of Na 3 Po 4 x 12H 2 O, 3 mg of NaBH 4 and water to 97 g were charged to a flask and the pH was adjusted to 9. 3 g of agarose and 200 g of hexanol were added and the mixture was heated with stirring to 95 ° C. After 95 DEG C. at 95 DEG C., the mixture was pre-emulsified for a few minutes with Ultra-turvax, 0.5 g of lauric acid was added and the pH was min. was adjusted to 8. The sample was then emulsified to about 20 μm, with an additional 100 g of hexanol being charged. The sample was cooled to room temperature. Distilled water was added and the mixture was acidified and the agarose gel particles passed to the aqueous phase. The aqueous phase was separated and agarose washed. In this way, a particle distribution of 65% by weight of the gel particles was obtained by centrifugation. narrow between 15 and 20 μm.

EXAMPLE 8 The procedure with the difference that 0.8 g of hexanoic acid replaced lauric acid. In this way, a narrow distribution with an average value of 20 μm was also obtained.

EXAMPLE 9 4 g of agarose, and 0.6 g of hydrophobicized silica particles were mixed and heated to 95 ° C. After 30 min. the sample was emulsified. An additional 200 g of hexanol was charged and the sample was cooled to room temperature and washed by centrifugation. In this way, a mainly particle size distribution of around 50 μm was obtained. according to Example 7, 96 g of distilled water, 200 g of hexanol (cab-o-sil Ts-120) were repeated, a narrow 461 149 8 EXAMPLE 10 An agarose solution was emulsified according to Example 7. At 95 ° C 400 g of extra hexanol were charged and 0.75 g of lauric acid before cooling the sample to room temperature. After washing the agarose gel particles, the sample was filtered to a dry cake and the dry matter content was determined to be 9.3 8. Correspondingly, the dry matter content of a sample without additional hexanol was determined to be 4.5%. The density of the aqueous hexanol was determined to be 0.830 with an aerometer while 0.816 for anhydrous hexanol.

This corresponds to about 9% water content in the hexanol.

EXAMPLE 11 An agarose solution was emulsified according to Example 7. At 95 ° C, a mixture of water and hexanol (about 70% water) was distilled off to a dry content of agarose in the agarose water droplets of 12%. The sample was then cooled to room temperature and further treated according to Example 7.

Agarose gel particles prepared according to the above examples can be used for biotechnological separation of the protein mixture.

In order to be able to work at higher flows, they can be hardened by crosslinking, for example according to the European patent application 203049. 'ÜÅ; I.

Claims (10)

10 15 20 25 30 35 9 461 149 Patent claims A method of preparing particles of gel-forming polysaccharides by dissolving the polysaccharide in water at a temperature above its solution temperature and emulsifying the solution in an organic phase to form a water in an oil emulsion, after which the emulsion is cooled so that the polysaccharide solidifies and separates. from the organic that the emulsification is carried out in the presence of inorganic, hydrophobic protective colloid. 2. As claimed, the amount of protective colloid is in the range from 0.1% to 50% based on the amount of polysaccharide solution. 3. A method according to claim 2, characterized in that the amount of protective colloid is in the range from 0.3% to 10% based on the amount of polysaccharide solution. 4. A method according to which the inorganic, hydrophobic protective colloid consists of a sparingly soluble metal oxide or a sparingly soluble metal phosphate, ganoic compound. 5. A method according to claims thereof, that washing. 6. A method according to claims 1 - 3, characterized in that the protective colloid consists of the silica hydrophase characterized therefrom, claim 1, thereof, claim 1, characterized as being hydrophobized by the addition of anionic ortho- 1 - 4, characterized in The signified protective colloid is removed by acidification and thereof, phobed with covalently bonded groups. 7. A method according to claim 1, characterized in that water from the droplets of saccharide solution before the emulsion cools. 8. A method according to claim 7, characterized in that the water is removed by distillation. 9. Characterized by the fact that the water is removed by adding an amount of organic phase which has a certain solubility for water. 10. Assume that the polysaccharide is agarose. removed by poly- according to claim 7, by further according to any one of the preceding claims, k e n e-