WO1989011493A1

WO1989011493A1 - Process for preparation of agarose particles

Info

Publication number: WO1989011493A1
Application number: PCT/SE1989/000281
Authority: WO
Inventors: Sten Porrvik; Gunnar Mattsson
Original assignee: Casco Nobel AB
Current assignee: Casco Adhesives AB
Priority date: 1988-05-25
Filing date: 1989-05-19
Publication date: 1989-11-30
Anticipated expiration: 1990-11-25
Also published as: SE8801952L; SE8801952D0; SE461149B

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

Process for preparation of agarose particles

Certain polysaccharides have become highly important as gel matrices for separating different biomolecules, such as proteins, nucleic acids etc. In this context, one of the more common gel-forming polysaccharides is agarose.

For preparing agarose gel particles, the normal procedure is first to prepare a solution of agarose in water at a temperature of 80-100°C, yielding a viscous aσueous solution. This solution is then emulsified at an elevated temperature in an organic phase in the presence of a suitable surfactant. This gives a water-in-oil emulsion with the viscous droplets of agarose-aqueous solution as discontinuous phase. By cooling the hot W/O-emulsion, the agarose molecules crystallise, with the formation of gel particles of a specific pore structure. The pore size can be affected by the content of agarose in the aqueous solution. The higher the agarose content is, the finer are the pores. The pore size is of vital importance since the gel particles are often used for separating biomolecules according to size, so-called molecule sieving. If an aqueous solution of different proteins is supplied to a column containing gel particles, a separation of the proteins according to size will take place in the sense that the smaller molecules will penetrate deeper into the network of the gel particles and, thus, be delayed in the column. The larger protein molecules will therefore leave the chromatographic column first. Protein molecules above a certain maximum size are completely unable to penetrate into the pores and, hence, will pass unimpededly through the column.

In the preparation of a W/O-emulsion of a polysaccharide solution, use has hitherto been made of different organic low-molecular-weight surfactants having a fairly high solubility in the continuous oil phase. Different hydrocarbons, such as toluene, carbon tetrachloride etc, have been used as organic phase. Examples of low-molecular-weight organic surfactants used are sorbitan sesquioleate, polyoxyethylene sorbitan monostearate and phosphate esters. In the preparation of W/O-emulsions from a polysaccharide solution by means of low-molecular-weight organic surfactants, a substantial spread in droplet size is generally obtained. To obtain good flow properties in chromatography columns, it is essential to have a narrow particle size distribution. This is the case especially when small gel particles are used for obtaining a sufficiently high chromatographic separation. Such columns provide a higher counterpressure, and it is then of vital importance to have a narrow size distribution. Otherwise, such a high pressure is built up in the column that the relatively soft gel particles are deformed, whereby the flow will cease almost completely.

To obtain a sufficiently narrow size distribution, the gel particles obtained from the W/O-emulsion are sieved. Sieving of these soft gel particles causes considerable technical problems. Also, agarose is a very expensive raw material. Since the sieving entails a limited yield within the desired particle size range, the particle size distribution initially obtained upon emulsification will considerably affect raw material costs in the preparation of agarose gel particles.

It is therefore of substantial importance in terms of economy to provide an emulsifying system giving a narrow particle size distribution for the W/O-emulsion of polysaccharide solution.

It is also of great importance that the emulsifying system makes it possible to prepare gel particles of different average particle size.

Finally, it is vital that the emulsifying system allows preparing polysaccharide gel particles of different dry solids content and, hence, different pore size. The present invention relates to a method for preparing particles of polysaccharides, using an inorganic protective colloid which satisfies to a considerable extent the requirements set out above, as appears from the accompanying claims.

In the method of the invention, the gel-forming polysaccharide is dissolved in water at a temperature above its dissolving temperature. The amount of polysaccharide should be within the range of from 0.5 to 20% by weight, preferably 2-15% by weight. The dissolving temperature of the polysaccharide varies with the type of compound used, the temperature being about 75°C for agarose, and a suitable temperature being about 80-100°C. In connection with the dissolution, an organic solvent having a limited water solubility is added, and the mixture is vigorously agitated so as to form a water-in-oil emulsion. The ratio of organic phase to aqueous phase should be within the range of from 0.5:1 to 10:1, preferably from 1:1 to 5:1. Upon emulsification, there is obtained, depending on the type and the amount of protective colloid, the amount of polysaccharide and the intensity of agitation, a droplet size within the range of from 3 to 200 μm, preferably from 5 to 100 μm, for the solution of polysaccharide. Before isolating the gel particles from the organic phase, the emulsion can be acidified, optionally after adding water, for dissolving the protective colloid. The dry solids content of the gel particles prepared is within the range of from 0.5 to about 50% by weight.

The protective colloid used according to the invention is characterised in that it comprises hydrophobated fine-grained inorganic compounds sparingly soluble in water. Normally, such inorganic, sparingly water-soluble compounds are too hydrophilic to be useful in these systems. The necessary hydrophobation can be achieved by adding small amounts of co-stabilisers adsorbed on the surface of the particles of the sparingly soluble inorganic compounds. It is also possible to make the particles suf ficiently hydrophobic by covalently bonding hydrophobic groups to the surface.

Examples of useful inorganic compounds sparingly soluble in water are different sparingly soluble salts of phosphate or polyphosphate, of sulphate, carbonate or silicate. It is also possible to use sparingly soluble oxides or fine-grained minerals. Specific examples of suitable inorganic compounds sparingly soluble in water are calcium phosphate, calcium sulphate, calcium carbonate, iron phosphate, magnesium hydroxide, aluminium oxide, aluminium hydroxide, silicon dioxide (silicic acid), iron hydroxide, barium sulphate, zinc oxide, fine-grained glass powder, bentonite, titanium dioxide etc. The amount of protective colloid should be within the range of from 0.1 to 50%, preferably from 0.3% to 10% based on the amount of the phase of polysaccharide solution.

Examples of suitable co-stabilisers for hydrophobating the particle surface of the sparingly soluble inorganic compounds are anionic, cationic organic compounds, alcohols, amines or derivatives of ethylene oxide or propylene oxide. Specific examples are carboxylic acids, mono- or diesters of phosphoric acid, alkyl sulphonic acids and quaternary ammonium compounds etc. As earlier mentioned, the particles of the sparingly soluble inorganic compounds may however also be made hydrophobic by bonding organic compounds to the surface of the particles. One example of such a case is treating particles of silicon dioxide with methyl silane to obtain hydrophobic methyl silanol groups on the particle surface. The hydrophobicity of the inorganic protective colloid varies with the type of polysaccharide and inorganic compound, the desired particle size and, primarily, the type of organic phase. By simple tests for a certain system, the amount of co-stabiliser can be easily determined. Examples of gel-forming polysaccharides are agar/agarose and dextran. Preferably, the invention relates to the preparation of agarose particles.

For the preparation of the water-in-oil emulsion, the organic phase may consist of different organic compounds, such as paraffin hydrocarbons, cycloalkanes, aromates, alcohols, esters, chlorinated hydrocarbons etc. Specific examples are C ₆-C ₁₆ alkanes, toluene, xylene, C₆-C₁₆ alcohols, carbon tetrachloride etc. For separating relatively small protein molecules, use is made of agarose gel particles with a higher agarose content, generally above 5% by weight, since a higher content entails smaller pores in the gel particles. It may however be difficult to prepare W/O-emulsions with the narrow particle size distribution which is characteristic of the system according to the invention if a polysaccharide-aqueous solution having a high polysaccharide content is used in the emulsification. A narrower particle size distribution is generally obtained when using agarose solutions having a lower dry solids content. According to a special embodiment of this invention, it is also possible also to prepare gel particles having a high dry solids content, generally within the range of from 5 to about 30% by weight, and a narrow particle size distribution. According to this variant, a W/O-emulsion having a lower dry solids content, preferably 0.5-5% by weight, is first prepared, and water is then removed from the droplets of polysaccharide-aqueous solution at a temperature above the temperature at which the polysaccharide solidifies so as to form a solid gel. This concentration by removing water can be carried out in different ways:

- by distillation, where a mixture of water and hydrocarbon is distilled from the W/O-emulsion;

- by diluting the organic phase with a substance increasing the solubility of the water in the continuous phase; - by adding more continuous phase if the continuous phase has a certain solubility for water, for instance hexanol; and

- by adding water-absorbent solids of inorganic or organic nature.

The invention will be described in more detail in the following Examples where parts and percentages indicate parts by weight and per cent by weight, respectively, unless otherwise stated. EXAMPLE 1

12 g agarose and 100 g distilled water were charged into a flask and heated under agitation to 95°C. After 30 min at 95°C, a solution, heated to 95°C, of 15 g nonionic surfactant (Tween 61) and 200 g toluene was added. The sample was emulsified with an Ultra-turrax to an average droplet size of 50 μm. The sample was thereafter cooled to room temperature, yielding solid gel particles of agarose-water. The particles were washed by centrifugation after they had settled and the toluene phase had been decanted. This test yielded a broad particle size distribution with only 40% by weight within the range of from 40 to 60 μm. Other tests with oil- soluble surfactants, such as phosphate esters, also gave broad distributions with a yield of about 40% in the range of 40-60 μm. EXAMPLE 2

The method according to Example 1 was repeated, however with the difference that the amount of Tween 61 was increased to 33 g. As a result, particles having an average particle size of about 20 μm were obtained, however with a broad particle size distribution. The yield within the range of from 15 to 20 μm was only about 30% by weight. Also other tests with oil-soluble surfactants provided a broad particle size distribution and a low yield in the range 10-20 μ. EXAMPLE 3

A colloid was pepared by mixing 10.2 g MgCl₂.6H₂O, 8 g 40% NaOH, 4 g lauric acid and 20 g toluene. The mixture was heated under agitation so as to melt the fatty acid.

3 g agarose, 97 g distilled water and 200 g toluene were charged into a flask and heated under agitation to 95°C. After 30 min at 95°C, the mixture was cooled to 75°C and mixed with a high-speed mixer of the Ultra-turrax type. By charging 8.5 g of the above-mentioned colloid, an emulsion with a droplet size of 50 μm was obtained. The sample was cooled to room temperature, yiedling solid gel particles of agarose-water. Distilled water was supplied and the mixture was acidified, thus dissolving the magnesium hydroxide. The aqueous phase was separated and the agarose gel particles were washed by centrifugation.

This test gave a narrow particle size distribution with 80% by weight in the range of from 40 to 60 μm. Further tests showed that it was possible to adjust the particle size distribution by the amount of colloid added.

If the amount of colloid was twice as large, a narrow particle size distribution was obtained with 65% between 15 and 20 μm. EXAMPLE 4 The method according to Example 3 was repeated, however with the difference that stearic acid was substituted for lauric acid. Also in this case, there was obtained a narrow particle size distribution with an average particle size of 50 μm. EXAMPLE 5

The method according to Example 3 was repeated, however with the difference that the colloid consisted of 4 g lauric acid, 11.3 g Ca(NO₃)₂×4H₂O, 2.6 g Na₃PO₄×12H₂O and 20 g toluene. 11 g of this colloid was charged, giving an average particle size of 50 μm with 70% by weight between 40 and 60 μm. EXAMPLE 6

3 g agarose, 97 g distilled water, 200 g toluene and 0.6 g Cab-O-Sil TS-720 (hydrophobated silica) were mixed and heated to 95°C. After 30 min, the sample was emulsified to 50 μm. It was then cooled to room temperature and water was added. The sample was washed by centrifugation. In this manner, a narrow particle size distribution was obtained with 80% by weight between 40 and 60 μm. In this case, it was not possible, after acidification, to completely wash off the hydrophobated silica particles from the surface of the agarose gel particles. EXAMPLE 7

1.7 g Ca(NO₃)₂ × 4H₂O, 1.5 g Na₃PO₄ × 12 H₂O, 3 mg NaBH. and water to 97 g were charged into a flask, and pH was adjusted to 9. 3 g agarose and 200 g hexanol were added, and the mixture was heated under agitation to 95°C. After 30 min at 95°C, the mixture was pre-emulsified with an Ultra-turrax for about a minute, 0.5 g lauric acid being added and pH being adjusted to 8. The sample was thereafter emulsified to about 20 μm, and another 100 g hexanol was added. The sample was cooled to room temperature. Distilled water was added and the mixture was acidified, the agarose gel particles passing into the aqueous phase. The aqueous phase was separated, and the agarose gel particles were washed by centrifugation. In this way, a narrow particle size distribution was obtained with 65% by weight between 15 and 20 μm. EXAMPLE 8

The method according to Example 7 was repeated, however with the difference that 0.8 g hexanoic acid was substituted for lauric acid. This gave a narrow particle size distribution with a mean of 20 um. EXAMPLE 9

4 g agarose, 96 g distilled water, 200 g hexanol and 0.6 g hydrophobated silica particles (Cab-O-Sil TS-720) were mixed and heated to 95°C. After 30 min, the sample was emulsified. Another 200 g hexanol was added and the sample was cooled to room temperature and washed by centrifugation. As a result, there was substantially obtained a narrow particle size distribution around 50 μm. EXAMPLE 10

An agarose solution was emulsified according to Example 7. At 95°C, an extra 400 g hexanol and 0.75 g lauric acid were added before the sample was cooled to room temperature. After washing the agarose gel particles, the sample was filtered so as to yield a dry cake, and the dry solids content was determined at 9.3%. Similarly, the dry solids content was determined for a sample without any extra hexanol at 4.5%. The density of the aqueous hexanol was determined at 0.830 by means of an aerometer and at

0.816 for non-aqueous hexanol. This corresponds to a water content of the hexanol of about 9%. 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 to a dry solids 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. The agarose gel particles obtained according to the Examples above can be used for biotechnical separation of the protein mixture. To be useful at higher flows, they can be hardened by cross-linking, for instance as described in European Patent Application 203049.

Claims

1. A method for preparing particles of gel-forming polysaccharides by dissolving the polysaccharide in water at a temperature above its dissolving temperature, and emulsifying the solution in an organic phase to form a water-in-oil emulsion, whereupon the emulsion is cooled such that the polysaccharide solidifies and is separated from the organic phase, c h a r a c t e r i s e d by carrying out the emulsification in the presence of an inorganic hydrophobic protective colloid.

2. Method as claimed in claim 1, c h a r a c t e r i s e d in that the amount of protective colloid is within the range of from 0.1% to 50% based on the amount of polysaccharide solution.

3. Method as claimed in claim 2, c h a r a c t e r i s e d in that the amount of protective colloid is within the range of from 0.3% to 10% based on the amount of polysaccharide solution.

4. Method as claimed in claim 1, c h a r a c t e r i s e d in that the inorganic hydrophobic protective colloid consists of a sparingly soluble metallic oxide or a sparingly soluble metallic phosphate which has been made hydrophobic by the addition of an anionic organic compound.

5. Method as claimed in claims 1-4, c h a r a c t e r i s e d in that the protective colloid is removed by acidification and washing.

6. Method as claimed in claims 1-3, c h a r a c t e r i s e d in that the protective colloid consists of silicon dioxide made hydrophobic with covalently bonded groups.

7. Method as claimed in claim 1, c h a r a c t e r i s e d in that water is removed from the droplets of polysaccharide solution before the emulsion is cooled.

8. Method as claimed in claim 7, c h a r a c t e r i s e d in that the water is removed by distillation.

9. Method as claimed in claim 7, c h a r a c t e r i s e d in that the water is removed by adding an additional amount of organic phase having a certain solubility for water.

10. Method as claimed in any one of the preceding claims, c h a r a c t e r i s e d in that the polysaccharide is agarose.