GB1600241A

GB1600241A - Ampholytic materials

Info

Publication number: GB1600241A
Application number: GB52432/76A
Authority: GB
Original assignee: UK Atomic Energy Authority
Current assignee: UK Atomic Energy Authority
Priority date: 1976-12-15
Filing date: 1976-12-15
Publication date: 1981-10-14

Description

(54) IMPROVEMENTS IN OR RELATING TO AMPHOLYTIC MATERIALS (71) We, UNITED KINGDOM ATOMIC ENERGY AUTHORITY, London, a British Authority, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to materials and particularly to ampholyte materials.

According to one aspect of the present invention there is provided an ampholyte material which is a composite material comprising a deformable gel having ampholyte properties (as hereinafter defined) retained within the pore structure of a porous rigid support material.

The deformable gel may be an ampholyte agent or an ampholyte agent may form part of the deformable gel.

By "ampholyte agent" we mean a substance which contains both insoluble basic and acidic chemical groups. The ratio of these two types of insoluble groups determines the net charge on the agent and thus the pH at which it has maximum capacity for protons (i.e. the maximum buffering capacity). Accordingly by "ampholyte properties" we mean having the ability to buffer hydrogen ion concentrations.

For biochemical applications the pH at which hydrogen ion concentration buffering is required is typically between 2.5 and 10.5 (i.e. at "non-extreme" pH values).

By "deformable gel" we mean a gel which itself is a non-rigid material (e.g. a zerogel). Such deformable gels include organic polymeric materials and certain inorganic materials, for example, silicic acid.

Preferably the porous rigid support material is in the form of discrete porous particles having an interconnected pore structure (for example those particles of inorganic material which may be prepared by a method as claimed in any one of Claims 1 to 9 or Claim 15 of our British Patent No. 1,421,531 (corresponding U.S. Patent is No. 3,943,072)), e.g. discrete porous particles of a natural earth, such as Celite or Keiselgulr.

The term "aerogel" has been used in the art to describe a rigid, preformed matrix containing pores and this term and the term "xerogel" are discussed in "An Introduction to Permeation Chromatography" by R. Epton and C. Holloway issued by Koch-Light Laboratories Ltd.

According to another aspect of the present invention there is provided a method for preparing a composite material of a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pores structure of a porous rigid support material comprising introducing a precursor for the gel into the pore structure of a porous rigid support material and treating the precursor to form the deformable gel in the pore structure.

It will be appreciated that the majority of the gel will be present in the internal pore structure of the porous rigid support material, but also it should be noted that some gel may be formed on the surface of the support material.

According to a further aspect of the present invention there is provided a method for preparing a composite material of a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material which method includes the step of treating an inactive deformable gel (as hereinafter defined) retained within the pore structure of a porous rigid support material to impart ampholyte properties to the deformable gel.

By "inactive deformable gel" we mean a deformable gel having little or no useful ampholyte properties.

Examples of organic polymeric materials which can be formed as inactive deformable gels in the pore structure of a porous rigid support material and subsequently treated to add further species thereby to impart ampholyte properties are: polysaccharide gels (e.g. agarose gels and cellulose gels) and synthetic polymer gels such as polymers of acrylates and polyvinyl alcohol polymer gels.

The inactive deformable gel can be treated to have ampholyte properties by modifying the inactive gel or by adding further species (e.g. acids and bases) thereto. For example, ampholyte properties may be imparted to an inactive deformable gel (e.g. an organic polymeric material such as cellulose or polysaccharide gel for example an agarose gel) by treating the inactive gel after formation in the pore structure to introduce acidic and basic groups. Another example is to form in the pore structure an inactive gel which contains either an acidic or basic component of an ampholyte and to treat the inactive gel to introduce the other component of the ampholyte (e.g. an inactive gel could comprise DEAE dextran (an amine) and acid groups could be added, or the inactive gel could comprise CMC cellulose (an acid) and amine groups could be added). It will be appreciated that DEAE and (MC are, respectively, abbreviations for "diethylaminoethyl" and "carboxymethyl".

An example of a composite material in accordance with the present invention is discrete porous particles of Celite (prepared by a method as claimed in B.P.

1,421,531 (U.S.P. 3,943,072)) having retained within the pore structure thereof an imine/glutamate gel.

(The word "Celite" is a Registered Trade Mark).

As hereinbefore disclosed the "deformable gel" may be an organic polymeric material.

Examples of organic polymeric materials which can be formed as insoluble ampholyte gels in the pore structure of a porous rigid support material are: copolymers of amino acids, acrylic acid derivatives, ethyleneimines, acidic polysaccharides and basic proteins. It will be appreciated that co-polymers of amino acids can provide basic and acidic groups, acrylic acid derivatives and acidic polysaccharides can provide acidic groups, and ethylene imines and basic proteins can provide basic groups, in ampholyte gels.

In view of the foregoing statements in this specification it will be appreciated that the porous rigid support material provides a rigid "skeleton" having dimensional stability as a support for the non-rigid deformable gel. Thus deformable gels which have, or can be treated to have, ampholyte properties, but which are difficult or inconvenient to handle because of their non-rigid nature (e.g.

hydrogels which will undergo dimensional changes when subject to pressures normally encountered in column operations (e.g. up to 3 atmospheres) and deform to cause an increase in back pressure), are incorporated into a composite material of the present invention which, due to the rigidity imparted by the porous rigid support material "skeleton", can be handled and used more easily.

Thus where the composite material comprises, for example, porous discrete particles with a deformable gel retained therein the composite materials can be loaded into, and used, conveniently in column systems.

We have found that if deformable gels having ampholyte properties are retained in accordance with the invention in discrete porous particles (for example porous particles of a natural earth (such as Celite or Keiselguhr) made by a method as claimed in our British Patent (U.S. Patent) hereinbefore mentioned) the composite material comprising deformable gel having ampholyte properties, and rigid support material possess desirable properties. Thus the particles of composite material tend to settle readily in aqueous media and can be used to form columns having good flow properties. Also the particles of composite material tend to be stable and not liable to release "fines".

The particles of composite materials can therefore be introduced into columns and used to function chromatographically with regard to systems containing selected chemical species (for example macromolecules such as proteins 1.

Ampholyte materials have weak ion exchange properties and therefore will be capable of sorbing macromolecules, such as proteins, from solution.

Examples of porous rigid support materials suitable for use in forming composite materials in accordance with the present invention are those disclosed in our British Patent No. 1,421,531 (U.S. Patent No. 3,943,072) hereinbefore mentioned. For example, using one type of the discrete porous particles disclosed therein we have prepared discrete porous particles of composite materials comprising porous rigid particles of Celite having retained therein a deformable gel having ampholyte properties. Discrete porous particles as disclosed in our aforementioned British Patent and being of materials other than Celite may, of course, be used in accordance with the present invention.

A number of methods may be used to prepare the ampholyte deformable gel in the pore structure of the porous rigid support material.

Thus, according to one embodiment of a method in accordance with the present invention a solution of a precursor for an ampholyte deformable gel is introduced into the pore structure of the porous rigid support material and the solution in the support material is subsequently treated with a precipitating agent to cause precipitation of an ampholyte gel from the precursor solution.

Thus, for example, a solution of an amine group containing molecule (such as an mine) and an acidic group containing molecule (such as monosodium glutamate) can be introduced into the pore structure of a porous rigid support material by soaking the support material in the solution and subsequently an ampholyte gel, containing both amine groups and acidic groups, produced by precipitation from the solution and cross-linking.

An inactive deformable gel suitable for subsequent treatment to produce ampholyte properties can be formed by precipitation (i.e. deposition) of rayon gel in the pore structure by introducing an aqueous solution of the cuprammonium complex of cellulose into the pore structure and subsequently treating the solution retained in the pore structure with dilute mineral acid to cause precipitation of rayon gel in the pores.

This rayon gel can be treated subsequently to formation in the pores to impart ampholyte properties.

Further examples of precipitation reactions which may be used to produce inactive gels suitable for subsequent treatment to produce ampholyte properties are (i) the regeneration of cellulose or cellulosic ion exchangers from solutions of the corresponding xanthates (e.g. by decomposition of the xanthates of cellulose, DEAE-cellulose or CMC-cellulose by aqueous mineral acids), (ii) decomposition of a silicate by mineral acid to give silicic acid gel, and (iii) precipitation of acidic polysaccharides with acid or calcium salts.

In another embodiment of a method in accordance with the present invention a precursor can be introduced into the pore structure of the porous rigid support material and subsequently polymerised to form a polymer gel in the pore structure, (e.g. acrylic acid derivatives may be introduced to the pore structure and subsequently polymerised to give gels of polymers and co-polymer of the derivatives -- e.g. acrylamide and the polymer gel treated to impart ampholyte properties.

In a further embodiment of a method in accordance with the invention crosslinking of the precursor can be used to form a gel. The cross-linking may be carried out with a chemical cross-linking chemical agent by diffusing the agent into the pore structure in order to react with the precursor. It is very desirable to carry out the cross-linking under conditions such that significant quantities of the precursor cannot diffuse out of the porous rigid support material whilst the cross-linking agent is diffusing into the porous rigid support material. This can be achieved by temporarily retaining the precursor in the support material (e.g. by precipitation) to hold it available in the pore structure thereof. for cross-linking and subsequently treating the temporarily retained precursor to cross-link it. It will be appreciated that by "temporarily retaining the precursor" we mean that the precursor is "localised" in the pore structure of the support substantially to prevent it diffusing out as the cross-linking agent diffuses in. Where the precursor has been introduced to the porous material in aqueous solution precipitation can be achieved. for example. by contacting the aqueous precursor solution with a water miscible organic solvent (e.g. acetone) capable of removing water from the aqueous solution thereby to precipitate precursor in the pore structure.

For example DEAE-dextran, polysaccharides and neutral polyols can be precipitated from aqueous solution using acetone as the water miscible organic solvent and cross-linked by use of epichlorhydrin. Examples of substances which can be used to produce a deformable gel in a composite material in accordance with the present invention by precipitation and cross-linking are dextran, DEAEdextran. dextran sulphate, CMC-cellulose, acrylamide, agarose and P.V. alcohol.

Cross-linking agents for use in accordance with the present invention can be, for example, epichlorhydrin, bis epoxides or dihalo components for polyols and. for example, dialdehydes for proteins.

Where the precursor, precipitation mechanism and cross-linking agent are such that the cross-linking of the precursor to form the gel is slow in comparison with the rate of precipitation, the precursor and cross-linking agent can be introduced into the pore structure of the porous rigid support material together in one solution (i.e. because precipitation will be effected before cross-linking occurs).

As hereinbefore disclosed the majority of the gel will be present in the internal pore structure of the porous rigid support material.

An important feature of the invention is that there is produced a composite material in which there is the minimum of deformable gel outside of the internal pore structure of the porous rigid support material. Thus, where the porous rigid support material is in the form of discrete porous particles there is a minimum of deformable gel formed between the particles, and substantially all of the deformable gel formed is retained by the particles with the majority of the deformable gel being in the internal pore structure thereof, so that the resulting composite material is in the form of discrete particles such as to aid, inter alia, handling, column packing and column operation.

Loosely adhering deformable gel may be removed from the particles of composite material by washing and, if necessary, mechanical means, e.g. sieving.

To assist in maximising the amount of the deformable gel, or inactive deformable gel, retained in the pore structure of the porous rigid support material where, in accordance with an embodiment of the method of the invention a solution of precursor is contacted with the porous rigid support material to introduce precursor into the pore structure, we prefer that the volume of the solution of precursor contacted with the support material (e.g. by soaking the support material in the solution) is approximately equal to the volume required to fill the pore structure. It will be appreciated that to minimise the amount of deformable gel, or inactive deformable gel, formed outside the pore structure the volume of the solution should not exceed the volume required to fill the pore structure.

It will be appreciated that in general the viscosity of the solution of the precursor should be such that it does not prevent uptake thereof by the support material (e,g. by capillary action).

Also we prefer that the volume of any reagent solutions used to treat the precursor in the pore structure to form a gel is not substantially in excess of that required to immerse the porous rigid support material.

A column of an ampholyte material in accordance with the present invention (e.g. in particulate form) may be used for isoelectric focussing in which a particular macromolecule (e.g. a protein) is retained from solution by an ampholyte material which buffers the surrounding solution to the pH at which the macromolecule has no net change (i.e. its isoelectric point). Under these conditions the particular macromolecule will be selectively retained by the ampholyte material when an electric field is applied to the column and any of the particular macromolecules introduced to the column in solution will concentrate in the ampholyte material.

It will be appreciated that the present invention is not limited to composite material which can be used in aqueous solution and that composite materials can be prepared which may be used in non-aqueous systems.

It will be appreciated that the deformable gel and porous rigid support material should be substantially insoluble in fluid substances with which they may be contacted in use (e.g. solutions to be buffered, solutions containing chemical components to be sorbed, feed solutions containing proteins and eluting agents solutions).

According to a further aspect the present invention provides a method for treating a solution to buffer it and/or for sorbing chemical species from solution comprising contacting the solution with a composite material comprising a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore. structure of a porous rigid support material.

The invention also provides a composite material whenever prepared by a method in accordance with the invention.

Also the invention provides a composite material obtainable by a method in accordance with the invention.

Our British Patent Specification No. 1,421,531 discloses and claims, inter alia: "A method for producing discrete porous particles for the selective retention of macromolecules from a fluid substance containing said macromolecules, said discrete porous particles having interconnected porosity throughout, which method includes the steps of preparing discrete green particles from a mixture containing solid particles of a finely divided, substantially insoluble, sorptive (as defined in the Specification), inorganic material and a fugitive additive in solution.

said mixture being formed by mixing said solid particles of inorganic material with a fugitive additive and a solvent therefor, said fugitive additive being for subsequently providing a pore structure in the inorganic material and said inorganic material being substantially insoluble in the solvent for the fugitive additive, the preparation of the discrete green particles being such that, and the fugitive additive being selected such that, the fugitive additive is provided in solid form in the green particles, and heating the green particles to remove fugitive additive therefrom and cause sintering of inorganic material to give discrete porous particles, the fugitive additive and the amount thereof in the green particles being selected such that the discrete porous particles have an interconnected porosity throughout the discrete porous particles providing an extended surface area and the pore structure is such as will allow the macromolecules to permeate the discrete porous particles and be sorbed".

and also claims discrete porous particles made by the method of British Patent No.

1,421,531.

Discrete porous particles (for example those fabricated from a finely divided, substantially insoluble, sorptive inorganic material by a method as claimed in any one of Claims 1 to 9 or Claim 15 of our British Patent No. 1,421,531) for use in accordance with the present invention preferably have a porosity of > 20% and an interconnected porosity with pores > 2000 A such as to allow both deformable gel and macromolecules (e.g. proteins or enzymes) to occupy the pores.

"Celite" (Registered Trade Mark) as hereinbefore mentioned is a natural diatomaceous earth produced by Johns-Manville Corp.

It is believed that porous materials, having dimensional stability such as foam metal and plastic foams can be used as porous rigid support materials in accordance with the present invention.

It is believed that acidic and basic groups can be grafted onto an inactive polyol or polysaccharide gel by initiation with peroxide or radiation in the presence of Fe (II) with ethylene imine and acrylic acid.

The invention will now be further described, by way of example only, as follows: Examples 1 to 4 These Examples relate to the production of ampholyte ion exchange composites.

Four solutions were used: polyethylenimine (BDH 10%) (Example 1) and three solutions containing polyethylene imine (BDH 10 ) mixed in various ratios with monosodium glutamate (20 ). (Examples 24).

5 ml samples of the four solutions were dry mixed with 8 ml samples of porous Celite particles prepared by a method as claimed in our British Patent Specification No. 1,421,531) (U.S. Patent 3,943,072); (400700,*4) diameter and a 1:1 mixture of acetone/50% glutaraldehyde (10 ml) was added. This localised the solution in the particles. The reaction proceeded for 8 hrs at 20 before the particles were washed and their ion exchange capacity determined by back titration with N hydrochloric acid. This capacity ranged from 3.5 mequivale.nts/ml for the mixture containing no glutamate to 2.5 meq/ml for one containing 3 parts glutamate to 1 part imine.

The maximum hydrogen ion buffering capacity for the ampholyte composites was as shown in the following table:

Composite pH Ex 1 -- Iimine alone 5.5 Ex 3 23 3 parts imine: I part glutamate 5.0 Ex 3 3I 1 part imine: I part glutamate 4.7 Ex 4 4I 1 part imine: 3 parts glutamate 2.7 Examples 5-9 Further ampholyte composites were prepared as follows: Solutions of polyethylene imine (I part) and monosodium glutamate (2 parts) were prepared with between 150,o and 1.5% total dissolved solids. 6 ml of each solution was drv mixed with 9 ml of porous Celite particles (of the kind used in Examples l) and reacted with 10 ml of 1:1 acetone/50 , glutaraldehyde at 200 for 4 hours.

The ion-exchange capacity and protein absorbing capacity of the composite particles was investigated. Although the ion exchange capacity of the particles decreased with decreasing solids content the protein adsorbing capacity went through a maximum as illustrated by the data in the table below. This suggests that the 10", solids sample not only has microporosity for hydrogen ions but was macroporous and able to bind protein molecules.

The following table shows dissolved solids concentration of the starting solutions against the OD of a supernatant obtained after stirring the composite particles with 50 ml haemoglobin in tris-buffer (pH 8.3) (2 mg/ml).

Example l % dissolved No. solids OD of supernatant 5 15 0.847 6 10 0,397 7 5 0.421 8 3 0.852 9 1.5 0.584 By way of comparison porous Celite particles carrying no organic material gave a supernatant having an OD of 0.821.

WHAT WE CLAIM IS: 1. An ampholyte material which is a composite material comprising a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material.

2. An ampholyte material as claimed in Claim 1, wherein the deformable gel is an ampholyte agent.

3. An ampholyte material as claimed in Claim 1, wherein an ampholyte agent is part of the deformable gel.

4. An ampholyte material as claimed in Claim 1, 2 or 3, wherein the porous rigid support material is in the form of discrete porous particles.

5. An ampholyte material as claimed in Claim 4, wherein the discrete porous particles are those prepared by a method as claimed in any one of Claims 1 to 9 or Claim 15 of British Patent Specification No. 1,421,531.

6. An ampholyte material as claimed in Claim 4 or Claim 5, wherein the discrete porous particles are discrete porous particles of a natural earth.

7. An ampholyte material as claimed in any one of Claims 1 to 6, wherein a component of the deformable gel is an organic polymeric material.

8. An ampholyte material as claimed in any one of Claims 1 to 7, wherein the deformable gel contains a co-polymer of amino acids, an acrylic acid derivative, an ethylene imine, an acidic polysaccharide, or a basic protein.

9. An ampholyte material as claimed in any one of Claims I to 8, wherein the deformable gel is an imine/glutamate gel.

10. An ampholyte material as claimed in Claim 7 or 8, wherein a component of the deformable gel is an organic polymeric material comprising a polysaccharide or a synthetic polymer.

11. An ampholyte material as claimed in Claim 10, wherein the polysaccharide is an agarose or a cellulose gel.

12. An ampholyte material as claimed in Claim 10, wherein the synthetic polymer is a polymer of an acrylate or a polyvinyl alcohol gel.

13. An ampholyte material which is a composite material comprising a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material, wherein the majority of said deformable gel is present in the internal pore structure of the porous rigid support material.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. Examples 5-9 Further ampholyte composites were prepared as follows: Solutions of polyethylene imine (I part) and monosodium glutamate (2 parts) were prepared with between 150,o and 1.5% total dissolved solids. 6 ml of each solution was drv mixed with 9 ml of porous Celite particles (of the kind used in Examples l) and reacted with 10 ml of 1:1 acetone/50 ,Ó glutaraldehyde at 200 for 4 hours. The ion-exchange capacity and protein absorbing capacity of the composite particles was investigated. Although the ion exchange capacity of the particles decreased with decreasing solids content the protein adsorbing capacity went through a maximum as illustrated by the data in the table below. This suggests that the 10", solids sample not only has microporosity for hydrogen ions but was macroporous and able to bind protein molecules. The following table shows dissolved solids concentration of the starting solutions against the OD of a supernatant obtained after stirring the composite particles with 50 ml haemoglobin in tris-buffer (pH 8.3) (2 mg/ml). Example l % dissolved No. solids OD of supernatant 5 15 0.847 6 10 0,397 7 5 0.421 8 3 0.852 9 1.5 0.584 By way of comparison porous Celite particles carrying no organic material gave a supernatant having an OD of 0.821. WHAT WE CLAIM IS:

1. An ampholyte material which is a composite material comprising a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material.

14. An ampholyte material as claimed in Claim 13 in the form of discrete

particles.

15. A method for preparing a composite material of a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material comprising introducing a precursor for the gel into the pore structure of a porous rigid support material and treating the precursor to form the deformable gel in the pore structure.

16. A method as claimed in Claim 15, wherein a solution of a precursor for an ampholyte deformable gel is introduced into the pore structure of a porous rigid support material and the solution in the support material is subsequently treated with a precipitating agent to cause precipitation of an ampholyte gel from the precursor solution.

17. A method for preparing a composite material of a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material which method includes the step of treating an inactive deformable gel (as hereinbefore defined) retained within the pore structure of a porous rigid support material to impart ampholyte properties to the deformable gel.

18. A method as claimed in Claim 17, wherein an inactive deformable gel (as hereinbefore defined) is formed in the pore structure of a porous rigid support material by introducing an aqueous solution of a cuprammonium complex of cellulose into the pore structure and subsequently treating the solution retained in the pore structure with dilute mineral acid to precipitate rayon gel in the pore structure.

19. A method as claimed in Claim 17 or Claim 18, wherein the deformable gel is treated to impart ampholyte properties thereto after formation in the pore structure by mddifying the deformable gel, or adding further species to the deformable gel.

20. A method as claimed in any one of Claims 17 to 19, wherein to impart ampholyte properties to an inactive deformable gel (as herein before defined) the gel is treated to introduce acidic and/or basic groups thereto.

21. A method as claimed in Claim 15 or Claim 16 comprising introducing a solution of an amine group containing molecule and an acidic group containing molecule into the pore structure of the porous rigid support material by soaking the support material in the solution and subsequently producing an ampholyte gel, containing both amine groups and acidic groups, by precipitation from the solution and cross-linking.

22. A method as claimed in Claim 21, wherein the amine group containing molecule is an imine.

23. A method as claimed in Claim 21 or 22, wherein the acidic group containing molecule is monosodium glutamate.

24. A method as claimed in any one of Claims 17, 19 or 20, wherein an inactive deformable gel (as hereinbefore defined) comprising a cellulose or a cellulosic ion exchanger is formed in the pore structure of a porous rigid support material bv regeneration of the cellulose or cellulosic ion exchanger from a solution of the corresponding xanthate.

25. A method as claimed in any one of Claims 17, 19 or 20, wherein a silicic acid gel is precipitated in the pore structure by decomposing a silicate by mineral acid.

26. A method as claimed in any one of Claims 15, 16, 17, 19 or 20, wherein a precursor for a deformable gel is introduced into the pore structure of the porous rigid support material and is subsequently polymerised to form a polymer gel in the pore structure.

27. A method as claimed in any one of Claims 15, 16, 17, 19 or 20, wherein a precursor for a deformable gel is introduced into the pore structure of the porous rigid support material and is cross-linked to form a gel.

28. A method as claimed in Claim 27, wherein the precursor is temporarily retained in the pore structure by precipitation prior to cross-linking.

29. A method as claimed in Claim 27, wherein the precursor is introduced into the pore structure of the porous rigid support material in aqueous solution and the aqueous solution in the pore structure is contacted with a water miscible organic solvent capable of removing water from the aqueous solution thereby to precipitate precursor in the pore structure.

30. A method as claimed in any preceding claim, wherein the precursor for a deformable gel comprises dextran, DEAE-dextran. dextran sulphate, CMCcellulose, acrylamide, agarose or polyvinyl alcohol.

31. A method for preparing a composite material of a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material comprising contacting a solution of a precursor for the gel with the porous rigid support material to introduce precursor into the pore structure and treating the precursor to form and retain the deformable gel in the pore structure, the volume of the solution of precursor contacted with the support material being approximately equal to the volume required to fill the pore structure.

32. A method of treating a solution to buffer it and/or for sorbing chemical species from solution comprising contacting thetsolution with a composite material comprising a deformable gel having ampholyte properties (as hereinbefore defined) retained within the pore structure of a porous rigid support material.

33. An ampholyte material comprising a composite material substantially as hereinbefore described with reference to any one of the Examples 1 to 9.

34. A method for preparing a composite material substantially as hereinbefore described with reference to any one of the Examples I to 9.

35. An ampholyte material comprising a composite material prepared by a method as claimed in any one of Claims 15 to 31 and 34.