WO2005103070A1

WO2005103070A1 - Affinity chromatography using ionic liquids

Info

Publication number: WO2005103070A1
Application number: PCT/GB2005/001549
Authority: WO
Inventors: Adam John Walker
Original assignee: The University Of York
Priority date: 2004-04-21
Filing date: 2005-04-21
Publication date: 2005-11-03
Also published as: GB0622206D0; GB2429284A; GB2429284A8; GB2429284B; GB0408854D0

Abstract

The present invention relates to a composition for separating a target molecule from, or in, an ionic liquid the composition comprising: (iii) a liquid medium comprising an ionic liquid; and (ii) a binding agent that is bound to a support wherein the binding agent is specific for a target molecule.

Description

AFFINITY CHROMATOGRAPHY USING IONIC LIQUIDS

The present invention relates to a composition and method useful in the separation of a target molecule from, or in, a medium containing an ionic liquid.

The desirable combination of properties exhibited by certain ionic liquids, including high solvation capability, stabilisation of reactive solutes and environmentally benign nature, has resulted in their use as solvents for numerous chemically and biologically catalysed processes (Welton T., Chem REV.99, 2071-2083 (1999); Cull et al Biotechnol. Bioeng. 69, 227-233 (2000); Sheldon R., Chem. Commun, 2399-2407 (2001); Magnus et al, J.Solution Chem.13, 583-587 (1984)).

Unlike most conventional organic solvents, many ionic liquids do not denature enzymes upon exposure, even in the presence of water as a molecular lubricant (Van Rantwijk et al, TIBTECH 21, 131-138 (2003)). Numerous enzymes have shown activity in ionic liquids (Walker & Bruce, Tetrahedron 60, 561-568 (2004); EP 1205555). Consequently, ionic liquids have been widely studied as potential replacements for organic solvents in enzyme catalysed processes.

Whilst numerous enzymes have shown activity in ionic liquids, no satisfactory technique for removing them from the ionic liquid is yet available. Enzyme removal is desirable in multi-stage processes such as biocatalysis since the biocatalytic enzymes are often very expensive. Currently, enzymes are recovered from ionic liquids by dialysis which is both inefficient and laborious. Another method for recovering enzymes from an ionic liquid is by solvent or salt precipitation, however, this process often results in denaturation of the enzyme.

The present inventors have exploited affinity interactions to separate target analytes from, or in, ionic liquid solvents. According to a first aspect, the present invention provides a composition comprising a liquid medium and a binding agent that is bound to a support wherein the liquid medium comprises an ionic liquid.

The binding agent may have binding specificity to one or more target molecules, for example, it may be monospecific or multispecific. The binding agent may bind at one or more sites and/or in one or more binding modes. The binding agent may be homogeneous or heterogeneous. Recognition between the binding agent and the target may be dependent upon steric, chiral, conformational, geometric, kinetic or thermodynamic effects. The interaction between the binding agent and target may take the form of hydrogen bonding, van der Waals interactions, covalent or electrostatic interactions, or a mixture of any of the above.

The binding agent may have an area on its surface, which may be a protrusion or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of one or more target molecules. Thus the binding agent has the property of binding specifically to one or more target molecules.

The binding between a binding agent and a target may be reversible or irreversible.

The binding agent may be one of a specific binding pair (binding agent and target) which may include antigen-antibody, biotin-avidin, hormone-hormone-receptor, receptor ligand, enzyme-substrate, nucleic acid/sugar-nucleic acid/sugar binding protein. Preferably the binding agent is a ligand.

Suitable binding agents of the invention include proteins, for example, antibodies or antibody fragments, antigens, hormones, enzymes, coenzymes, receptors, recombinant proteins, DNA binding proteins, protein surface domains involved in molecular recognition; peptides; nucleic acid, for example DNA such as an oligonucleotide, or RNA; fatty acid; affinity tag; biotin; avidin; carbohydrate; lectin. The binding agent may be naturally derived or wholly or partially synthetically produced.

As used herein when considering the interaction between the binding agent and the support, the term "bound" may encompass the binding agent interacting in some manner to the support, for example the binding agent may be adsorbed or immobilised to the support.

The interaction between the binding agent and the support may be reversible or irreversible. Preferably the interaction is a permanent, irreversible linkage. Thus the interaction between the binding agent and support may be a covalent or non-covalent interaction. Covalent interactions may include, for example, disulphide linkages

(between for example cysteine residues provided on the binding agent e.g protein and thiol residues provided on the support), ester linkages (between an activated acid residue on the binding agent/support and an alcohol on the respective binding agent/support) and carbon bonds. Non-covalent interactions may include for example electrostatic (provided by a + or - charge on the respective binding agent and support), hydrogen bonding (e.g N-H, O-H where the H is provided on the binding agent or support) and other irreversible interactions. Alternatively the surface of the support may be modified, for example by the use of a linker attached either to the binding agent or the support, to form a permanent interaction with the binding agent.

The support may be a support molecule or scaffold. The support may be a solid or semi-solid support surface. Preferably, the support is a solid support surface.

The support may comprise a polymer, for example agarose, sepharose, dextran, cellulose, xylan, lignin, nylon, polystyrene, polyethylene, polypropylene, polymethylmethacrylate, polytetrafluoroethylene and halogenated derivatives of any of the above, DNA, RNA, polyvinyl alcohol, polyvinyl chloride, polyester, polycarbonate, polyphosphate, polyethylene glycol, polybutadiene polyamide, polypeptide, polyacrylamide and derivatives, polyhema and derivatives, or any combination or copolymer of the above.

The support may be a solid support, for example, a glass or silica support.

The support may be in the form of beads for example polymer beads or glass beads. Alternatively, the solid support may comprise a microtitre plate or a glass slide, or may be composed of a gel, fibres, filaments, membrane, dendrites, resins, micelles, nanoparticles or phase interfaces.

The support may form part of a column, filtration device, mesh or gel.

The liquid medium may comprise an ionic liquid and a co-solvent (for example water). The co-solvent may comprise more or less than 10% by volume of the total, for example less than 10%, preferably less than 5% and more preferably less than 1% co-solvent, for example less than 0.5%, 0.25% or 0.1% co-solvent. Alternatively, the ionic liquid may consist of an ionic liquid.

The term "ionic liquid" may relate to liquids which may have a melting point not higher than 100°C.

The ionic liquid used in all aspects of the invention may be made up of anions and cations or alternatively consist of zwitterions carrying both a positive and a negative charge on the same molecule. Most commonly the ionic liquid will comprise an anion and a cation. The ionic liquid may be in a pure context or may comprise a homogeneous mixture with a co-solvent, a eutectic mixture, clathrate, emulsion or bi- or multi-phasic system. In the latter, the support and interactions may be used to maintain one or more target compounds (which may be catalysts, products or substrates) in one of the phases specified. Preferably, the ionic liquid is composed of cationic and anionic portions according to the following general formula:

[A]_n ⁺[Y]n^"

wherein n = 1 or 2 and wherein A represents the cationic portion and Y represents the anionic portion.

The cations utilised in the ionic liquids of the invention are typically composed of a quaternary nitrogen-based ion which may be aromatic, aliphatic, cyclic or acyclic.

Preferably, the cation is based on a nucleus selected from ammonium or phosphonium cations, pyrazolium cations, imidazolium cations, triazolium cations, pyridinium cations, pyridazinium cations, pyrimidinium cations, pyrazinium cations and triazinium cations. The cation nucleus may be substituted at any carbon or nitrogen atom by any alkyl, alkenyl, alkoxy, alkenedioxy, allyl, aryl, arylalkyl, aryloxy, amino, aminoalkyl, thio, thioalkyl, hydroxyl, hydroxyalkyl, oxoalkyl, carboxyl, carboxylalkyl, haloalkyl or halide function including all salts, ethers, esters, pentavalent nitrogen or phosphorus derivatives or stereoisomers thereof. Any of these functions may include a functional group selected from the group consisting of alkenyl, hydroxyl, amino, thio, carbonyl and carboxyl groups.

By "alkenyl" is meant any alkenyl group, preferably an alkenyl group with a carbon chain length of between 2 and 20 carbon atoms. The alkenyl group may be a straight chain, branched or cyclic group.

Preferred cations are those based on an ammonium or imidazolium nucleus.

The anion may be selected from, but is not necessarily limited to, halogenated inorganic anions, nitrates, sulphates, carbonates, sulphonates and carboxylates. The alkyl groups of the sulphonates and carboxylates may be selected from Ci - C₂₀ alkyl groups and may be substituted at any position with any alkyl, alkenyl, alkoxy, alkeneoxy, aryl, arylalkyl, aryloxy, amino, aminoalkyl, thio, thioalkyl, hydroxyl, hydroxyalkyl, carbonyl, oxoalkyl, carboxyl, carboxyalkyl or halide function, including all salts, ethers, esters, pentavalent nitrogen or phosphorus derivatives or stereoisomers thereof. For example, the anion may be selected from bis(trifluoromethylsulphonyl)imide, carbonate, hydrogen carbonate, sulphate, hydrogen sulphate, silicate, phosphate, hydrogen phosphate, dihydrogen phosphate, metaphosphate, methanesulphonate, trifluoromethanesulphonate, ethylenediaminetetraacetate, chloride, bromide, iodide, hexafluorophosphate, tetrafluoroborate, trifluoroacetate, pentafluoropropanoate, heptafluorobutanoate, oxalate, formate, acetate, propanoate, butanoate, pentanoate, hexanoate, heptanoate, octanoate, nonanoate, decanoate, benzoate, benezenedicarboxylate, benzenetricarboxylate, benzenetetracarboxylate, chlorobenzoate, fluorobenzoate, pentachlorobenzoate, pentafluorobenzoate salicylate, glycolate lactate, pantothenate, tartrate, hydrogen tartrate, mandelate, crotonate, malate, pyruvate, succinate, citrate, fumarate, phenylacetate.. An especially preferred anion is an organic carboxylate. When the anion is required to include a labile proton then glycolate, tartrate and lactate functional groups are preferred. All of these anions incorporate acid and hydroxyl functional groups.

In a further aspect, the invention provides a method for the preparation of a composition according to the first aspect of the invention, the method comprising: i) providing a binding agent within a liquid medium comprising an ionic liquid; and ii) contacting said binding agent with a support.

The support surface may be introduced into a chromatography column or onto a surface or interacting medium.

In a further aspect the invention provides a chromatography column comprising a composition according to the first aspect of the invention. The support surface may comprise a surface within the chromatography column itself or it may be separate to the chromatography column. The chromatography column may be a microcolumn.

The composition of the invention can be used to separate a target molecule from an ionic liquid or to separate a target molecule from other molecules in an ionic liquid. Thus, in a preferred aspect of the invention, the binding agent is specific to a target molecule to be separated, for example, the binding agent is complementary in structure to a target molecule to be separated.

The composition of the invention may be useful for the separation of any target molecule from, or in, an ionic liquid.

A target molecule may include a biological molecule and may include the product or substrate of a chemical or biological reaction. Examples of target molecules include any biological molecule (including proteins, for example, antibodies or antibody fragments, antigens, hormones, enzymes, coenzymes, receptors, recombinant proteins, DNA binding proteins, protein surface domains involved in molecular recognition), peptide, nucleic acid (for example DNA such as an oligonucleotide, or RNA), polymer (homogeneous or heterogeneous), fatty acid, affinity tag, biotin, avidin, carbohydrate, lectin, dye, pharmaceutical agent, small molecule e.g. toxin, substances of abuse such as cocaine, nicotine, opioids e.g. heroin, or explosives. Small molecules may include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Thus the composition of the first aspect of the invention may include a target molecule such as defined herein.

Where the separated target molecule is an enzyme, this can then be re-used in further biocatalysis reactions. The present invention thus allows for the re-cycling of molecules, such as enzymes.

In a further aspect, the invention provides a method of separating a target molecule from, or in, an ionic liquid , the method comprising:

(i) providing a composition comprising a liquid medium and a binding agent wherein (i) the liquid medium comprises an ionic liquid; (ii) the binding agent is bound to a support; and • (iii) the binding agent is specific to a target molecule to be separated; ii) contacting said binding agent with said target molecule to form a reaction product therewith; and iii) removal of the target molecule from the support.

The contact between the binding agent and the target molecule may take place within the liquid medium or at an interface therein or between the liquid medium and the support.

Preferably, in the method of the invention the binding agent binds specifically to one or more target molecules. Interactions which bind the binding agent to the target molecule may include hydrogen bonding, van der Waals interactions, covalent or electrostatic interactions, or a mixture thereof.

The reaction product formed may be an adduct or a complex. The removal of the target molecule from the support may occur by washing said support with the same or a different solvent. The target molecule may be eluted. Elution of the target molecule may cause it to precipitate, change the nature of its interaction with the binding agent, change its structure or activity or cause a further separation between targets.

A first washing of the support surface may be used to remove the ionic liquid from the support surface and the reaction product. In this case, a second washing step will be used to elute the target molecule from the support surface. The solutions or solvents useful in the first and second washing steps are likely to be different, for example, the first wash may be performed using an aqueous solvent such as water or a water miscible ionic liquid, and the second wash may be performed using an aqueous solution containing a bond-disrupting agent such as imidazole. Alternatively, the first wash may be performed using an ionic liquid that is different, or is of a different concentration, to the ionic liquid in the liquid medium. In a further alternative, the first washing step may be used to elute the separated target molecule from the support surface. The washing steps may be perfoπned individually or continuously.

Where the target molecule to be eluted is water soluble, washing may be carried out using an aqueous eluent. Where the target molecule to be eluted is insoluble in water, the washing step may be carried out using an ionic liquid or an alternative molecular solvent. Nonetheless, elution may be performed using any solvent, dependent upon the specific requirements of the individual system in question.

If being eluted from the support surface, the separated target molecule may subsequently be stored in an appropriate solvent, for example, an aqueous solvent or an ionic liquid. This solvent may or may not be identical to that used for elution.

The binding agent may form a reaction product or complex with the target molecule via any specific linkage, which may or may not be reversible. The interaction may include hydrogen bonding, van der Waals, electrostatic or covalent interactions between the binding agent and the target molecule. The hydrogen bonding may be between charged regions of the target molecule and specifically oriented, counter polarised groups on the binding agent.

In a preferred method of the invention, the target molecule to be separated is specifically and reversibly bound to a binding agent e.g ligand which is immobilised on a solid support. This facilitates the specific recovery of the target molecule from dilute solutions or in the presence of large numbers of contaminants, including the separation of active and denatured forms of the same ligand.

In a specific embodiment, the target molecule is an enzyme. This method thereby allows the separation of an enzyme from an ionic liquid which can be used in a further reaction or process, for example a biocatalysis reaction.

In a further embodiment of the invention, the target molecule a hydrophobic molecule, for example, a molecule that is soluble in an ionic liquid but not in water.

The target molecule may be labelled to allow it to be detected and/or analysed or quantified. The label may comprise, for example, an enzyme, a fluorescent label or a radioisotope which is readily detectable.

The target molecule to be separated may be synthesised in the liquid medium prior to its separation from the liquid medium or from one or more other molecules in the same liquid medium. Alternatively, the target molecule may be stored in the liquid medium prior to its separation therefrom.

In a further aspect, the present invention provides the use of an ionic liquid in the separation of molecules such as hydrophobic molecules and/or biomolecules. The use may be in the separation of a biomolecule(s) from an ionic liquid or in the separation of a biomolecule from one or more other biomolecules in an ionic liquid. Preferably, the ionic liquid comprises a ligand bound to a solid support.

Affinity chromatography is a separation technique, enabling the purification of almost any compound on the basis of its chemical structure or biological function. Thus in a preferred aspect, the present invention provides the use of an ionic liquid in affinity chromatography.

In a further aspect, the invention provides the use of a supported ligand, for example in a chromatography column, in the separation of a target molecule from, or in, an ionic liquid, by means of specific target-ligand interactions.

The application of ionic solvent technology to affinity chromatography offers significant advantages in several areas. A major application of the technique involves the extraction of a desired molecule from solution; since, for example, in the case of biological molecules this will usually be aqueous, there exists little scope or necessity for the introduction of ionic liquids. However, the use of an aqueous medium imposes restrictions upon the application of affinity chromatography, which ionic liquids may overcome.

In a further aspect, the invention provides the use of affinity chromatography in the separation of a target molecule from, or in, an ionic liquid.

The specific ligands often employed in affinity chromatography can be organic molecules, whose interaction with an aqueous mobile phase is consequently limited. Ionic liquids offer the unique capability of solvating both the biomolecular target and the affinity ligand, thus permitting homogeneous interaction between the two and increasing the efficiency of the technique. Furthermore, the ability of ionic liquids to solvate a much wider range of analytes than water may facilitate the extension of affinity chromatography to numerous hydrophobic target species (not necessarily biomolecules), which are not currently purified by this technique. Furthermore, the supports generally used in affinity chromatography (such as agarose) are in many cases limited in their application due to their ease of breakdown (which can lead to leaching of support or ligand), their enabling of only limited ligand densities and their incompatibility with many interesting and useful chemistries. Using ionic liquids as affinity chromatographic media may facilitate the use of a wider range of support surfaces, by reducing or removing any disadvantages affecting such supports in conventional media such as water. This could enable the extension of many current technologies into otherwise inaccessible applications.

The pharmaceutical agent, oxycodone, has been prepared by a mixture of enzymatic and chemical catalysis, in a single ionic liquid solvent (Walker & Bruce, Tetrahedron, 60, 561-568, 2004). This process involved an initial enzyme catalysed step, followed by an independent chemically-catalysed reaction upon the biotransformation product. This process suffers from the destructive effect of the chemical catalyst upon the enzyme, preventing the expensive biocatalyst from being reused and significantly depleting the overall yield. Affinity chromatography may be used to improve the process using some or all of the following steps: i) dissolve the substrate and components for biocatalysis, for example enzyme(s) and enzyme cofactor(s), in a medium comprising an ionic liquid; ii) perform biotransformation reaction in the ionic liquid medium; iii) run reaction solution through an affinity chromatography column, having one or more ligands bound thereto, to remove the enzyme(s) from the reaction solution; iv) add a chemical catalyst to the enzyme-free reaction solution from (iii). This allows any final reaction step(s) not requiring the biocatalyst to be performed. v) wash the affinity chromatography column to remove the ionic liquid. Liberate the enzyme with an appropriate eluent for recycling. Ionic liquids have been shown to be capable of eliciting the dissolution of large concentrations of biopolymers, such as proteins and nucleic acids. They have additionally been found to stabilize these polymers, both in temporal and thermal terms. This has resulted in ionic liquids being suggested as suitable storage media for high concentrations of biomolecules. However, the problem of how to remove the stored material from the ionic liquid has not been addressed as appropriate methodologies have not as yet been devised. Affinity chromatography, due to its selectivity, efficiency and use of a single common solvent, can be used to remove the stored material from the ionic liquid.

Enzyme activity in ionic liquids exhibits a complex relationship to solvation environment and protein folding, which has not been studied in detail. The selectivity of affinity chromatographic ligands for specific conformations of a given protein renders affinity chromatography a potentially invaluable tool for the investigation of enzyme secondary structure in ionic liquids, providing vital information necessary for the design of appropriate solvents for biocatalytic processes. Affinity chromatography, therefore, offers a major complementary technology to the use of ionic liquids in biocatalysis.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. An embodiment of the invention will now be described by example only;

EXAMPLE

Removal of morphine dehydrogenase from solution in the combined catalytic preparation of oxycodone

This method requires the preliminary preparation of a synthetic ligand capable of selectively and reversibly binding moφhine dehydrogenase, and the immobilisation thereof upon a solid support, in the form of chromatography beads.

Lyophilized moφhine dehydrogenase (MDH, 100 μg) is dissolved in 10 mL dry (<100 ppm H₂O) ionic liquid with vortexing. The resultant solution is centrifuged at 13,000 φm for 5 minutes and the supernatant filtered through 0.2 micron syringe filters. Anhydrous codeine free base (150 mg) is added, with 1 equivalent (380 mg) NADP sodium salt. The vessel is shaken at 110 φm until the reaction is complete. The immobilized MDH-selective ligand is then added on solid chromatographic beads (1 g). The beads are shaken in the ionic liquid for 10 minutes, after which they are removed by filtration. The enzyme-free ionic liquid solution of codeinone is retained for additional reaction steps. The recovered beads are transferred to a glass column and the bound MDH is eluted with an aqueous solution of imidazole. MDH recovered from the resultant solution is lyophilized and recycled for use in subsequent repeats of the reaction.

Claims

1. A composition for separating a target molecule from, or in, an ionic liquid the composition comprising: i) a liquid medium comprising an ionic liquid; and ii) a binding agent that is bound to a support wherein the binding agent is specific for a target molecule.

2. A composition as claimed in claim 1 wherein the liquid medium further comprises a co-solvent.

3. A composition as claimed in claim 1 wherein the co-solvent comprises less than 10%) by volume of the liquid medium.

4. A composition as claimed in claim 1 wherein the liquid medium comprises an ionic liquid comprising cationic and anionic portions according to the following general formula:

wherein nm = pq and wherein A represents the cationic portion and Y represents the anionic portion.

5. A composition as claimed in claim 4 wherein the cationic portion is composed of a quaternary nitrogen or phosphorus-based ion that is aromatic, aliphatic, cyclic or acyclic.

6. A composition as claimed in claim 5 wherein the quaternary nitrogen- or phosphorus-based ion is based on a nucleus selected from ammonium cations, phosphonium cations, pyrazolium cations, imidazolium cations, triazolium cations, pyridinium cations, pyridazinium cations, pyrimidinium cations, pyrazinium cations and triazinium cations

7. A composition as claimed in claim 4 wherein the ionic liquid includes an ion which contains a functional group selected from the group consisting of alkenyl, hydroxyl, amino, thio, carbonyl and carboxyl groups.

8. A composition as claimed in claim 4 wherein the anionic portion is selected from halogenated inorganic anions, nitrates, sulphates, carbonates, sulphonates and carboxylates.

9. A composition as claimed in claim 1 wherein the binding agent is one of a specific binding pair comprising binding agent and target molecule.

10. A composition as claimed in claim 9 wherein the specific binding pair is selected from antigen-antibody, biotin-avidin, hormone-hormone-receptor, receptor ligand, enzyme-substrate, nucleic acid/sugar-nucleic acid/sugar binding protein.

11. A composition as claimed in claim 1 wherein the binding agent is selected from the group consisting of proteins (for example, antibodies or antibody fragments, antigens, hormones, enzymes, coenzymes, receptors, recombinant proteins, DNA binding proteins, protein surface domains involved in molecular recognition) peptides, nucleic acid (for example DNA such as an oligonucleotide, or RNA) fatty acid, affinity tag, biotin, avidin, lectin.

12. A composition as claimed in claim 1 wherein the support surface is a solid or semi-solid support.

13. A composition as claimed in claim 12 wherein the semi-solid support is a polymer.

14. A composition as claimed in claim 13 wherein the polymer is agarose, sepharose. dextran, cellulose, xylan, lignin, nylon, polystyrene, polyethylene, polypropylene, polymethylmethacrylate, polytetrafluoroethylene and halogenated derivatives of any of the above, DNA, RNA, polyvinyl alcohol, polyvinyl chloride, polyester, polycarbonate, polyphosphate, polyethylene glycol, polybutadiene polyamide, polypeptide, polyacrylamide and derivatives, polyhema and derivatives, or any combination or copolymer thereof.

15. A composition as claimed in claim 12 wherein the solid support comprises glass or silica.

16. A composition as claimed in claim 12 wherein the support is part of a column, filtration device, mesh or gel.

17. A composition as claimed in claim 1 wherein support comprises a gel, fibres, filaments, dendrites, resins, micelles, nanoparticles or phase interfaces.

18. A composition as claimed in claim 1 wherein the target molecule is a biological molecule.

19. A composition as claimed in claim 1 wherein the target molecule is the product or substrate of a biological or chemical reaction.

20. A composition as claimed in claim 1 wherein the target molecule is selected from the group consisting of proteins (for example, antibodies or antibody fragments, antigens, hormones, enzymes, coenzymes, receptors, recombinant proteins, DNA binding proteins, protein surface domains involved in molecular recognition), peptides, nucleic acid (for example DNA such as an oligonucleotide, or RNA), polymer (homogeneous or heterogeneous), fatty acids, affinity tags, biotin, avidin, carbohydrates, lectin, dyes, pharmaceutical agents, small molecules (e.g. toxin, substances of abuse such as cocaine, nicotine, opioids e.g. heroin, or explosives).

21. A method for the preparation of a composition as claimed in claim 1, the method comprising: i) providing a binding agent within a liquid medium comprising an ionic liquid; and ii) contacting said binding agent with a support.

22. A chromatography column comprising a composition according to claim 1.

23. A method of separating a target molecule from, or in, an ionic liquid, the method comprising:

(i) providing a composition comprising a liquid medium and a binding agent wherein (i) the liquid medium comprises an ionic liquid; (ii) the binding agent is bound to a support; and (iii) the binding agent is specific to a target molecule to be separated; ii) contacting said binding agent with said target molecule to form a reaction product therewith; and iii) removal of the target molecule from the support.

24. A method as claimed in claim 23 wherein step (iii) comprises washing the support to elute the target molecule from the support.

25. A method as claimed in claim 24 wherein a first wash is performed using water.

26. A method as claimed in claim 25 wherein a second wash that is performed using an aqueous solution containing a bond-disrupting agent.

27. A method as claimed in claim 26 wherein the bond-disrupting agent is imidazole.

28. Use of a composition as claimed in claim 1 in the separation of a target molecule from, or in, an ionic liquid.

29. Use as claimed in claim 28 wherein the target molecule is a biological molecule.

30. Use as claimed in claim 28 or 29 wherein the separation process is affinity chromatography.

31. Use of affinity chromatography in the separation of a target molecule from,^; or in, an ionic liquid.