Ionic Liquids for Natural Product Extraction Adam Walker Bioniqs Ltd.

1. Ionic Liquids for Natural Product Extraction Adam Walker Bioniqs Ltd.

2. Ionic Liquids - Introduction BMIm PF6 (mp -79°C) NaCl (mp 801°C) • Ionic liquids are salts, with melting points below ambient temperature • They are composed solely of anions and cations – no water is present • Their regular, ordered structure gives them very different properties to conventional liquids • Ionic liquids based on imidazole have been well studied as novel solvents

3. Ionic Liquids - Properties • Extremely good solvents for many compounds – organic, inorganic, mineral, polymeric and biological • Non-volatile – do not evaporate • Non-flammable • Stabilize sensitive compounds • Many possible combinations of ions – can design the solvent for a particular application

4. Applications • Solvents for chemical and biochemical reactions • Extraction media for natural product isolation • Cleaning agents • Storage media for unstable compounds • Lubricants • Electrochemical/battery materials

5. “First Generation” Ionic Liquids - Disadvantages • Expensive in comparison with molecular solvents. • Very few commercially available. • Viscous • Poorly biodegradable and potentially toxic.

6. “Second Generation” Ionic Liquids • Bioniqs has developed new types of simple ionic liquids, based on ammonium salts. • These retain the advantages of ionic liquids, but also offer: • - reduced viscosity. • - low cost. • - straightforward preparation from sustainable precursors. • - minimal toxicity and environmental impact. • - recyclability and biodegradability.

7. Example Applications • Stabilization of enzymes for biocatalysis • Dissolution and processing of biomass • Natural product isolation and purification • Reactor decontamination

8. Stabilization of Biomolecules Activity of alcohol dehydrogenase enzyme after storage in water compared to standard (purple) and optimized (red) ionic liquids. H2O AEFF-390 AAFF-015 Enzyme in water deactivated after 10 days at room temperature. Enzyme in AEFF-390 retains activity after 90 days at room temperature.

9. Biomass Processing • Ionic liquids can dissolve numerous polymers, including biological macromolecules such as:- • Cellulose (up to 10% wt. solutions) • Lignin • Proteins • DNA/RNA • Bioprocessing requires solvent to dissolve substrate without denaturing enzyme. • Ionic liquid can be designed accordingly.

10. Natural Product Extraction & Processing Morphine (free base) – solubility (gL-1) • Solubility of natural products in ionic liquids often very much higher than in conventional solvents. • Can be used to extract high concentrations from natural sources, using small extractor volumes.

11. Natural Product Extraction & Processing • Dissolution of penicillin in ionic liquids – varies between solvent formulations • Solubility limit ranges from 20-270g/L • Can design solvent to optimize extraction efficiency

12. Natural Product Extraction & Processing • With support from MMV, Bioniqs has developed ionic liquids for artemisinin extraction, from both wet and dry plant material. • Extraction up to 4 times faster than in hexane • Solubility in ionic liquids up to 110 gL-1 @ 25°C. • Artemisinin may be recovered by adding water, precipitating and filtering off. • Residual solvent content of recovered artemisinin <0.001%. • Solvent may then be recycled and re-used Image provided courtesy of www.pp.clinet.fi

13. Solubility of two nitroaromatic pollutants in ionic liquids compared to water. Ionic liquid may be designed to dissolve one compound even in presence of structurally similar materials. Decontamination & Cleaning

14. Decontamination & Cleaning ILs designed to remove specified contaminant (amoxicillin) All ILs tested dissolve contaminant to >300g/L; several to >500g/L

15. Ecotoxicity and Biodegradation • ILs have been widely touted as “green” solvents • recyclable • non-flammable • non-volatile • BUT early imidazole-based ILs were • poorly biodegradable • toxic to micro-organisms • may form hazardous hydrolysis products • Biodegradability must be “built in” to an ionic liquid at the design stage.

16. Biodegradation of Ionic Liquids • Ordinary soil organisms can grow on Bioniqs ionic liquids and completely degrade them within 48 hours (above). • Degradation products are water, carbon dioxide and ammonium ion. • No significant toxic effects at concentrations below 80mM.

17. Summary • Ionic liquids are not a panacea, but can offer major benefits in certain key areas:- • Can now compete on comparable cost footing with conventional solvents for bulk applications. • Reduced pollution and waste • Improved process safety • Enhanced efficiency of reaction and extraction processes • Facilitate improved processes. • Increased solubilities – smaller volumes • Improved turnover rates and yields • Stabilization of sensitive molecules • Application to poorly soluble substrates

18. Acknowledgements

19. Further Information Bioniqs Ltd. Biocentre York Science Park Heslington York YO10 5DG United Kingdom 0(+44)1904 561538 office@bioniqs.com www.bioniqs.com