1. Centrifugal Partition Chromatographic (CPC) Technology: Application for Natural Product Isolation Pilot Plant Corporation 118 Veterinary Road Saskatoon, SK Canada Udaya N. Wanasundara

2. 1 – Introduction to Counter-Current Chromatography (CCC) & CPC 2 – CPC Instrumental setup & components 3 – The advantages of CPC technology 4 – Applications of CPC technology 5 – About POS Pilot Plant Corporation Topics Covered in Presentation

3. Topic 1: Introduction to Counter-Current Chromatography (CCC) & CPC

4. Counter Current Chromatography (CCC)  A separation technique that involves two liquid phases flowing in opposite direction.  Analogous to other chromatographic methods: The stationary phase and the mobile phase result in a relative countercurrent motion.  Both stationary and mobile phases are liquids.  Separation is based on the partition co-efficient (  ) of the components in mixture.

5. For a given substance “A” the partition coefficient is defined as:  A = [A] upper phase/[A] lower phase  A is a constant at any given temperature, and unaffected by the presence of other substances or concentration of the solute in question. Partition Coefficient [A]

6. The Basis of CPC Technology When a compound is dissolved in a mixture of two liquids, the compound will concentrate more in one of the liquids. The compact CPC unit uses over one thousand extraction cells in a high speed spinning disk pack to separate and purify the desired compound. Each cell increases the purity of the desired compound until a high purity product is created.

7. Summary of CPC Technology CPC is derived from CCC. A new liquid chromatographic technique utilizing liquid-liquid partition and counter-current distribution of solutes between 2 immiscible liquid phases. No solid support for the stationary phase is involved. Stationary phase solvents are retained in the column by the action of centrifugal force. CENTRIFUGAL PARTITION CHROMATOGRAPHY

8. Topic 2: CPC Instrument Setup & Components

10. CPC Rotor Elements Partition DiscPartition Disc Pack Partition Channels

11. The Inner Workings of the CPC

12. Topic 3: The Advantages of CPC Technology

13. CPC HPLC No columnExpensive columns High recoveryIrreversible adsorption High throughputPoor loadability Retention of fragileLoss of biological activity Compounds (molecular integrity)(denaturation) Volume ratio of stationary/mobileRatio is low very high (better resolution)

14. Topic 4: Applications of CPC Technology

15. CPC Technology has Applications in the Following Industries NutraceuticalFine Chemicals PharmaceuticalBio-Medical BiotechnologyFats and Oils Fermentation

16. Compounds That Can Be Isolated in High Purity by CPC Technology SaponinsAlkaloids ChlorophyllsTannins CarotenoidsPhospholipids Fat soluble vitaminsMono/Oligo-saccharides AnthocyaninsLignans Phenolic compoundsSynthetic Compounds Other active compounds present in plants (e.g., herbs and spices) Much more!

17. Fractionation of Tea Polyphenols by CPC Isolation of tea catechins by CPC technology Solvent system: Chloroform/methanol/n-propanol/water (9:12:2:8 v/v) Purity was determined by HPLC Source: Okuda et al., J. Liqud Chromato. (1990)

18. Purification of Ginsenosides from Ginseng Isolation of Ginsenosides by CPC technology Solvent system: Ethyl acetate/1-butanol/water Purity was determined by TLC Source: Le et al., J. Liqud Chromato. (1995)

19. Methods Developed by POS Bio-Separation Unit POS has developed several procedures to obtain high purity bio-active compounds: e.g.High value Fatty Acids Natural Tocopherols

20. 20406080100 120 140 DPA = 84.9% DHA = 84.6% C14:0 = 11.7% Response Starting oil: DPA = 15.2% DHA = 39.7% After CPC separation Time (min) 20 40 60 80 100 120 140 Response DPA = 90.8%DHA = 98.7% Purification of DHA by CPC Pre-purified DHA to 66.4% and DPA to 24.3% by urea CPC separation

21. mV 0.0 200 400 600 800 1000 1200 1400 Minutes 1030507090110130 C18:2=98%GLA = 98.3% Purification of GLA from Borage Oil by CPC BORAGE OILFA (%) C16:010.3 C16:10.33 C18:04.34 C18:116.9 C18:236.1 C18:3 (GLA)21.8 C18:30.20 C18:40.17 C20:14.11 C20:20.19 C22:00.22 C22:12.91 C24:11.84 CPC Fraction VI

22. mV Minutes 0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00100.00110.00120.00130.00140.00150.00160.00 GLA = 99.0% Pre-purified GLA to 40% by salt solubility method CPC separation High Purity GLA from Borage Oil by CPC Fraction VI

23. mV 0.0 400 800 1200 1600 Minutes 20.0040.0060.0080.00100.00120.00 C20:4n6 = 83.3% C18:2 = 12.9% C16:0 = 11.0% C18:1 = 85.6% Purification of Arachidonic Acid By CPC

24. mV 20.00 40.00 60.00 80.00 100.00 120.00 Minutes 40 60 80 100 120 140 160  -Tocopherol Purity = 97%  -Tocopherol Purity = 99%  -Tocopherol Purity = 97% Separation of Tocopherols by CPC Starting material had 70% tocopherol mixture

25. Topic 5: About POS Pilot Plant Corporation

26. POS Pilot Plant Corporation  POS is a private confidential contract research organization  Successfully serving clients for over 20 years in the nutraceutical, lipids, bio-technology, pharmaceutical and related industries.  5 large pilot plant areas and 11 fully equipped laboratories  90 staff covering scientific, technical, project management, operational and administrative functions.

27. POS Pilot Plant Corporation (continued)  POS helps create superior new products more quickly using cost effective methods.  Unique facilities and capabilities not available anywhere else.  Take new products ideas from conception laboratory bench scale pilot scale custom manufacturing all in one facility.  POS is part of Innovation Place: A world class research park and agricultural biotechnology cluster.  Up to date and comprehensive web site at www.pos.ca

28. POS’s CPC Capability  Lab Scale CPC: (1.5L volume, 5-25 ml/injection)  Pilot Scale CPC: (5.4L, 25-100 gm/injection)  New Product Development  Production of high purity products for analytical standards, clinical trials, market development and other purposes.  All services available on a contract basis with flexible working arrangements.  Creation of superior products, faster without capital expenditures on equipment or staffing.

29. Summary CPC technology is a new purification technique which can be used to produce very high purity chemical components with distinct advantages. POS serves clients by developing CPC methods for isolation and purification of chemical components and custom manufacturing of these materials. POS has developed a number of novel processes to separate bio-active compounds.

30. How to Contact Us: POS Pilot Plant Corporation 118 Veterinary Road Saskatoon, SK, S7N 2R4 CANADA http://www.pos.capos@pos.ca Toll Free: (800) 230 2751 Fax: (306) 975 3766 Your Bioprocessing and BioSeparation Partner