Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

What CPC can bring to your natural compounds purification?

174 views

Published on

Workshop presented by Gregoire Audo from Gilson Purification at the annual meeting of the American Society of Pharmacognosy (ASP 2018) in Lexington, Kentucky.

Published in: Science
  • Be the first to comment

  • Be the first to like this

What CPC can bring to your natural compounds purification?

  1. 1. WHAT CPC COULD BRING TO YOUR NATURAL COMPOUNDS PURIFICATION PROCESS ?
  2. 2. GILSON BY THE NUMBERS Founded by Dr. Warren Gilson in 1957 # OF EMPLOYEES # OF ORGANIZATIONS # OF PATENTS GRANTED IN THE GLOBAL TOP We’re globally accessible to our customers through a complete network of experts. From product to customer-facing organizations, we’re investing in the life science industry. Since 1957, we’ve been making lab life easier for researchers by pioneering key advances to our lab devices. Our customers have made us a top reference company in the analytical & life science industries. 550+ 14 750+ 100
  3. 3. GILSON GROUPE : CORE PRODUCTS Automated liquid handling HPLC Preparative Pipette CPC, Flash and HPLC preparative compact
  4. 4. CENTRIFUGAL PARTITION CHROMATOGRAPHY (CPC) BASICS
  5. 5. CPC DEFINITION Centrifugal Partition Chromatography also names Counter Current Chromatography Preparative, pilot, and industrial scale liquid/liquid partition chromatography for purification of all kinds of molecules in solutions with the specificity to do not use solid support as silica Silica free preparative chromatography
  6. 6. CPC HISTORY/TERMINOLOGY Separatory funnel RLCC HSCCC CPC DCCC : Droplet Counter Current Chromatography RLCC : Locular Liquid Counter Current Chromatography HSCCC : High Speed Counter Current Chromatography CPC : Centrifugal Partition Chromatography Craig DCCC
  7. 7. CPC PURIFICATION WORKFLOW
  8. 8. CPC SETUP A CPC system is a preparative LC column analogous to a prep HPLC column or flash cartridges. A preparative-scale pump and an injector are required, and an optional detector and collector can be added. CPC Setup
  9. 9. CPC SYSTEM Rotary Seals CPC Rotor (or Column) From pump To detector/Collector
  10. 10. CPC COLUMN • High pressure resistance rotating chromatography column (rotor) • Discs composed of twin cells o Better retention of stationary phase o Allows for higher flow rates for faster separations
  11. 11. CPC CELLS Heavier Mobile Phase Centrifugal Force Lighter Stationary Phase Descending Mode Laboratoire Gilson purification, GEPEA Saint Nazaire
  12. 12. CPC GENERAL PRINCIPLE • Liquid-liquid partition chromatography o two immiscible liquid phases o Done by mixing two or more solvents • One phase is the stationary phase: maintained by centrifugal force • The other phase is the mobile phase: pumped through the liquid stationary phase • Compounds separated according to their partition coefficient KD between the phases Same principle as prep HPLC but no solid support Silica in HPLC Biphasic System in CPC
  13. 13. CPC GENERAL PRINCIPLE KD > 1 KD = 1 KD < 1 lower upper D A AK ][ ][= For a substance dissolved in a biphasic system, at a fixed temperature, the partition coefficient can be defined as follows: Concentration of A in upper phase Concentration of A in lower phase Separatory funnel Lighter upper phase Heavier lower phase
  14. 14. CPC GENERAL PRINCIPLE 1. Separatory funnels in series 2. Loading of stationary lightest phase in all separatory funnels 3. Injection of sample (mixture of three molecules) 4. Loading of mobile phase in the first funnel, shaking, separation 5. Transfer of lower mobile phase from the first to the second funnel, loading of fresh mobile phase in the first funnel 6. Step 5 repeat until the last funnel Heaviest phase: mobile phase Time KD > 1 KD = 1 KD < 1
  15. 15. CPC GENERAL PRINCIPLE 1. Separatory funnels in series 2. Loading of stationary lightest phase in all separatory funnel 3. Injection of sample (mixture of three molecules) 4. Loading of mobile phase in the first funnel, shaking, separation 5. Transfer of lower mobile phase from the first to the second funnel, loading of fresh mobile phase in the first funnel 6. Step 5 repeat until the last funnel Heaviest phase: mobile phase KD > 1 KD = 1 KD < 1 Time
  16. 16. FROM SEPARATORY FUNNELS TO CPC Heaviest Mobile Phase Lightest stationary phase Heaviest mobile phase Cells in series linked by ducts 3 CPC : disk with engraved cells 2 Craig system CPC Rotor or column: Stacked disks 1 Separatory funnels
  17. 17. CENTRIFUGAL PARTITION CHROMATOGRAPHY (CPC) NATURAL PRODUCTS APPLICATIONS EXAMPLES
  18. 18. CPC IN BATCH MODE
  19. 19. BATCH MODE : PURIFICATION OF EPA AND MYRISTIC ETHYL ETHER FROM MICROALGAE Crude extract CPC 250+ PLC 2250 ELSD detection CPC Fractions Mass (g) Yield (%) Purity GC Comments R3 0,939 23,39 % 99 EPA fractions R5 0,404 10,06 % 98 MYR fractions Total 4,015 g 77,69 %
  20. 20. PURIFICATION OF PIPERINE FROM BLACK PEPPER EXTRACT WITH CPC 250 SYSTEM PARAMETERS CPC Column CPC 250 Elution Flow-rate 10 mL/min CPC 250 + PLC 2050 RESULTS Mass Injected Separation Time Solvent Consumption HPLC Purity @ 254 nm 1g 50 min 500 mL 95%
  21. 21. PURIFICATION OF ANTHOCYANINS FROM BLACKCURRANT USING CPC 1000 COLUMN Peaks HPLC Areas of Crude Extract: • 40.3% anthocyanin A • 18.6% anthocyanin B • 6.6% anthocyanin C • 31.6% anthocyanin D PARAMETERS CPC Column CPC 1000 Elution Flow-rate 30 mL/min CPC 1000 + PLC 2250 RESULTS Mass Injected Separation Time Solvent Consumption HPLC Purity @ 530 nm 5g 80 min 2500 mL 97.6% A 97.4% B
  22. 22. PARAMETERS CPC Column CPC 250 Solvent system Az U Elution Flow-rate 12 mL/min CPC 250 + PLC 2050 RESULTS Mass Injected Separation Time Solvent Consumption HPLC Purity @ 254 nm 1,7g 60 min 720 mL >98% PURIFICATION OF CAULERPENYNE FROM CAULERPA TAXIFOLIA WITH CPC 250 MeOH-CH2Cl2 Crude extract Pure Caulerpenyne 268mg Laboratoire Gilson purification, Université de Nice, JCA
  23. 23. CPC PRO, EXTRACTOR
  24. 24. PRODUCTION OF VANILLIN FROM VANILLA OIL WITH CPC 250 PRO SYSTEM Fractions 9 to 10 Vanillin RESULTS Mass Injected Mass of vanillin Separation Time Solvent Consumption HPLC Purity @ 280 nm 70mL = 6;35g 314mg (6 to 8) 200mg (9 to 10) 23 min 950 mL 99,04% (6 to 8) 100% (9to 10) CPC 250 PRO + PLC 2050
  25. 25. BATCH MODE : REMOVAL OF NON DESIRABLE MOLECULE FROM VEGETAL MOSS Laboratoire Gilson purification, Robertet CPC 1000 PRO+ PLC 2250 Overload of the CPC column. Removal of indesirable molecule from the crude
  26. 26. CPC MS
  27. 27. 6 fractions automatically collect based on MS XIC channel of Artemisinin. PARAMETERS CPC Column CPC 250 Elution Flow-rate 10 mL/min RESULTS Mass Injected Separation Time Solvent Consumption HPLC Purity @ 280 nm 5g 40 min 800 mL 90% Triggering fraction collection! CPC 250 + PLC 2050 + VERITY® 1900 MS XIC 284-287 PURIFICATION OF ARTEMISIN FROM ARTEMISIA ANNUA EXTRACT WITH CPC 250 SYSTEM COUPLED TO MS DETECTOR
  28. 28. HPLC- DAD-UV profiles of the collected fractions Cafeine Theobromine The isolation of the two alkaloids was clearly improved with MS detection 12 fractions automatically collected based on MS screening (m/z 195 caffeine, m/z 181 theobromine) PURIFICATION OF THEOBROMINE AND CAFEINE FROM COCOA BY-PRODUCTS WITH CPC 250 COUPLED TO MS CPC 250 + PLC 2050 + VERITY® 1900 Laboratoire Gilson purification, Robertet
  29. 29. CPC CANNABINOIDS PURIFICATION
  30. 30. PURIFICATION OF CBD FROM CANNABIS OIL WITH CPC 250 SYSTEM Sample CBD Extract Mass/Volume injected 1 gr in 10 mL Flow rate 12 mL/min Detection 270, 266,215, Scan 200-600CBD CBD
  31. 31. CPC A EFFICIENT TOOLS FOR CANNABINOIDS PURIFICATION It allows to obtain different final product/fraction from cannabis oil as for example : Ultra pure cannabinoids standard from mg to multi gram scales as CBD, CBN, THC, CBDA …  Production of pure cannabinoids as CBD up to multi kg scale  Production of enriched CBD extract (50-70%), THC Free up to multi kg scale  ...
  32. 32. CENTRIFUGAL PARTITION CHROMATOGRAPHY (CPC) CONCLUSION
  33. 33. CPC VS. FLASH VS. PREP CPC PREP HPLC FLASH Silica No Yes Yes Sample Simple to complex Simple Simple to complex Solvent 5X less time Resolution Based on selectivity Based on efficiency Based on gradient Cost No consumable Prep HPLC column Cartridge Flexibility No stationary phase to replace Need to change column Need to change column Scale-Up High injection capacity Low injection capacity High injection capacity
  34. 34. CPC TECHNOLOGY FEATURES/ BENEFITS Features Benefits One column for several usages Cost effective techniques : No column to replace or silica to recycle, less solvent consumption No silica inside the column Preserve the integrity of the molecule : no irreversible absorption or denaturation of fragile molecule 100% liquid chromatographic process Complex sample injection in one step : complex natural extract, synthetic compounds, fermentation broth … Scalability of the technique High injection capacity : from milligrams to multi kilograms of sample
  35. 35. GILSON PURIFICATION
  36. 36. GILSON CPC RANGE BUNDLED WITH PLC PURIFICATION SYSTEMS • Direct control by Gilson Glider CPC (GGC) software • All-in-one system with pump, automated injection valve, automated backflush valve, detector, fraction collector and software • ELSD and MS detectors in option Complete purification system for CPC, prep HPLC and flash
  37. 37. GILSON CPC RANGE ONE HOUSING FOR ALL CPC COLUMNS MODEL INJECTION CAPACITY MAX ELUTION FLOW RATE MAXIMUM PRESSURE CPC 100 Up to 1g 15 mL/min 100 bar (1450 psi) CPC 250 Up to 6g 15 mL/min 100 bar (1450 psi) CPC 1000 Up to 30 gr 50 mL/min 80 bar (1160 psi) CPC 250 PRO Up to 30 gr 80 mL/min 100 bar (1450 psi) CPC 1000 PRO Up to 100 gr 350 mL/min 80 bar (1160 psi) General features o Controllable by built-in keyboard and/or Glider Software o Leak detector o Balancing detector
  38. 38. GILSON GLIDER CPC (GGC) SOFTWARE  SAG : Solvent system automatic generation  Direct control of CPC device
  39. 39. • CPC 5L + SKID avec pompe d’elution, pompe d’injection, collecteur de fraction, detecteur et vannes • Système cGMP, ATEX (option) • Premier prototype de nouvelle génération en cours de montage GILSON PURIFICATION : INSTRUMENTS
  40. 40. GILSON PURIFICATION : CONTRACT SERVICES Proposed our know-how and expertise in preparative and industrial chromatography to purify and / or enriched molecules of interests from complex mixtures as biological matrices, natural extract, synthetic mixture or fermentations booth Proposal:  Overall supply from feasibility to production through industrial intensification of the process  Use of innovative techniques as Centrifugal Partition Chromatography to reduce costs and solve problems related to the use of traditional techniques  Full project support to provide turnkey solutions instrument / method.  Unlock problems related to the use of traditional techniques
  41. 41. • Provide standard CPC line • Servicing for purification Feasibility studies Scale up and production studies • Provide global solution for purification COMPLETE SOLUTION SERVICES INSTRUMENTATION ENGINEERING A team of engineers to provide custom systems APPLICATION LAB A team of experts in purification Complete range Automated system Complete system GILSON PURIFICATION : CONTRACT SERVICES
  42. 42. THANKS YOU! Gregoire Audo, Gilson purification gaudo@gilson.Com

×