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

1. WHAT CPC COULD BRING TO
YOUR NATURAL COMPOUNDS
PURIFICATION PROCESS ?

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. GILSON GROUPE : CORE PRODUCTS
Automated liquid
handling
HPLC Preparative
Pipette
CPC, Flash and HPLC
preparative compact

4. CENTRIFUGAL PARTITION
CHROMATOGRAPHY (CPC)
BASICS

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. 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. CPC PURIFICATION WORKFLOW

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. CPC SYSTEM
Rotary Seals
CPC Rotor (or Column)
From pump
To detector/Collector

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. CPC CELLS
Heavier
Mobile Phase
Centrifugal
Force
Lighter
Stationary Phase
Descending Mode
Laboratoire Gilson purification,
GEPEA Saint Nazaire

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. 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. 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. 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. 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. CENTRIFUGAL PARTITION
CHROMATOGRAPHY (CPC)
NATURAL PRODUCTS APPLICATIONS EXAMPLES

18. CPC IN BATCH MODE

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. 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. 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. 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. CPC PRO, EXTRACTOR

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. 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. CPC MS

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. 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. CPC CANNABINOIDS
PURIFICATION

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. 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. CENTRIFUGAL PARTITION
CHROMATOGRAPHY (CPC)
CONCLUSION

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. 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. GILSON PURIFICATION

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. 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. GILSON GLIDER CPC (GGC) SOFTWARE
 SAG : Solvent system automatic generation
 Direct control of CPC device

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. 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. • 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. THANKS YOU!
Gregoire Audo, Gilson purification
gaudo@gilson.Com