POS Pilot Plant Corporation

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Centrifugal Partition Chromatographic
(CPC) Technology Application for Natural Product
Isolation
Udaya N. Wanasundara
Pilot Plant Corporation 118 Veterinary
Road Saskatoon, SK Canada
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Topics Covered in Presentation
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
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Topic 1Introduction to Counter-Current
Chromatography (CCC) CPC
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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.

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Partition Coefficient

• For a given substance A the partition
  coefficient is defined as
• aA A upper phase/A lower phase
• aA is a constant at any given temperature, and
  unaffected by the presence of other substances or
  concentration of the solute in question.

A
A
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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.

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Summary of CPC Technology
CENTRIFUGAL PARTITION CHROMATOGRAPHY

• 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.

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Topic 2 CPC Instrument Setup Components
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(No Transcript)
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CPC Rotor Elements
Partition Disc
Partition Disc Pack
Partition Channels
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The Inner Workings of the CPC
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Topic 3 The Advantages of CPC Technology
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The Advantages of CPC Technology
CPC HPLC No column Expensive
columns High recovery Irreversible
adsorption High throughput Poor
loadability Retention of fragile Loss of
biological activity Compounds (molecular
integrity) (denaturation) Volume ratio of
stationary/mobile Ratio is low very high (better
resolution)
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Topic 4Applications of CPC Technology
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CPC Technology has Applications in the Following
Industries
Nutraceutical Fine Chemicals Pharmaceutical B
io-Medical Biotechnology Fats and
Oils Fermentation
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Compounds That Can Be Isolated in High Purity by
CPC Technology
Saponins Alkaloids Chlorophylls Tannins Car
otenoids Phospholipids Fat soluble
vitamins Mono/Oligo-saccharides Anthocyanins L
ignans Phenolic compounds Synthetic
Compounds Other active compounds present in
plants (e.g., herbs and spices) Much more!
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Fractionation of Tea Polyphenols by CPC
Isolation of tea catechins by CPC
technology Solvent system Chloroform/methanol/n
-propanol/water (91228 v/v) Purity was
determined by HPLC
Source Okuda et al., J. Liqud Chromato. (1990)
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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)
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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
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Purification of DHA by CPC
Starting oil DPA 15.2 DHA
39.7 After CPC separation
Response
DHA 84.6 C140 11.7
DPA 84.9
20
40
60
80
100
140
120
Pre-purified DHA to 66.4 and DPA to 24.3 by
urea
Response

CPC separation
DPA 90.8
DHA 98.7
20
40
60
80
100
120
140
Time (min)
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Purification of GLA from Borage Oil by CPC
BORAGE OIL FA () C160 10.3 C161 0.33 C180
4.34 C181 16.9 C182 36.1 C183
(GLA) 21.8 C183 0.20 C184 0.17 C201 4.11 C20
2 0.19 C220 0.22 C221 2.91 C241 1.84
1400
1200
CPC
1000
mV
Fraction VI
800
600
400
200
C18298
GLA 98.3
0.0
10
30
50
70
90
110
130
Minutes
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High Purity GLA from Borage Oil by CPC
Fraction VI
Pre-purified GLA to 40 by salt solubility method
mV

CPC separation
GLA 99.0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
110.00
120.00
130.00
140.00
150.00
160.00
Minutes
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Purification of Arachidonic Acid By CPC
1600
1200
mV
800

400
C204n6 83.3 C182 12.9
C160 11.0 C181 85.6

0.0
20.00
40.00
60.00
80.00
100.00
120.00
Minutes
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Separation of Tocopherols by CPC
?-Tocopherol Purity 97
Starting material had 70 tocopherol mixture
120.00
100.00
mV
?-Tocopherol Purity 97
?-Tocopherol Purity 99
80.00
60.00
40.00
20.00
40
60
80
100
120
140
160
Minutes
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Topic 5 About POS Pilot Plant Corporation
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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.

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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

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POSs 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.

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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.

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How to Contact Us
POS Pilot Plant Corporation 118 Veterinary
Road Saskatoon, SK, S7N 2R4 CANADA http//www.pos
.ca pos_at_pos.ca Toll Free [phone] Fax
[phone]
Your Bioprocessing and BioSeparation Partner