Post on 17-Jan-2016
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Centrifugal Partition Chromatographic (CPC)Technology: Application for Natural Product Isolation
Pilot Plant Corporation118 Veterinary Road
Saskatoon, SKCanada
Udaya N. Wanasundara
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
Topic 1:Introduction to Counter-Current Chromatography (CCC) & CPC
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.
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]
[A]
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.
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
Topic 2:CPC Instrument Setup &
Components
CPC Rotor Elements
Partition Disc Partition Disc PackPartition Channels
The Inner Workings of the CPC
Topic 3:The Advantages of CPC Technology
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 activityCompounds (molecular integrity) (denaturation)
Volume ratio of stationary/mobile Ratio is lowvery high (better resolution)
Topic 4:Applications of CPC Technology
CPC Technology has Applications in the Following Industries
Nutraceutical Fine Chemicals
Pharmaceutical Bio-Medical
Biotechnology Fats and Oils
Fermentation
Compounds That Can Be Isolated in High Purity by CPC Technology
Saponins Alkaloids
Chlorophylls Tannins
Carotenoids Phospholipids
Fat soluble vitamins Mono/Oligo-saccharides
Anthocyanins Lignans
Phenolic compounds Synthetic Compounds
Other active compounds present in plants(e.g., herbs and spices)
Much more!
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)
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)
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 40 60 80 100 120 140
DPA = 84.9%DHA = 84.6%C14:0 = 11.7%
Res
po
nse Starting oil: DPA = 15.2%
DHA = 39.7%
After CPC separation
Time (min)
20 40 60 80 100 120 140
Res
po
nse
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
mV
0.0
200
400
600
800
1000
1200
1400
Minutes10 30 50 70 90 110 130
C18:2=98% GLA = 98.3%
Purification of GLA from Borage Oil by CPC
BORAGE OIL FA (%)
C16:0 10.3C16:1 0.33C18:0 4.34C18:1 16.9C18:2 36.1C18:3 (GLA) 21.8C18:3 0.20C18:4 0.17C20:1 4.11C20:2 0.19C22:0 0.22C22:1 2.91C24:1 1.84
CPCFraction VI
mV
Minutes
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
GLA = 99.0%
Pre-purified GLA to 40%by salt solubility method
CPC separation
High Purity GLA from Borage Oil by CPC
Fraction VI
mV
0.0
400
800
1200
1600
Minutes
20.00 40.00 60.00 80.00 100.00 120.00
C20:4n6 = 83.3%C18:2 = 12.9%
C16:0 = 11.0%C18:1 = 85.6%
Purification of Arachidonic Acid By CPC
mV
20.00
40.00
60.00
80.00
100.00
120.00
Minutes
40 60 80 100 120 140 160
-TocopherolPurity = 97%
-TocopherolPurity = 99%
-TocopherolPurity = 97%
Separation of Tocopherols by CPC
Starting material had 70% tocopherol mixture
Topic 5:About POS Pilot Plant Corporation
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.
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
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.
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.
How to Contact Us:
POS Pilot Plant Corporation118 Veterinary Road
Saskatoon, SK, S7N 2R4CANADA
http://www.pos.ca pos@pos.caToll Free: (800) 230 2751 Fax: (306) 975 3766
Your Bioprocessing and BioSeparation Partner