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