Making liquid stationary phases

available for

high purity chromatography

purification at all scales

Liquid/Liquid Solid/Liquid

Activestationaryphase

mobilephase

The difference is the amount of

active stationary phase

Key benefits of liquid stationary phases

• High mass and volume injection loadings

• Improved handling of sample solubility

issues

• Ease and cost of scale-up

• Extremely low solvent usage

• Total sample recovery

• Reduced sample preparation

• New elution strategies

What is HPCCC?

High Performance Countercurrent Chromatography

• Crude material is partitioned between two immiscible layersof solvent phases

• Centrifugal rotation around 2 axis creates a planetarymotion that causes rapid mixing and the separation ofphases every revolution

• The stationary phase (SP) is retained by hydrodynamicforce field effect

• The mobile phase (MP) is pumped through the column

• Many successive liquid-liquid extractions occur enablingpurification of the crude material to occur

A complimentary and orthogonal liquid

chromatography technique

Using selectivity (A) rather than efficiency (B)

We achieve this by providing

High Performance CCC instruments

• These allow high resolution purifications at

high mobile phase flow rates

• Provide a range of instruments from

milligram to multi-kilo

Wide range of application

Typical HPCCC Applications Provides orthogonality to existing separation processes

Small molecule purification

Wide range of targets: synthetics, natural products, peptides

Preparative separations

Highly predicatable scale up, high throughput, low solvent use

“Difficult” samples

Crude samples, problematic solubility

Low concentration component isolation

Impurities or natural extract

1mg of each standard

loaded in 1ml (total 5mg)

Mixed Polarity Synthetic Standards

SS11 + 0.1%TFA

RP mode

analytical coil

pindolol propranolol

verapamil

acetanilide ketoprofen

1mg of each standard

loaded in 1ml(3 mg total)

Non-polar Synthetic Standards

SS14

NP mode

analytical coil

carbazaprinetrimerapramine

verapamil

30mg of each standard

loaded in 1ml total

Non-polar Synthetic Standards – 30 times scale-up

SS14

NP mode

analytical coil

verapamil

trimerapraminecarbazaprine

1mg of each standard

loaded in 1ml(3 mg total)

Polar Standards

SS01 + TFA

NP mode

analytical coil

nifenazonetryptophan

mandelic acid

6mg of each standard

loaded in 6ml total

Polar Standards – 6 times Scale-up

SS01 + TFA

NP mode

preparative coil

nifenazone

tryptophanmandelic acid

Preparative separations

Synthetic compound application• DE Centrifuge: Midi

• Type of separation: Hydrophobic (Non-polar)

• Crude loading per injection: 25 grams

• Target compound isolated per injection: 6 grams (average)

• Purity: > 92%

• Recovery: >95%

• Separation time: 25 minutes

• Total quantity of crude processed: 9 kg

• Total solvent used: 468 litres

Natural product application

• DE centrifuge: Maxi

• Type of separation: Hydrophilic (polar)

• Crude loading per injection: 160 grams

• Target compound isolated per injection: 23.6 grams

• Purity: >95%

• Recovery: >90%

• Separation time: 25 minutes

• Total quantity of crude processed: 6.7 kg

• Total solvent used: 456 litres

Difficult separations

Separation of GlucosinolatesBrunel-CCC - 980ml Capacity, 3.63mm bore, N=1200rpm, F=40ml/min

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35

Time (mins)

Op

tica

l Den

sity

(23

5nm

)

JH Sample - 5g in 10ml

GR

GI

Johns Hopkins - Repeatability

0

0.2

0.4

0.6

0.8

1

1.2

0 200 400 600 800 1000 1200

Mobile Phase Volume (mL)

Run 5

Run 6

Run 7

Run 8

Run 9

Run 10

•52.6g from 0.59kg

•34 runs

•47%w/w sample conc

•17g/run sample loading

•98.5% pure

•3 days

Low concentration components

Impurity isolation for identification purposes

0 20 40

0 20 40

Analytical and Preparative

runs

min0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

0

500

1000

1500

2000

2500

88% yield, 98% purity based on HPLC-UV

min0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

-200

0

200

400

18%

Starting material

Final sample

Courtesy of Pfizer UK

Minor impurity removal

20 40

Analytical run, Preparative

run

92% yield, 99.9% purity on HPLC (impurity not detected)

min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

3.4

%

Courtesy of Pfizer UK

Reduced Sample Preparation

Viscous syrup

Crude extract including precipitates

Loading capacity up to 50%w/w depending on solubility

Scale up

Scale-up is simply volumetric

• You use the ratio of the column volumes that

you are scaling between

• For example, 20ml column to 120ml column

would be 1:6

• For complete scale-up simply multiply

1. the sample volume by this ratio

2. The mobile phase flow rate by this ratio

Performed and optimised at analytical scale

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 2 4 6 8 10 12

Time, min

15.7%, Rs = 1.1

12.5 %, Rs = 1.4

9.9 %, Rs = 1.5

5.1 %, Rs = 1.6

2.5 %, Rs = 1.7

1.26 %, Rs = 1.8

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 2 4 6 8 10 12

Time, min

52.5 mg/ml BA - 25 mg/ml PC, Rs = 1.4

42.0 mg/ml BA - 20 mg/ml PC, Rs = 1.4

36.5 mg/ml BA - 15 mg/ml PC, Rs = 1.6

21.0 mg/ml BA - 10 mg/ml PC, Rs = 1.5

10.5 mg/ml BA - 5 mg/ml PC, Rs = 1.6

DE Mini

Directly transferred to the kilo/pilot scale

Maxi separation - 4.6 L - 20 and 33% of column volume (Vc)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time, min

Maxi- 0.92 L- 20% of Vc

Maxi- 1.51 L- 33% of Vc

Mini separation - 5.4 ml column - 10, 20 and 33% of column volume (Vc)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time, min

Mini-1.78 ml= 33% of Vc

Mini-1.07 ml= 20% of Vc

Analytical to semi-preparative scale-upMinor impurity removal to obtain high purity product - Scale-up x26, from 300 mg

on a 37 mL coil to 7.8g on a 950 mL coil

20 40

Analytical run, Preparative

run

92% yield, 99.9% purity on HPLC (impurity not detected)

min0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

3.4

%

Courtesy of Pfizer UK

Analytical to semi-preparative scale-up

0 20 40

0 20 40

Analytical and Preparative

runs

Impurity isolation for identification purposes - Scale-up x26, from 200 mg on

a 37 mL coil to 5.2g on a 950 mL coil

min0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

0

500

1000

1500

2000

2500

88% yield, 98% purity based on HPLC-UV

min0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

-200

0

200

400

18%

Starting material

Final sample

Courtesy of Pfizer UK

Literature Scale-up Examples

honokiolglucoraphinin(GR)

30L420LTotal Solvent Usage

342Total # runs

150g8kgTotal crude processed

20min25minCycle Time

20g23.6gTarget

Compound/injection

5%5%Column volume loading

50g crude in 190mL115g crude in 230mLLoading/injection

Hep:EtOAc:MeOH:Water (1.0:0.4:1.0:0.4)

Propanol:CH3CN:AS:Water (1.0:0.5:1.2:1.0)

Phase System

O

OH

OHSS

NO

HO

OHOSO3H

n

n=2 GR, glucoraphininn=1 GI, glucoiberin

OHOH

OH

OH

honokiol magnolol

1) Sutherland, I.A., J.

Chrom. A, 1151 (2007),

6-13;

2) 2) Fisher, D., Garrard,

I.J., Heuvel, R. van den,

Chou, F.E., Fahey, J.W.,

J. Liq. Chrom. Rel.

Tech., 28 (2005), 1913-

1922;

3) 3) Chen, L. et al., J.

Chrom. A, 1142 (2007),

115-122.

DE MAXI

Understanding HPCCC

CCC mechanism of separation is partitioning

Test tubes A columnHPCCC

Instrument

Key concepts in HPCCC

Mechanism of separation• Separation of compounds in HPCCC is based on liquid-liquid

distribution

• Purification occurs because of the different solubility of the

components in the liquid mobile and stationary phases

Compound retention determined by the distribution ratio, D

D can be calculated by partitioning studies

D=[ stationary phase ]

[mobile phase ]

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60 70 80 90

Elution Volume, VR

Retention is highly predictable

VC= 17.6mL

VS=13.4mL

Stationary phase fraction, Sf= 0.75For,

D=1

D=2

D=4VM V'R

VR

VR= V

C+(D - 1)V

S

Reverse phase (RP) - Stationary phase (SP) is upper phase (UP)

- Mobile phase (MP) is lower phase (LP)

Advantages: Direct analysis of fractions without need to vac down

Normal phase (NP) - Stationary phase (SP) is lower phase (LP)

- Mobile phase (MP) is upper phase (UP)

Advantages: Easier concentration of fractions

• NP is a good starting point for method development.

• If unsuccessful, switching to RP can potentially improve resolution.

• Advanced run modes: elution-extrusion, pH zone refining

HPCCC Run Modes

Method Development

Solvent System SelectionHEMWat solvent systems are suitable for the majority of applications

• 4 components – Heptane(or hexane), ethyl acetate, methanol & water

• Forms 2 immiscible phases

• Denser lower phase (LP) comprising mostly methanol and water

• Lighter upper phase (UP) of heptane and ethyl acetate

• Butanol, ethyl acetate and water provides separation in more polar

applications

• Additives - Buffers, acids bases expand the selectivity of HEMWat

solvent systems

• No pH-column stability issues as inert tubing no solid

HEMWat Solvent SystemSS Heptane EtOAc MeOH Butanol Water

1 0 0 0 5 5

2 0 1 0 4 5

3 0 2 0 3 5

4 0 3 0 2 5

5 0 4 0 1 5

6 0 1 0 0 1

7 More 1 19 1 0 19

8 Polar 1 9 1 0 9

9 1 6 1 0 6

10 1 5 1 0 5

11 1 4 1 0 4

12 1 3 1 0 3

13 2 5 2 0 5

14 1 2 1 0 2

15 2 3 2 0 3

16 5 6 5 0 6

17 1 1 1 0 1

18 6 5 6 0 5

19 3 2 3 0 2

20 2 1 2 0 1

21 5 2 5 0 2

22 3 1 3 0 1

23 4 1 4 0 1

24 5 1 5 0 1

25 Less 6 1 6 0 1

26 Polar 9 1 9 0 1

27 19 1 19 0 1

28 1 0 1 0 0

Upper Layer Solvent Composition

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Solvent System No

% V

olu

me

Methanol

Ethyl Acetate

Heptane

Butanol

Water

Lower Layer Solvent Composition

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Solvent System No

% V

olu

me

I.J.Garrard. L.Janaway, D.Fisher, J. Liq. Chrom. Rel. Tech., 30 (2007), 151-163

Provides separation

for the majority of

compounds

Good solubility of a

wide range of

materials

10 20 30 min

0.0

1.0

2.0

3.0

4.0

5.0mAU(x100)

0.0

25.0

50.0

75.0

CExtract-280nm,4nm (1.00)

14.388

17.791 18.56219.986 20.156

21.119 34.15935.171

36.41436.607

36.70937.194

37.626

10 20 30 min

0.0

0.5

1.0

1.5

2.0

2.5

mAU(x100)

0.0

25.0

50.0

75.0

CExtract-280nm,4nm (1.00)22.056 35.172

35.577

10 20 30 min

0.0

0.5

1.0

1.5

2.0

2.5

mAU(x100)

0.0

25.0

50.0

75.0

CExtract-280nm,4nm (1.00)19.938 20.163

27.28627.568 27.710 27.959

31.806 31.919 36.496

13

15

17

*

*

*

*

*

*

*

*

*

Solvent System Selection

Using the correct SS is key to obtaining an effective and efficient separation

Separation of Caffeine, feruli

acid and umbelliferone:

Analytical coil

Reverse phase

1ml/min flow rate

34 min run followed by 4

min extrusion (pump SP @

8ml/min)

Automated HPCCC Solvent System Screening

• Automated, unattended operation

using any standard analytical HPLC

systems (Agilent, Shimadzu, etc.)

• On-demand mixing of solvent

systems using quaternary HPLC

pumps

• 2 to 3 hours/solvent system screen:

– 5 solvent systems

– normal and/or reverse phase

separation modes

– any pH can be used

Automated Solvent System Screening Applied

to a set of 5 b-Blockers – RP, pH9.5

min5 10 15 20 25 30

mAU

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29Z01.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29Z02.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29Z03.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29Z04.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29A.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29B.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29Y01.D)

min5 10 15 20 25 30

mAU

0

DAD1 B, Sig=280,8 Ref=550,100 (06-29-09\06_29Y02.D)

Solvent

System

21

19

17

15

13

11

09

07

*

*

*

*

*

*

*

*

* propranolol

atenololpindolol

metoprolol alprenolol

Range of HPCCC equipment

DE HPCCC Instrument Range

Spectrum Midi Maxi

up to 2 5 – 40 500-1500

Flow rate (mL/min) 0.5 – 10 30 – 50 500 – 1500

1600 1400 850

Coil Volume (mL) 20 and 140 38 and 940 4600 or 18000

Loading (grams/run)

Rotational Speed 240g (RPM)

Integrated solutions

DE Solutions & Capabilities

Who are we?

• UK manufacturer of High Performance Countercurrent Chromatography instruments

• Instruments are sold and supported internationally from UK and US offices plus a

network of specialist international distributors

• Instrument range – Analytical, Preparative and Pilot plant/ Manufacturing scales

• Customer education & training, sample feasibility studies available internationally

Products and engineered solutions for

end-user use

DE Capabilities provided

• Product development and engineering design

• Feasibility studies

• Gram to Kilo separations to GLP

• Training

• Demonstration

A diverse range of customers

Key benefits of liquid stationary phases

• High mass and volume injection loadings

• Improved handling of sample solubility

issues

• Ease and cost of scale-up

• Extremely low solvent usage

• Total sample recovery

• Reduced sample preparation

• New elution strategies

Thank you for your attention

Any questions?