What differentiatesDynamic Extractions?

Making liquid stationary phasesavailable for

high purity chromatographypurification at all scales

Liquid/Liquid Solid/Liquid

Activestationary

phase

mobilephase

Stagnant mobilephase

Silica skeleton

The difference is the amountof active stationary phase

Key benefits of liquid stationary phases

• High injection loadings

• Improved sample solubility

• Reproducibility and ease of scale-up

• New elution strategies

• Total sample recovery

• Little or no sample preparation

We achieve this by providingHigh Performance CCC instruments

• These allow high resolution purifications athigh mobile phase flow rates

• Provide a range of instruments frommilligram to muilti-kilo

Key applications• Where you have a target to focus upon, either product or

impurity

• All types of molecule

– Synthetics (small or large molecules)

– Peptides

– Natural products

• The first option for lead candidates and analogs where youwant to avoid redeveloping chromatography as you move upin scale

• Where you recognise that solubility of your compounds willbe problematic

HPCCC is complimentary and orthogonal

Understanding HPCCC

CCC mechanism of separation is partitioning

Test tubes A column HPCCCequipment

Essential HPCCC theory

Distribution Ratio

• Purification occurs because of the different solubility ofthe components in the liquid mobile and stationaryphases

• This is determined by the Distribution Ratio (D) = CS/CM

• CS is the concentration of the component in the stationaryphase

• CM is the concentration of the component in the mobilephase

Volume

Vm

DVs

VsS

amp

leIn

ject

ion

So

lven

tF

ron

t(D

=0

)

Sam

ple

Elu

tio

n(D

)

Syst

emV

olu

me

(D=

1)

Ex

tra-

coil

Vo

lum

e(V

ext)

Co

nce

ntr

atio

n

Highly predictable• A component with a low

distribution constant has ahigher concentration in themobile phase and elutes early

• A component with a D=1 willelute after one column volume ofmobile phase has been pumpedthrough the column

• A component with a highdistribution constant has ahigher concentration in thestationary phase elutes late

• A component with a D=5 willelute after 5 column volumes

Sf = 90% 1 MP : 9 SP

Sf = 50% 1 MP : 1 SP

Sf = 33% 2 MP : 1 SP

What is stationary phaseretention?

0

0 . 0 5

0 . 1

0 . 1 5

0 . 2

0 . 2 5

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0

1

2 3

4

5

6

7

89

1 0

0

0 . 0 5

0 . 1

0 . 1 5

0 . 2

0 . 2 5

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0

1

2

3

4

5

6

7

89

1 0

0

0 . 0 5

0 . 1

0 . 1 5

0 . 2

0 . 2 5

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0

2

35 7

91 0

18

0

0 . 0 5

0 . 1

0 . 1 5

0 . 2

0 . 2 5

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0

R e t e n t i o n v o l u m e ( m L )

57

91 0

2

UV signal (254 nm )

Sf = 90%

Sf = 70%

Sf = 30%

Sf = 50%

VS =108 mL

VS = 84 mL

VS = 60 mL

VS = 36 mL

Showing the importance of stationaryphase retention

Stationary phase retentionincreases as rotational speedof the CCC instrument isincreased

Experimental conditions:– The same CCC instrument was used throughout– The mobile phase flow rate was constant– The solvent system was constant– The injected sample had constant volume and

concentration– The only variable that was altered was the

rotational speed of the CCC instrument

Defining Normal and Reverse Phase

Upper Phase (Organic)

Lower Phase (Aqueous)

Periphery

(Heavy End)

Centre

(Light End)

Normal Phase

Lower Phase (Aqueous Stationary)

Upper Phase (Organic mobile)

X

Reverse Phase

Upper Phase (Organic Stationary)

Lower Phase (Aqueous mobile)

X

Method Development

Volume

Vm

DVs

Vs

Sam

ple

Inje

ctio

n

So

lven

tF

ron

t(D

=0

)

Sam

ple

Elu

tio

n(D

)

Syst

emV

olu

me

(D=

1)

Ex

tra-

coil

Vo

lum

e(V

ext)

Co

nce

ntr

atio

n

Important Considerations in Developing aSeparation Method • Elution Time is

determined by D

• To keep run times to aminimum, D valuesshould be less than 5.0

• To produce the bestseparations, D valuesshould differ by > 0.5

Selectivity is the power of HPCCC

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

0 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

Dis

trib

uti

on

Rati

o

Benzyl Alcohol

Para-Cresol

No Heptane EtOAc MeOH Butanol Water

1 0 0 2 2

2 0 0.4 0 1.6 2

3 0 0.8 0 1.2 2

4 0 1.2 0 0.8 2

5 0 1.6 0 0.4 2

6 0 2 0 0 2

7 0.1 1.9 0.1 0 1.9

8 0.2 1.8 0.2 0 1.8

9 0.29 1.71 0.29 0 1.71

10 0.33 1.67 0.33 0 1.67

11 0.4 1.6 0.4 0 1.6

12 0.5 1.5 0.5 0 1.5

13 0.57 1.43 0.57 0 1.43

14 0.67 1.33 0.67 0 1.33

15 0.8 1.2 0.8 0 1.2

16 0.91 1.09 0.91 0 1.09

17 1 1 1 0 1

18 1.09 0.91 1.09 0 0.91

19 1.2 0.8 1.2 0 0.8

20 1.33 0.67 1.33 0 0.67

21 1.43 0.57 1.43 0 0.57

22 1.5 0.5 1.5 0 0.5

23 1.6 0.4 1.6 0 0.4

24 1.67 0.33 1.67 0 0.33

25 1.71 0.29 1.71 0 0.29

26 1.8 0.2 1.8 0 0.2

27 1.9 0.1 1.9 0 0.1

28 2 0 2 0 0

MorePolar

MoreNon-Polar

This set of phase systems covers 80% plus of thetarget molecules that we are asked to separate 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

Using HPLC data to determine initialsolvent system selection

Normal Phase Reverse Phase

0 – 20 2 – 7 1 - 5

20 – 40 8 – 13 5 - 10

40 – 60 13 – 19 10 - 16

60 – 80 19 – 25 16 - 22

80 – 100 25 – 27 23 - 27

Substitute acetonitrile for methanol or Toluene for Hexane

Compound(s) Elution PointFrom C18 rp-

HPLC column (% Acetanitrile)Solvent System operating windows giving a D value between 1.0 and 3.0

Sample solubility

Samples partition between either themobile or stationary phase

New elution strategies

30mg of each standardloaded in 1ml total

Elution extrusion

Difference of partitioncoefficient only 0.3

min1 1 .2 1 .4 1 .6 1 .8

mAU

-300

-200

-100

0

100

200

300

DAD 1 C, Sig=210,8 Ref=360,100 (H:\DATA\2005 \SD51115A\061-0101 .D)

Dual mode elution

Reconstructed chromatogram for normal CCC

-50

450

950

1450

1950

2450

0 5 10 15 20

Fraction number

Am

ou

nt

of

co

mp

ou

nd

Standard mode elution - 200 mginjection Resolution: 0.25

Reconstructed chromatogram of Dual CCC

-50

450

950

1450

1950

2450

2950

3450

3950

30 35 40 45 50 55 60

Fraction number

Am

ountofpro

duct

Dual mode elution - 250 mginjection Estimated resolution: 0.85

min1 1 .2 1 .4 1 .6 1 .8

mAU

-400

-200

0

200

400

600

800

1000

1200

DAD 1 C, Sig=210,8 Ref=360,100 (H:\DATA\2005\SD51117A\022 -0201 .D)

96% pure, 95 mg (82% recovery)

pH-zone refining : Regioisomersseparation Urology

NO2

NO2

0

500

1000

0 10 20 30 40 50 60 70

pKa prediction: 7.70 +- 0.7 pKa prediction: 7.90 +- 0.7

200 mg of mixture -> 65 mg of each compound 99% pure

Reconstructed chromatogram

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 20 40 60 80 100

Fraction number

UV

are

a

Sample Preparation

Viscous syrup

Crude extract including precipitates

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

Applications

Typical application areas• Where you have a target to focus upon

• Throughput is an issue regardless of scale

• Solubility of your sample is problematic

• You want to avoid the time consumingredevelopment of LC

• Polar compounds not easily retained in solidphase chromatography

• Purified target compounds from crude synthesisreaction mixtures

Minor impurity removal to obtain high purityproduct

20 40

Analytical run, Preparativerun

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

Impurity isolation for identification purposes

0 20 40

0 20 40

Analytical and Preparativeruns

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

Comparing chromatography techniques

HPCCC HSCCC HPLC

Stationary phaseLower phase of hexane-ethyl

acetate-methanol-water (1:0.4:1:0.4,v/v)

Upper phase of light petroleum-ethylacetate-tetrachloromethane-

methanol-water (1:1:8:6:1, v/v)

Zorbax Eclipse XDB-C18 column250x9.4mm ID 5 m

Mobile phase Upper phase Lower phase Methanol-water (70:30, v/v)

Sample capacity per run g 43 2.0 1.96x10-2

Run time min 45 450 40

Productivity mg/min 431 4.44 0.49

Purity of isolated compounds >99.9% >98.5% >99.0%

Solvent consumption L/g 1.39 1.93 5.10

Separation of GlucosinolatesB ru n e l-C C C - 9 8 0 m l C a p a c ity , 3 .6 3 m m b o re , N =1 2 0 0 rp m , F =4 0 m l/m in

0

0 .2

0 .4

0 .6

0 .8

1

1 .2

0 5 10 15 20 25 30 35

T im e (m in s)O

pti

cal

Den

sit

y(2

35n

m)

JH S am ple - 5g in 10m l

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

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

Mixtures of synthetic standards

• Sample 1 - Pindolol, Propranolol, Acetanilide,Verapamil and Ketoprofen (Mixed Polarity)

• Sample 2 - Carbamazepine, Trimipramineand Verapramil (Non-Polar)

• Sample 3 - Nifenazone, Tryptophan and

Mandelic acid (Very Polar)

Overlay of HPLC analyses of sample 1

1mg of each standardloaded in 1ml(total 5mg)

Separation of sample 1 - Solvent system 11 +TFA - RP mode - Analytical column

HPLC analysis of peaks from HPCCC run withsolvent system 11 + TFA

1mg of each standardloaded in 1ml(3 mg total)

Separation of sample 2 - Solvent system 14 -NP mode - Analytical column

HPLC analysis of Peaks 1 to 3 from HPCCCseparation of sample 2

30mg of each standardloaded in 1ml total

30 x Scale–up of sample 2 separation - Solventsystem 14 - NP mode - Analytical column

HPLC Analysis of Peaks 1 – 3 from Scale – Up

1mg of each standardloaded in 1ml(3 mg total)

Separation of sample 3 - Solvent System01 TFA - Analytical column

HPLC Analysis of Peaks 1 to 3 from HPCCCSeparation

6mg of each standardloaded in 6ml total

6 x Scale–up - Sample 3 separation -Preparative column

Scale up

Scale-up is simply volumetric

• You use the ratio of the column volumes thatyou are scaling between

• For example, 20ml column to 120ml columnwould 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 mgon a 37 mL coil to 7.8g on a 950 mL coil

20 40

Analytical run, Preparativerun

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 Preparativeruns

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

Range of HPCCC equipment

HPCCC instrument range

HPCCC product range scale of operation

• Spectrum – Analytical to semi-prep

– 5mgs to 2 grams per injection

– Up to 10 – 15 grams per day processed

• Midi – Analytical to prep

– 5 to 40 grams per injection

– Up to 400 grams per day processed

• Maxi – Pilot to production

– 1,500 grams per injection

Spectrum HPCCC

• Injection loading – 5 – 2,000 mgs

• Typical flows from 0.5 to 10 ml/min

• Run times 5-35 minutes

• Speed of rotation 1600rpm - 240g

• 20ml (analytical) &140ml(semi-prep) columns

• Temperature controlled

Midi HPCCC• Injection loading – 25 – 40

grams

• Typical mobile phase flow -30 to 50 ml/min

• Run times 5 - 35 minutes

• Speed of rotation1400rpm –240g

• 38ml (analytical) and 940ml(preparative) columns

• Temperature controlled

DE Maxi HPCCC• Injection loading – 500 –

1500 grams

• Typical mobile phase flow- 500 to 1500 ml/min

• Run times 5 - 35 minutes

• 4,600ml or 18,000mlcolumns

• Speed of rotation 850rpm- 240g

• Custom design

DE Solutions & Capabilities

Who are we?

A UK based company focused on the development of novelpurification technology, for use by the internationalpharmaceutical industry by providing improved solutions totheir existing and future liquid chromatography challenges.

This technology has been substantially developed andgreatly enhanced over the last 7 years, via substantialresearch grants and equity funding.

We have been working with the industry over the last 3years, primarily in the UK & US, working with customersshowing them how our technology and products can benefitthem in their work.

Products and engineered solutions forend-user use

DE Capabilities provided

• Feasibility studies

• Gram to Kilo separations to GLP

• Training

• Demonstration

A diverse range of customers

Key benefits of liquid stationary phases

• High injection loadings

• Improved sample solubility

• Reproducibility and ease of scale-up

• New elution strategies

• Total sample recovery

• Little or no sample preparation

Thank you for your attention

Any questions?