What differentiates Dynamic Extractions?

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1

2 What differentiates Dynamic Extractions?

3 Making liquid stationary phases available for high purity chromatography purification at all scales

4 The difference is the amount of active stationary phase mobile phase Liquid/Liquid Active stationary phase Solid/Liquid Stagnant mobile phase Silica skeleton

5 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

6 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 muilti-kilo

7 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 you want to avoid redeveloping chromatography as you move up in scale Where you recognise that solubility of your compounds will be problematic

8 HPCCC is complimentary and orthogonal

9 Understanding HPCCC

10 CCC mechanism of separation is partitioning Test tubes A column HPCCC equipment

11 Essential HPCCC theory

12 Distribution Ratio Purification occurs because of the different solubility of the components in the liquid mobile and stationary phases This is determined by the Distribution Ratio (D) = C S /C M C S is the concentration of the component in the stationary phase C M is the concentration of the component in the mobile phase

13 Concentration Extra-coil Volume (V ext ) Highly predictable Sample Injection V m Solvent Front (D=0) DV s V s Volume Sample Elution (D) System Volume (D=1) A component with a low distribution constant has a higher concentration in the mobile phase and elutes early A component with a D=1 will elute after one column volume of mobile phase has been pumped through the column A component with a high distribution constant has a higher concentration in the stationary phase elutes late A component with a D=5 will elute after 5 column volumes

14 What is stationary phase S f = 90% 1 MP : 9 SP retention? S f = 50% 1 MP : 1 SP S f = 33% 2 MP : 1 SP

15 Showing the importance of stationary phase retention Stationary phase retention increases as rotational speed of the CCC instrument is increased UV signal (254 nm ) Sf = 90% 1 0 V S =108 ml 1 0 V S = 84 ml Sf = 70% 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 V S = 60 ml Sf = 50% V S = 36 ml Sf = 30% R e t e n t i o n v o l u m e ( m L )

16 Defining Normal and Reverse Phase Upper Phase (Organic) Lower Phase (Aqueous) Periphery (Heavy End) Centre (Light End)

17 Normal Phase X Upper Phase (Organic mobile) Lower Phase (Aqueous Stationary)

18 Reverse Phase Upper Phase (Organic Stationary) Lower Phase (Aqueous mobile) X

19 Method Development

20 Concentration Important Considerations in Developing a Separation Method Extra-coil Volume Sample (V ext ) Injection V m Solvent Front (D=0) DV s V s Volume Sample Elution (D) System Volume (D=1) Elution Time is determined by D To keep run times to a minimum, D values should be less than 5.0 To produce the best separations, D values should differ by > 0.5

21 More Polar More Non-Polar Selectivity is the power of HPCCC No Heptane EtOAc MeOH Butanol Water This set of phase systems covers 80% plus of the 0.20 target molecules that we are asked to separate 0.00 Distribution Ratio Solvent System No 52.5 mg/ml BA - 25 mg/ml PC, Rs = mg/ml BA - 20 mg/ml PC, Rs = mg/ml BA - 15 mg/ml PC, Rs = mg/ml BA - 10 mg/ml PC, Rs = mg/ml BA - 5 mg/ml PC, Rs = 1.6 Benzyl Alcohol Para-Cresol Time, min

22 Using HPLC data to determine initial solvent system selection Compound(s) Elution PointFrom C18 rp- Solvent System operating windows giving a D value between 1.0 and 3.0 HPLC column (% Acetanitrile) Normal Phase Reverse Phase Substitute acetonitrile for methanol or Toluene for Hexane

23 Sample solubility

24 Samples partition between either the mobile or stationary phase

25 New elution strategies

26 Elution extrusion 30mg of each standard loaded in 1ml total

27 Dual mode elution Dual mode elution mg injection Estimated resolution: 0.85 mau DAD1 C, Sig=210,8 Ref=360,100 (H:\DATA\2005\SD51115A\ D) Standard mode elution mg injection Resolution: 0.25 Reconstructed chromatogram for normal CCC min Difference of partition coefficient only 0.3 Amount of product Reconstructed chromatogram of Dual CCC Fraction number 96% pure, 95 mg (82% recovery) DAD 1 C, Sig=210,8 Ref=360,100 (H:\DATA\2005\SD51117A\ D) mau 1200 Amount of compound Fraction number min

28 ph-zone refining : Regioisomers separation Urology pka prediction: pka prediction: NO 2 NO UV area Reconstructed chromatogram Fraction number 200 mg of mixture -> 65 mg of each compound 99% pure

29 Sample Preparation

30 Viscous syrup

31 Crude extract including precipitates Loading capacity up to 50%w/w depending on solubility

32 Applications

33 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 consuming redevelopment of LC Polar compounds not easily retained in solid phase chromatography Purified target compounds from crude synthesis reaction mixtures

34 Minor impurity removal to obtain high purity product Analytical run, Preparative run 3.4 % min % yield, 99.9% purity on HPLC (impurity not detected) Courtesy of Pfizer UK

35 Impurity isolation for identification purposes Analytical and Preparative runs mau Starting material % Final sample min mau % yield, 98% purity based on HPLC-UV min Courtesy of Pfizer UK

36 Comparing chromatography techniques HPCCC HSCCC HPLC Stationary phase Lower phase of hexane-ethyl acetate-methanol-water (1:0.4:1:0.4, v/v) Upper phase of light petroleum-ethyl acetate-tetrachloromethanemethanol-water (1:1:8:6:1, v/v) Zorbax Eclipse XDB-C18 column 250x9.4mm ID 5 m Mobile phase Upper phase Lower phase Methanol-water (70:30, v/v) Sample capacity per run g x10-2 Run time min Productivity mg/min Purity of isolated compounds >99.9% >98.5% >99.0% Solvent consumption L/g

37 Separation of Glucosinolates B ru n e l-c C C m l C a p a c ity, m m b o re, N = rp m, F = 4 0 m l/m in Optical Density (235nm) JH S a m p le - 5 g in 1 0 m l 0.8 GR GI Johns Hopkins T i m e (m i n s) Repeatability Run 5 Run 6 Run Run 8 Run 9 Run Mobile Phase Volume (ml) g from 0.59kg 34 runs 47%w/w sample conc 17g/run sample loading 98.5% pure 3 days

38 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

39 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

40 Mixtures of synthetic standards Sample 1 - Pindolol, Propranolol, Acetanilide, Verapamil and Ketoprofen (Mixed Polarity) Sample 2 - Carbamazepine, Trimipramine and Verapramil (Non-Polar) Sample 3 - Nifenazone, Tryptophan and Mandelic acid (Very Polar)

41 Overlay of HPLC analyses of sample 1

42 Separation of sample 1 - Solvent system 11 + TFA - RP mode - Analytical column 1mg of each standard loaded in 1ml (total 5mg)

43 HPLC analysis of peaks from HPCCC run with solvent system 11 + TFA

44 Separation of sample 2 - Solvent system 14 NP mode - Analytical column 1mg of each standard loaded in 1ml (3 mg total)

45 HPLC analysis of Peaks 1 to 3 from HPCCC separation of sample 2

46 30 x Scale up of sample 2 separation - Solvent system 14 - NP mode - Analytical column 30mg of each standard loaded in 1ml total

47 HPLC Analysis of Peaks 1 3 from Scale Up

48 Separation of sample 3 - Solvent System 01 TFA - Analytical column 1mg of each standard loaded in 1ml (3 mg total)

49 HPLC Analysis of Peaks 1 to 3 from HPCCC Separation

50 6 x Scale up - Sample 3 separation Preparative column 6mg of each standard loaded in 6ml total

51 Scale up

52 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

53 Performed and optimised at analytical scale %, Rs = %, Rs = %, Rs = %, Rs = %, Rs = %, Rs = Time, min mg/ml BA mg/ml BA mg/ml BA mg/ml BA mg/ml BA - 25 mg/ml PC, Rs = mg/ml PC, Rs = mg/ml PC, Rs = mg/ml PC, Rs = mg/ml PC, Rs = DE Mini Time, min

54 Directly transferred to the kilo/pilot scale Mini separation ml column - 10, 20 and 33% of column volume (Vc) 1.2 Mini-1.78 ml= 33% of Vc Mini-1.07 ml= 20% of Vc Time, min Maxi separation L - 20 and 33% of column volume (Vc) 1.2 Maxi L- 20% of Vc Maxi L- 33% of Vc Time, min

55 Analytical to semi-preparative scale-up Minor 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 Analytical run, Preparative run 3.4 % min 40 92% yield, 99.9% purity on HPLC (impurity not detected) Courtesy of Pfizer UK

56 Analytical to semi-preparative scale-up Impurity isolation for identification purposes - Scale-up x26, from 200 mg on a 37 ml coil to 5.2g on a 950 ml coil Analytical and Preparative runs Starting material mau 18% min Final sample mau % yield, 98% purity based on HPLC-UV min Courtesy of Pfizer UK

57 Range of HPCCC equipment

58 HPCCC instrument range

59 HPCCC product range scale of operation Spectrum Analytical to semi-prep 5mgs to 2 grams per injection Up to 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

60 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

61 Midi HPCCC Injection loading 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

62 DE Maxi HPCCC Injection loading grams Typical mobile phase flow to 1500 ml/min Run times 5-35 minutes 4,600ml or 18,000ml columns Speed of rotation 850rpm - 240g Custom design

63 DE Solutions & Capabilities

64 Who are we? A UK based company focused on the development of novel purification technology, for use by the international pharmaceutical industry by providing improved solutions to their existing and future liquid chromatography challenges. This technology has been substantially developed and greatly enhanced over the last 7 years, via substantial research grants and equity funding. We have been working with the industry over the last 3 years, primarily in the UK & US, working with customers showing them how our technology and products can benefit them in their work.

65 Products and engineered solutions for end-user use

66 DE Capabilities provided Feasibility studies Gram to Kilo separations to GLP Training Demonstration

67 A diverse range of customers

68 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

69 Thank you for your attention Any questions?