Is there a Preparative Chromatography Green, Economic and Productive?

Transcription

1 Is there a Preparative Chromatography Green, Economic and Productive? Granada, 23 Febrero 2012 Alberto Méndez Applications Specialist alberto_mendez@waters.com 2012 Waters Corporation 1

2 Why CO 2? CO 2 reaches supercritical state at 31.1 C and 73.8 bar Its physical state can be easily manipulated CO 2 is non toxic, non flammable CO 2 is chemically pure, stable and non-polar solvent, also compatible with LC detectors Carbon Dioxide as a Green Solvent Recovered from industrial and fermentation plants Avoids the production of CO 2 that would have been generated from disposal of the solvents it replaces. Less time and energy are used to evaporate fractions to get to pure analytes Waters Corporation 2

3 Supercritical Fluid Applications Chromatography (SFC) Analysis & Purification Chiral Separation Normal Phase SF Particle Design (RESS & SAS) Polymerization Cristallization Impregnation Supercritical CO 2 Extraction (SFE) Bioactive compounds Nutraceuticals Spices and aromatic Decaffeination Decontamination or cleaning Valuable molecules from waste Reaction Hydrogenation Hydroformylation Carboxylation 2012 Waters Corporation 3

4 What is a Supercritical Fluid? Gas - like low viscosity confers high diffusivity and limits pressure drop Liquid - like high density ensures good solvent properties High diffusion coefficient enhances kinetic performance of SFC 2012 Waters Corporation 4

5 HPLC 2012 Waters Corporation Courtesy of C. West, E. Lesellier, ICOA University of Orléans 5

6 SFC : Unified Chromatography 2012 Waters Corporation Courtesy of C. West, E. Lesellier, ICOA University of Orléans 6

7 What is SFC? A chromatographic technique similar to HPLC in both hardware and operational principle CO 2 in combination with one or more organic solvent, most commonly alcohols, is used as the mobile phase SFC is a normal phase separation technique by nature. It complements industry dominating RPLC SFC is popular for chiral separations because of its resolution and speed SFC is compatible with most LC-type detectors 2012 Waters Corporation 7

8 SFC & LC Similarities Unique Features Compressible mobile phase Cooling device for CO 2 pump Pressure Resistant UV Flow Cell Back Pressure Regulator 2012 Waters Corporation 8

9 SFC Advantages Higher speed and throughput Better peak shape Better resolution Faster re-equilibration Shorter cycle time More samples per day SFC complements RP-LC Normal phase technique MS compatible Different selectivity Different elution order Better retention of polar compounds. SFC is proven better for separating isomers and enantiomers, and structurally related compounds. Not a total replacement for HPLC, but it handles ~80% of all small molecules 2012 Waters Corporation 9

10 SFC vs RPLC - Orthogonality SFC A B C D E F G H H RPLC G A E F B C D Paracetamol and 7 related process impurities 2012 Waters Corporation 10

11 HPLC or SFC? Normal Phase SFC SFC is a Normal Phase replacement o Reduces the usage of solvents like Hexane o Less dry down requirements/bottlenecks Chiral - SFC Predominant separation technique for Chiral compounds is SFC Stacked injections of separated isomers enables increased throughput o 30mg injections, 115 sec intervals o 6g purified in 6 hrs and 3.8L solvent uv uv Time (min) Time (min) 2012 Waters Corporation 11

12 HPLC or SFC? Biomolecule HPLC Peptides, oligos and small protiens Large Scale Chiral SFC Achiral/Small Molecule can be either SFC or LC System type dependant on o Scale o Requirements Software, MS? o Infrastructure - Availability of CO2 o Current process/bottleneck o Budget o Customer preference 2012 Waters Corporation 12

13 The SFC Purification Advantages SFC can be used with modifier (normally MeOH) gradient to perform generic analysis of diverse compounds of a wide range of polarity and functionality Fractions are collected in a minimal volume of modifier for easy recovery of purified compounds. Elimination of water in collected fractions alleviates evaporation bottleneck and save time and energy Cost of CO 2 and operation is a fraction of RP-LC Faster, higher efficiency chromatography reduces time per sample Mass trigger-based collection offers specificity and possible structural identification for target compounds, ensures higher success rate of overall process in terms of integrity, recovery as well as purity Waters Corporation 13

14 Fast Separation Analysis H (µm) Van Deemter curve H = f(u) u opt Only 25% efficiency loss 3 x u opt Generic conditions 2-EP, 150 x 4.6mm, 5µm. CO 2 -MeOH, 3.5mL/min 10mL/min Oven 40 C 200bar UV 220nm u (mm/s) AU min 4 Steroids 1. Androstenedione 2. Mestanolone 3. Testosterone 4. Stanozolol 1.90 min Minutes AU I II III IV V VI I II III IV 0.85 min Benzodiazepines I. Midazolam II. Flunitrazepam III. Lormetazepam IV. Clorazepate V. Nitrazepam VI. Oxazepam 2.75 min Minutes V VI 2012 Waters Corporation Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva 14

15 Superoptimal Flow Rate Conditions Column: 21.2 x 150 mm Cyano Flow rate: 50 ml/min optimal 200 ml/min, superoptimal Modifier: 15% MeOH, isocratic Temp: 35 C Detection: 280 nm Pressure: 100 bar Sample: 87 mg/ml Ibuprofen / Ketoprofen Injection: 2 ml Throughput : 13.9 g/hr for each 200 ml/min, with 45 sec interval Process : 668 g/day total compound, 11.7 kg 200 ml/min sample/kg sorbent/day 50 ml/min 2012 Waters Corporation 15

16 Stacked Injection Mode Normal injections Time (min) Time (min) Time (min) Increase throughput without compromising efficiency Works in isocratic mode Time (min) Time (min) Stacked injections 2012 Waters Corporation 16

17 Method Robustness - Reproducibility Precision of Retention Times for all 4 peaks : RSD < 0.4% Conditions Flow Rate: 200 ml/min Gradient: 5-50% MeOH, 10%/min Outlet Pressure:100 bar Temp.: 35ºC Detection: 280nm Sample: Standard mix in MeOH concentration: 40mg/ml Each Injection: 2.0 ml 1,300 1,250 1,200 1,150 1,100 1,050 1, Superimposition of 88 injections! Pfizer 2012 Waters Corporation 17

18 SFC Purification : kg/day Throughput Overall throughput 60 g/h; 1.45 kg/day, 5.4 kg sample/kg sorbent/day Flow rate: 400 ml/min Modifier: 50% Isopropanol Temp: 35 C Pressure: 100 bar Column: 50 x 250 mm Whelk-O Detection: 254 nm Sample: 670 mg/ml CBZ derivative Injection: 3ml Collection: 120 s interval SPW 0.20 STH Wyeth 2012 Waters Corporation 18

19 Large Chiral Preparative SFC: Cost Analysis 1 kg of racemic mixture (API) 2.5 kg alcohol enantiomers SFC HPLC HPLC SFC Equip. time (hr) Time (hr) Column i.d. (inch) Flow rate (ml/min) Cycle time (min) Rotovap time (hr) Column i.d. (cm) Total solvent (L) Collection (L) , , Total Solvent (L) Total ($) From: T Blackwell, Rhodia ,850 From: William Leonard, J. DaSilva, D. Henderson & C.J. Welch, Merck Less solvent consumption Cost of CO 2 is low Collected in less solvent Collected in volatile solvent Less post-purification endeavor SFC is Green Chromatography 2012 Waters Corporation 19

20 SFC is the Choice for Chiral Separation Mixture of diastereisomers HPLC: 34.5 min On average, SFC is 3-10 times faster than normal phase HPLC for chiral separation AU Minutes SFC: 4.3 min Source: Craig White, Lilly, UK 2012 Waters Corporation 20

21 Preparative SFC : Typical Scale Guide System Type Max Flow ml/min Column I.D./mm Loading/ mg Capacity g/24 hr Day Method Station Analytical Investigator Semi-Prep /Analytical Prep 80 Prep or Prep 200 Prep Prep 350 Prep Prep 100 Mass Directed Prep or *Flow rates are listed in ml/min of CO2, HPLC chromatographers will use ml/min, SFC chromatographers will use g/min. The values translate to approximately the same number. Prep 80, 200 and 350 are bulk preparative systems typically dedicated to chiral purification Chiral purification uses a stacked injection technique, with increases the throughput Smaller scale chiral purification is often performed on the Investigator also 2012 Waters Corporation 21

22 Analytical and Small Scale Prep Semi-Prep Investigator II Method Station II with fraction collection Analytical method collection: less than 1 gram of specific compounds Flow rate up to 15ml/ min Max column ID 10mm I.D. Sample load up to 10mg TUV detector ChromScope Software only Fraction collection In this case 6 fraction collection positions are available 2012 Waters Corporation 22

23 SFC Chiral Preparative System: Similar configuration as before, added collection capability More CO2 containers for greater throughput Cyclone separators are a safe way to collect at high flows Recycling recommended with flow rates at ml/min 2012 Waters Corporation 23

24 Large Scale SFC Purification SFC Benchtop 80 3 g/hour SFC Prep g/hour SFC Prep g/hour 2012 Waters Corporation 24

25 Open Bed Collection Large set of compounds such as library, or multiple components in one sample such as natural products Software has to be able to track sample, collection and so on. Collection media have to be compatible with downstream dry-down device Waters Corporation 25

26 Similar Strategy For MS Directed SFC Injection/collection Waters 2767 Sample Manager Detection Waters 3100 Mass Detector Waters 2998 PDA Software MassLynx/FractionLynx OpenAccess Add MS functionality if and when it s necessary 2012 Waters Corporation 26

27 Schematic of SFC-MS Prep Waters Corporation 27

28 Proprietary gas/liquid separator (GLS) enables Open bed collection Gas/liquid separator ensures minimum CO 2 at collection but also preserves peak integrity Waters Corporation 28

29 The Key Innovation: Gas-Liquid Separator (GLS) Upon exiting GLS, the flow is mainly in liquid form. Peak integrity is well maintained before and after GLS across the gradient. Less than 1.5 s peak dispersion due to GLS. Time variance across gradient is less than 1.5 s. UV MS triggered collection portion Collector tip Same portion at collector tip 10% Co-solvent 30% Co-solvent 50% Co-solvent 2012 Waters Corporation 29

30 The Effectiveness of GLS e+2 3.0e+2 2.8e+2 2.6e+2 2.4e+2 2.2e Standard 2.0e+2 1.8e+2 AU 1.6e+2 1.4e+2 1.2e+2 1.0e+2 8.0e+1 6.0e+1 4.0e+1 Carbamazepine Collection 2.0e Time Carbamazepine Ketoprofen Injection Recovery Purity Recovery Purity Average RSD% Ketoprofen Collection 2012 Waters Corporation 30

31 Multiple Modes of Triggering Intelligent collection using different signals with uncompromised performance. Can be extended to include other detectors as well. Recovery% UV Triggering MS Triggering UV+MS Triggering Caffeine 90.5± ± ±1.2 Sulconazole 90.2± ± ±0.5 Bendroflumethazide 94.7± ± ± Waters Corporation 31

32 Focused Gradient: SFC 5-55% in 10 min Just like its LC counterpart, applying a narrow, focused gradient in SFC, retention time, resolution, and peak width of the target compound(s) can be maneuvered for ensuing purification % in 10 min 2012 Waters Corporation 32

33 AutoPurify in SFC Screening Title Purification Title 2.25e+2 SFC MS Resolution AU 2.0e e+2 1.5e e+2 1.0e+2 7.5e+1 5.0e+1 2.5e Re-analysis % Time SFC MS Prep 100 Intensity (%) Time (min) All systems under MassLynx/FractionLynx SFC MS Resolution 2012 Waters Corporation 33

34 Which Instrument is Right for You? Compounds achiral chiral library singleton Analytical screening LC and SFC SFC scale SFC-MS Prep100 Autopurification LC-MS Prep100 Prep LC Investigator: up to 100mg Prep 80: 100mg-100g Prep 200: 100g-500g Prep 350: 500g-kg 2012 Waters Corporation 34

35 Conclusions The purification challenges we are facing today require an integrated solution that encompasses different technology and platform. SFC expands the purification arsenal and offers complementary selectivity to RPLC based solutions. The SFC advantages include: small collection volume in volatile organic solvent, shorter dry-down time, low operational cost, and shorter batch processing time. The combination of the two techniques will result in an increase in success rate. Waters is committed to providing customers with a complete system solution that includes hardware, software and columns. References R. Chen, LCGC Europe EApplication Note Alert, Feb A. Mich, B. Matthes, R. Chen, and S. Buehler, LCGC Europe: The Application Notebook , March Waters Corporation 35