A Comparison of Compendia Normal Phase LC Methods Run Under Supercritical Fluid Chromatography Conditions

Transcription

1 A Comparison of Compendia Normal Phase LC Methods Run Under Supercritical Fluid Chromatography Conditions Stéphane Dubant Waters ESAC April 5th Waters Corporation 1

2 Agenda Introduction and Benefits of Supercritical Fluid? SFC and small particles Normal Phase Achiral Applications Chiral Applications Summary 2011 Waters Corporation 2

3 CO 2 Phase Diagram Dipole T P Moment CO A pure substance is considered to be in a supercritical state when its T and P are respectively higher than its critical T and P. At these conditions CO 2 has high diffusivity, low density and low surface tension 2011 Waters Corporation 3

4 Benefits of Carbon Dioxide (CO 2 ) Chemically pure, stable and non-polar solvent Non-toxic and non-flammable Can be easily removed from fractions Physical state can be easily manipulated Widely available By-product from commercial processes (i.e. fermentation) Considered Green Prevent/minimize waste Maximize usage of materials Reduce toxicity Use renewable feedstock Minimize energy use 2011 Waters Corporation 4

5 Carbon Dioxide as a Green Solvent Commercially produced carbon dioxide is recovered from industrial plants which produce hydrogen or ammonia from natural gas, coal, or other hydrocarbon feedstock Fermentation processes: Captured from breweries and other alcohol production facilities Corn-to-ethanol plants have been the most rapidly growing source of feed gas for CO 2 recovery. Commercially available carbon dioxide would have been vented into the atmosphere without dual-use as a solvent. Further, it replaces the carbon dioxide that would have been generated from production and disposal of the solvents it replaces Waters Corporation 5

6 Properties of Supercritical Fluids Diffusivity (cm 2 /s) Viscosity (g/cm x s) Gas Supercritical Fluid Liquid Like Gas Like U opt 1.63D d p m Liquid < High Diffusivity & Low Viscosity! 2011 Waters Corporation 6

7 What s SFC good for? Any compound soluble in methanol or less polar solvent is a candidate for SFC. Solubility is the big concern for a lot of new users. Log P is a measure for water solubility. Demonstrated SFC Region LogP RPLC Practically insoluble in Water <0.1mg/mL Data from Actelion Corp. Web Site 2011 Waters Corporation 7

8 SFC Analytical Overview SFC covers a wide a range of compounds in both functionality and polarity. Compatible with most LC type detectors, including UV-Vis, ELSD, CD and MS. SFC is a normal phase technique. SFC readily lends itself as an useful analytical tool for many industries: petrochemical and polymer, environmental, food, natural products, many stages of pharmaceutical from discovery to process. SFC can withstand the rigors of method validation in the pharmaceutical industry Waters Corporation 8

9 SFC and small particles 2011 Waters Corporation 9

10 Diffusivity ~ Speed/Efficiency Optimal Linear Velocity: U opt 1.63D d p m L U Retention time: t R = ( 1+ K' ) Efficiency: L N 2 d p High diffusivity (D m ), higher optimal linear velocity U opt, same number of peaks can be separated in less time. Same L, SFC is 3-10 times faster because of U opt ; or, for the same analysis time, 3-10 times longer columns can be used in SFC, N is therefore 3-10 times larger Waters Corporation 10

11 Remember: Smaller Particles the Enabler of Productivity 2011 Waters Corporation 11

12 SFC Isobaric Results 2.1 x 50mm Columns 2,50E-03 2,00E-03 5µm H [cm] 1,50E-03 1,00E µm 2.5µm H [cm} H [cm} H [cm} H [cm} fitted H [cm] fitted H [cm] fitted H [cm] fitted H [cm] 5,00E µm 0,00E ,5 1 1,5 2 u [cm/sec] 2011 Waters Corporation 12

13 High Resolution Separation in SFC Instrument Conditions 2.1 x 150 mm VIRIDIS TM Hybrid 1.7 µm Mobile phase A = Carbon Dioxide B =Methanol C Back 130 Bar (1,885 psi) UV 230 nm 2 µl injection Time %A %B Curve Waters Corporation 13

14 18 Compounds Mix - 6 Overlaid Injections 0.32 AU Benzoylpyridine Cholecalciferol Hydroxyflavanone Nitroaniline Caffeine Theophylline Carbamazepine Estradiol ,4'-Biphenol Cortisone Prednisone Acetaminophen Sulfisoxazole Consistent peak widths High resolution <0.4% RSD in RT Hydrocortisone Sulfamethizole Prednisolone Sulfaphenazole Aminophenyl Sulfone Minutes 2.1 x 150 mm Viridis Hybrid C A=Carbon Dioxide B=Methanol UV 230 nm (compensated nm) 2 µl injection Gradient 2% B to 16% B over 7 minutes 2011 Waters Corporation 14

15 SFC as a Normal Phase Replacement for achiral applications 2011 Waters Corporation 15

16 Normal Phase Method Conversion Anthralin Many Normal Phase chromatography applications require use of environmentally unfriendly and hazardous mobile phases. Utilizing properties of CO 2 as NP solvent in an SFC application removes the unfriendly and hazardous concerns Normal Phase HPLC SFC Chromatography AU Anthralin INSTD Anthralin: Rt = 2.1 USP Tailing = 1.23 USP Resolution = AU Anthralin INSTD Anthralin: Rt = 1.6 USP Tailing= 1.05 USP Resolution = Minutes Minutes 2011 Waters Corporation 16

17 Method Conversion Impact Analysis Anthralin We almost eliminated flammable and toxic solvents Utilized Standard HPLC Column We reduced the cost from $0.92/analysis to $0.04/analysis = 95% savings!! Analysis time reduced by 20% Resolution adequate for the method s purpose 2011 Waters Corporation 17

18 Chromatographic Assay of Tolbutamide AU Tolbutamide Normal Phase HPLC Taolazamide USP Method Column x 300 mm, silica column (L3) Flow - Isocratic 1.5 ml/min Mobile phase - hexane, water-saturated-hexane, tetrahydrofuran, alcohol, and glacial acetic acid (475:475:20:15:9) Minutes *Cost per run ~ $1.40 AU Tolbutamide Tolazamide SFC Column - Viridis Hybrid, 3.0 x 100 mm, 1.7 µm at 50 C Flow Rate ml/min, Backpressure Bar/1740 psi Mobile Phase - 95% Carbon Dioxide: 5% Methanol/IPA (1/1) containing 0.2% TFA Detection - UV /PDA at 254 nm *Cost per run ~ $ Minutes 1/10 th the run time *Solvent cost per run 2011 Waters Corporation 18

19 Chromatographic Purity of Estradiol Currently, the United States Pharmacopeias (USP) method for the estimation of chromatographic purity of Estradiol utilizes a 4.6 mm 250 mm silica column that uses a mobile phase consisting of 2,2,4-trimethylpentane, n-butyl chloride and methanol (45:4:1) at 2 ml/min. The results were compared against results obtained with a method developed on an ACQUITY UPSFC System using identical sample preparation. The SFC method conditions were as follow: Column: 2.0 x 150 mm Viridis Hybrid, 1.7 µm Back pressure: 130 bar (1,885 psi), Temperature: 45 C UV Detection /PDA at 280 nm Gradient Table Time Flow %A %B Curve (min) (ml/min) Waters Corporation 19

20 Chromatographic Purity of Estradiol AU Normal Phase HPLC Cost per run ~ $ Compound RT %Area S/N Unk. Impurity Unk. Impurity Not Found Unk. Impurity Unk. Impurity Not Found Unk. Impurity Unk. Impurity Estradiol Main Impurity Minutes SFC Cost per run ~ $0.05 Compound RT %Area S/N Unk. Impurity Unk. Impurity Unk. Impurity Unk. Impurity Unk. Impurity Unk. Impurity Estradiol Main Impurity Waters Corporation 20

21 Chiral Analysis 2011 Waters Corporation 21

22 Chiral HPLC vs. Chiral SFC A sample of BINOL was separated using both NPLC and SFC techniques. The key parameters for both methods were as follows: NPLC SFC Flow rate (ml/min) 2 4 Mobile phase hexane:methanol=98:2 CO 2 :methanol=75:25 Pressure (bar) n/a 120 Temperature ( C) ambient 40 Column Sample Conc. (mg/ml) CHIRALPAK AS-H ( mm, 5 µm) 2 Injection volume (µl) Waters Corporation 22

23 Enantiomeric Separation of Binol Rs= AU Minutes Flow rate: 4 ml/min Co-solvent: 25% Methanol Pressure: 120 bar Temperature: 40 C Column: ChiralPak AS-H ( mm, 5µm) 2011 Waters Corporation 23

24 Enantiomeric Separation of Binol Normal Phase LC Chromatogram 0.06 Rs=1.67 AU SFC Chromatogram AU Rs= Speed: 9x improvement Resolution: 51% increase 0.40 Cost/Analysis: $ Minutes 2011 Waters Corporation 24

25 Enantiomeric Separation of Benzyl mandelate Benzyl mandelate (Figure 1) is an important synthetic intermediate for pharmaceutical synthesis. A racemic mixture of R- and S-benzyl mandelate (0.20 mg/ml in methanol for each enantiomer) was separated using a Waters ACQUITY UPSFC system. OH OH O (R) O (S) O O Structures of R- and S-benzyl mandelate 2011 Waters Corporation 25

26 Enantiomeric Excess Determination of Benzyl Mandelate SFC chromatogram of R- and S- benzyl mandelate at 0.20 mg/ml of each enantiomer. AU 0.20 R S R S = Minutes SFC chromatogram of R-benzyl mandelate at 2 mg/ml. AU R 0.02% impurity S Minutes 2011 Waters Corporation 26

27 Diastereoselective Separation of Permethrin Twenty five percent of currently used pesticides are chiral. Chirality plays an important role in the potency, toxicity, metabolism, and environmental fate of these chiral pesticides. As a result, there has been an increasing demand for stereo-selective separation techniques and analytical assays to evaluate enantiomeric purity of pesticides Waters Corporation 27

28 Diastereoselective Separation of Permethrin Permethrin is a synthetic chemical widely used as an insecticide and an insect repellent. Permethrin has four stereoisomers, arising from the two stereo-centers in the cyclopropane ring. Cl Cl (R) O O Cl (R) O O O Cl O (1R, trans) (1R, cis) Cl Cl (S) O O Cl (S) O O Cl (1S, trans) (1S, cis) Great effort has been put into developing HPLC methodology, both normal phase LC and reverse phase LC, for separating permethrin, but with moderate success. Our study describes baseline resolution of all four permethrin isomers in less than 6 min using an ACQUITY UPSFC system Waters Corporation 28

29 Permethrin with SFC Key experimental parameters. NPLC Flow rate (ml/min) 1 4 Mobile phase hexane:ethanol=90:10 CO 2 :methanol:dea=95:5:0.2 Back Pressure (bar) n/a 120 Temperature ( C) ambient 40 Column CHIRALCEL OJ-H ( mm, 5 µm) Sample Conc. (mg/ml) 2 Injection volume (µl) 10 SFC CHIRALCEL OJ-H ( mm, 5 µm) Retention time, retention factor (K ), selectivity (α), resolution (Rs) and USP tailing factor of permethrin enantiomers obtained under SFC conditions Peak RT (min) K α Rs USP Tailing Factor Waters Corporation 29

30 Diastereomeric Separation of Permethrin Chromatogram of Permethrin obtained with a CHIRALCEL OJ-H column under NPLC conditions AU Minutes 0.06 AU Minutes Chromatogram of Permethrin obtained with a CHIRALCEL OJ-H column under SFC conditions Waters Corporation 30

31 Summary Using a SFC system, the chromatographic purity of several achiral compounds has been estimated. SFC has also provided considerable improvement over standard LC chiral analysis. These SFC methods were faster (up to 10 times) than the current normal phase method from the USP and eliminated the usage of some nasty normal phase solvents. The SFC technology considerably reduces the cost of running those analysis. Required sensitivity levels can now be achieved with the UPSFC methods with impurities as low as 0.01% of the main peaks being easily detected Waters Corporation 31

32 Thank you for your attention and Questions? 2011 Waters Corporation 32