Fundamentals and Advancements in Analytical SFC
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UltraPerformance Convergence Chromatography Supercritical Fluid Separations With The Power & Performance of ACQUITY ©2012 Waters Corporation 1 Agenda What is a Supercritical Fluid ? What is Supercritical Fluid Chromatography ? Introduction To UPC2™ ACQUITY UPC2™ Performance ACQUITY UPC2™ Applications Summary ©2012 Waters Corporation 2 What Is A Supercritical Fluid ? "A supercritical fluid is any substance at a temperature and pressure above its critical point, where distinct liquid and gas phases do not exist. It can effuse through solids like a gas, and dissolve materials like a liquid. In addition, close to the critical point, small changes in pressure or temperature result in large changes in density, allowing many properties of a supercritical fluid to be "fine-tuned". Supercritical fluids are suitable as a substitute for organic solvents in a range of industrial and laboratory processes. Carbon dioxide and water are the most commonly used supercritical fluids, being used for decaffeination and power generation, respectively."* * http://en.wikipedia.org/wiki/Supercritical_fluid ©2012 Waters Corporation 3 Pressure – P (bar) CO2 Phase Diagram Critical T = 31.1 oC Critical P = 74 bar/1070 psi -75 -25 25 75 125 Temperature - T (oC) * Adapted from http://en.wikipedia.org/wiki/File:Carbon_dioxide_pressure-temperature_phase_diagram.svg ©2012 Waters Corporation 4 Properties of Supercritical Fluids Diffusivity & Viscosity More Like A Gas – Diffusivity promotes chromatographic mass transfer/resolving power – Lower viscosity promotes chromatographic speed Diffusivity (cm2/s) Gas Supercritical Fluid Liquid 10 -1 10-4 - 10-3 < 10 -5 Viscosity (g/cm x s) 10 -4 10-4 - 10-3 10 -2 Solvating Power Like A Liquid ©2012 Waters Corporation 5 What is Supercritical Fluid Chromatography (SFC) ? Chromatographic Technique Similar to HPLC Mobile Phase = Supercritical Fluid + One or More Co-Solvents – CO2 = most common supercritical fluid – MeOH = most common co-solvent ©2012 Waters Corporation 6 SFC Characteristics Normal Phase Technique – – – – Polar stationary phase/non-polar mobile phase Elution order from lowest to highest polarity Strong solvent = more polar solvent = MeOH Weak solvent = less polar solvent = CO2 Compatible With Common LC Detectors – UV-VIS, ELSD, MS Utility For Many Industries/Market Segments – Applicable to wide range of compounds/varied functionality & polarity – Petrochem, pharma, polymer, environmental, food, natural products Ideal For Prep Applications – Easy removal of CO2 from fractions Method Of Choice For Chiral Separations ©2012 Waters Corporation 7 SFC vs. GC vs. LC Better Mass Transfer Than LC – More efficient separations Better Solvation Than GC – Useful for broader range of analytes – Not restricted to volatile analytes Easily Manipulated Density – For fine tuning separations ©2012 Waters Corporation 8 SFC vs. LC Separation Speed & Efficiency http://www.soton.ac.uk/~gjl/images/SFC_figure2.jpg Greater Diffusivity – Higher optimal flow rates – Shorter run times – Same resolving power Lower Viscosity – Longer columns can be used – Increased separation efficiency/equivalent run times ©2012 Waters Corporation 9 Which Compounds Work? Candidates for SFC – Normal phase LC separations – Separations that are problematic for LC and GC – Compounds in need of derivatization for GC – Compounds soluble in methanol or less polar solvents Demonstrated SFC Region -10 -8 -6 -4 RPLC -2 0 LogP 2 4 6 8 10 12 Insoluble In Water <0.1mg/mL Data from Actelion Corp. Web Site ©2012 Waters Corporation 10 Do Small Particle, i.e. < 2 µm, Column Chemistries Provide The Same Benefits To SFC As They Do To Traditional Reversed Phase LC? ©2012 Waters Corporation 11 SFC Column Efficiency Curves 5.0 µm XBridge™ HILIC, 3.0x50 mm 3.5 µm XBridge™ HILIC, 3.0x50 mm 2.5 µm XBridge™ HILIC, 3.0x50 mm 1.7 µm ACQUITY BEH , 3.0x50 mm ©2012 Waters Corporation 12 SFC Resolution Comparison Column 1 5 µm Particles SFC Separations Column 2 1.7 µm Particles ©2012 Waters Corporation 3.0 x 100 mm Column 2 mL/minute CO2/Methanol Gradient 13 Introduction & Performance ©2012 Waters Corporation 14 A World Without UPC2 ©2012 Waters Corporation 15 Why Is SFC A Niche Technique ? Considered Exotic – Not taught as part of analytical chemistry curriculum No Major Chromatography Company Presence – "Frankensystems" comprised of with modules from multiple vendors Problems With Robustness – Poor precision – Baseline noise/poor sensitivity – Unsuitable for regulated environments Systems Don't Provide A Complete Solution – Hardware/Software/Chemistry/Support ©2012 Waters Corporation 16 The ACQUITY UPC2 Designed Holistically For Optimum Performance Built Upon Proven UPLC Technology – Quantifiable increases in productivity SFC Capability With LC Familiarity Reproducible & Robust – Can withstand rigors of method validation Complete Solution – Hardware, software, chemistry, service & support ©2012 Waters Corporation 17 ACQUITY UPC2 System Features UPC2 Binary Solvent Manager – Blends & delivers CO2 & co-solvent – 4 selectable co-solvents – 0.010 to 4.000 mL/min flow rate range UPC2 Fixed Loop Sample Manager – Partial or full loop injection modes ACQUITY Column Manager – Multi column selector for selectivity screening UPC2 Convergence Manager – Incoming CO2 management & system pressure control – Key to precision, ruggedness & reducing baseline noise Empower™ & MassLynx™ Compatible ©2012 Waters Corporation 18 UPC2™ COMPOUND DIVERSITY Gradient Separation Diverse Polarity Mix – 18 components, – Amines, vitamins, dyes, steroids, antibacterials Exceptional Resolution Exceptional Precision – Retention time repeatability < 0.4 % RSD, N = 6 Column: Flow: Col Temp: Detection: Inj Vol: Gradient: ABPR: ©2012 Waters Corporation ACQUITY UPC2 Hybrid, 2.1 x 150 mm ,1.7µm 1.2mL/min 45 C UV 230nm 2µL 2 – 16% MeOH in 7 minutes 130 bar 19 UPC2 ™ Gradient Performance Overlay Of Of 10 10 Injections Injections Overlays 0.5% Difference in Solvent Composition Overlay Of 10 Injections Precise & Accurate Co-Solvent Addition – Essential to the separation of closely related compounds, e.g. isomers, plant extracts, & essential oils of natural products ©2012 Waters Corporation 20 UPC2 ™ Injector Linearity Excellent Partial Loop Linearity – 1 – 10 μL ©2012 Waters Corporation 21 UPC2 ™ Detector Sensitivity Low Noise For Enhanced Sensitivity ©2012 Waters Corporation 22 UPC2 ™ Column Chemistries BEH BEH 2-Ethylpyridine CSH Fluoro-Phenyl HSS SB ©2012 Waters Corporation 23 Applications Lipids ©2012 Waters Corporation 24 UPC2 MS Cell Membrane Polar Lipids Rapid Screening Separation PC No Derivatization Direct Injection of Extraction Solvent LPC SM CER ©2012 Waters Corporation PG PE LPE 25 UPC2 & Lipidomics Mouse Heart Extract/MS Detection Glycerolipids PC (NH4+ adducts) MAG TAG PE Cardiolipins LPC SM LPE DAG ©2012 Waters Corporation PS PG 26 Applications Mixed Tocopherols ©2012 Waters Corporation 27 UPC2 ™ Mixed Tocopherols Column: ACQUITY UPC2 BEH 3.0 x 100 mm, 1.7 μM Flow Rate: 2.5 mL/min Column Temp: 50 oC Detection: UV 293 nm (compensated 500 -600 nm) Inj Vol: 1 μL ABPR: 1885 psi Gradient: 2 – 5 % Methanol in 1.5 minutes ©2012 Waters Corporation 28 Applications Organic Light Emitting Diodes ©2012 Waters Corporation 29 UPC2 ™ Organic Light Emitting Diodes Ir(fppy)3 Area, 0.4% RSD RT, 0.1% RSD F α-NHP Area, 0.4% RSD RT, 0. 0.1% RSD F N N TCTA Area, 1.1% RSD RT, 0.1% RSD Ir F Balq (a) Area, 1.1% RSD RT, 0.1% RSD F N F F 7 min UPC2 Analysis –10x reduction vs. NPLC 4 Components/7 Impurities – Resolves NPLC co-elution ©2012 Waters Corporation 30 UPC2 ™ OLEDS Ir(fppy)3 Degraded Sample Via MS F F N N Ir F F N F F Ir(fppy)3 Isomer ? Ir(fppy)3 Ir(fppy)3 – F Isomers ? Ir(fppy)3 – F ? Ir(fppy)3 -2F ? ?? ©2012 Waters Corporation 31 Applications Chiral Separations ©2012 Waters Corporation 32 UPC2™Enantiomeric Excess Determination of Benzyl Mandelate (R) & (S) Benzyl Mandelate 0.20 mg/mL each "Pure" (R) Benzyl Mandelate 2.0 mg/mL 0.02% Impurity ©2012 Waters Corporation 33 Normal Phase LC Method Conversion & GC Alternative ©2012 Waters Corporation 34 NPLC To UPC2™ Method Conversion USP Impurity Analysis For Estradiol UPC2 – 3x reduction in run time ACQUITY UPC2 Solvent Cost/Run ~ $0.05 – 2 more impurities found – > 10x reduction in solvent cost – No halogenated solvent waste 30.856 0.0 020 0.0 018 Normal Phase HPLC Solvent Cost/Run ~ $5.89 0.0 016 0.0 014 0.0 012 AU 0.0 010 0.0 008 35.819 0.0 006 20.850 10.855 6.237 0.0 002 26.632 0.0 004 0.0 000 -0.0 00 2 -0.0 00 4 0.0 0 5.0 0 ©2012 Waters Corporation 10 .00 1 5.0 0 2 0.0 0 25. 00 M in ute s 30 .00 35 .00 40 .00 4 5.0 0 5 0.0 0 35 Biodiesel Glyceride Analysis 100 GC-FID Derivatized Biodiesel standard FAMEs 80 FAMEs pA 60 40 20 Triglycerides Petroleum Diesel Hydrocarbons Monoglycerides 0 0 4 8 ACQUITY UPC2 ELSD Underivatized Biodiesel standard LSU 16 20 24 UPC2 2000 1500 12 Minutes Diglycerides FAMEs & HCs 1000 – No derivatization – 2.5x reduction in analysis time – Glycerides well separated from FAMES 500 Triglycerides Mono & Diglycerides 0 0.0 ©2012 Waters Corporation 3.0 Minutes 6.0 9.0 36 Summary ©2012 Waters Corporation 37 Analyze a Diverse Range of Compounds ©2012 Waters Corporation 38 Green Cost Effective Solution ©2012 Waters Corporation 39 ACQUITY UPC2 ™ Merges Proven UPLC Technology With SFC Improves Resolution, Sensitivity & Speed Direct Replacement For NP LC Method Of Choice For Chiral Separations Reduces Cost Of Operation Reduces Use Of Toxic Solvents Complete System Solution – Hardware, software, chemistry support ©2012 Waters Corporation 40 ©2012 Waters Corporation 41
