1 Liquid Chromatography 2 Lecture Date: April 14 th , 2008 Outline of Topics  UHPLC – ultra-high pressure liquid chromatography (also referred to as UPLC TM , as sold by Waters) – Smaller particle packed columns  Monolithic stationary phases: – Dionex ProSwift TM – Phenomenex Onyx TM  2D LC  Micro-HPLC – Eksigent Technologies 8-channel HPLC – NanoStream 24 column HPLC – Other examples  Preparative and Simulated Moving Bed (SMB) LC 2 10 min 1980’s to present day 3.5 - 5µm spherical micro-porous 1500-4000 psi (106.4-283.7 bar) 50,000 - 80,000 plates/meter 3.9 x 300mm Early 1970’s 10µm Irregular micro-porous 1000-2500 psi (71-177 bar) 25,000 plates/meter 3.9 x 300mm 10 min Particle Size Evolution Late 1960’s 40µm pellicular non-porous coated 100-500 psi (7.1-35.5 bar) 1000 plates/meter 1m columns 10 min Diagrams from Waters Inc. J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A. Smaller Particles  Smaller particles provide increased efficiency  With smaller particles this efficiency increase extends over a wider linear velocity  This provides the ability for both added resolution and increased speed of separation  Particles are central to the quality of the separation The evolution of the van Deemter plot Diagram from Waters Inc. 3 Faster Chromatography Can Reduce Resolution * 50 mm column * Higher Flow Rates 2.0 mL/min 0.0 1 2 3.0 mL/min. 1 2 Time in Minutes 3.0 1 0.4 0.1 2 3.3 0.3 0.3 Peak Rs RT %RSD Area %RSD 1 0.8 0.3 2 2.3 0.6 0.4 Peak Rs RT %RSD Area %RSD Fails Rs Goal of 3 Limitation 5um Reversed Phase Column “Compressed Chromatography” Run time is reduced, but resolution is lost! Diagram from Waters Inc. UPLC Separations Diagram from Waters Inc. 4 Achieving Speed without Compression Peak Capacity = 153 AU 0.00 0.05 0.10 Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 2.1 x 50 mm, 5 µm Peak Capacity = 123 AU 0.00 0.05 0.10 Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 2.1 x 50 mm, 1.7 µm 6x Faster 3x Sensitivity AU 0.00 0.05 0.10 Minutes 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Diagram from Waters Inc. HPLC and UPLC TM 2.1x100mm 4.8µm HPLC 0.30 AUFS Time in Minutes 0.0 10.0 Rs = 4.71 Rs = 9.15 2.1x100mm 1.7µm ACQUITY UPLC More Resolution ACQUITY UPLC TM 0.30 AUFS 10.0 Rs = 1.86 Rs = 2.30 8 Diuretics + impurity Diagram from Waters Inc. 5 10.0 0.30 AUFS Rs = 4.71 Rs = 9.15 2.1x100mm 1.7µm ACQUITY UPLC 0.33 AUFS Time in Minutes 0.0 3.5 Rs = 3.52 Rs = 1.82 2.1x30mm 1.7µm ACQUITY UPLC Scaled Gradient Same Resolution as HPLC, Less Time ACQUITY UPLC TM ACQUITY UPLC TM HPLC and UPLC TM Diagram from Waters Inc.  Requires improvements in the whole column: – Sub 2 µm particles  Porous for optimum mass transfer  New bridged hybrid particle required for pressure tolerance (up to 15000 psi)  Sizing technology for narrow particle size distribution – Column hardware  New frit technology to retain particles  New end fittings for high pressure/low dispersion operation – Packing technology  New column packing processes to optimize stability Technology Requirements 6 Creating a New Particle Technology Advantages Disadvantages Inorganic (Silicon) • Mechanically strong • High efficiency • Predictable retention • Limited pH range • Tailing peaks for bases • Chemically unstable Polymer (Carbon) • Wide pH range • No ionic interactions • Chemically stable • Mechanically ‘soft’ • Low efficiency • Unpredictable retention Hybrid (Silicon-Carbon) Particle Technology Diagram from Waters Inc. Bridged Ethane-Silicon Hybrid Particles Anal. Chem. 2003, 75, 6781-6788 Si Si Si S i Si O O O O O Si Si C CC C C C Bridged Ethanes in Hybrid Matrix - Strength - Good Peak Shape - Wider pH Range Diagram from Waters Inc. 7 If N ↑ 3x, then R s ↑ 1.7x Explaining UHPLC with the Resolution Equation System Selectivity Retentivity Efficiency                k kN R s 11 4   NR s  J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.  In UHPLC systems, N (efficiency) is the primary driver  Selectivity and retentivity are the same as in HPLC  Resolution, R s , is proportional to the square root of N: Improving Resolution with Smaller Particles If d p ↓ 3X, then N ↑ 3X, and R s ↑ 1.7X J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.  For now, assume a constant column length  From the van Deemter equation, we know that efficiency (N), is inversely proportional to particle size (d p ): p s d R 1  8 Relationship between Peak Width and Efficiency for Constant Column Length J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A. 2 1 W N  W Height 1   Efficiency (N) is inversely proportional to the square of Peak Width W:  Peak height is inversely proportional to peak width  Outcome – narrower peaks are taller, and easier to detect If d p ↓ 3X, then N ↑ 3X, and R s ↑ 1.7X and sensitivity ↑ 1.7X Back Pressure at Constant Column Length If d p ↓ 3X, then P ↑ 27X J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.  Back Pressure is proportional to Flow Rate (F) and inversely proportional to the square of particle size (d p ): p d FP 1  p d F 1   Optimal flow rate is inversely proportional to particle size: 9 Summary of Effects at Constant Column Length Resolution Improvement Speed Improvement Sensitivity Improvement Back Pressure 1.7 vs. 5 µm particles 1.7x 3x 1.7x 25x 1.7 vs. 3 µm particles 1.3x 2x 1.3x 6x Fixed Column Length: Flow Rate Proportional to Particle Size AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00 4.8 µm, 0.2 mL/min, 354 psi AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 Theory: 1.7X Resolution 3X Faster 1.7X Sensitivity 25X Pressure 1.5X Resolution 2.6X Faster 1.4X Sensitivity 22X Pressure 1.7 µm, 0.6 mL/min, 7656 psi 2.1 x 50 mm columns Diagram from Waters Inc. 10 HPLC UPLC™ Cycle time (min) 27 3 # of Samples Run per Year 10,000 90,000 Productivity Improvements  UPLC™ gives 70% higher resolution in 1/3 the time  Target resolution is obtained 1.7x (+70%) faster  Method development up to 5x faster  Assume that an HPLC is running about 67% of the year, or 4,000 hr: Diagram from Waters Inc. Novel UHPLC Applications: High Resolution Peptide Mapping AU 0.00 0.02 0.04 0.06 0.08 AU 0.00 0.02 0.04 0.06 0.08 Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 UPLC™ 1.7 µm Peaks = 168 P c = 360 2.5X increase HPLC 4.8 µm Peaks = 70 P c = 143 Diagram from Waters Inc. [...]... reequilibration times column flow rates from 20 0 nl/min up to 20 ul/min 15 High Throughput HPLC: Eksigent Express 800 56 Chromatograms 8 10 Minutes 6 1500 5 Absorbance (mAU) Absorbance 7 4 3 2 1000 500 0 0 1 10 20 30 40 50 Time (sec) 0.0 2. 5 5.0 7.5 10.0 Time (min) 50 x 300 mm; 5 mm Luna C18 (2) Gradient: 65  95 % ACN in 25 s Hold for 20 s; Equilibrate: 20 s 12 mL/min Another Example: The Nanostream PLC... • Cost Solvent mixing problems Lack of variety in commercial columns at 1.7 um Baseline ripple – real data: DA D1 A , Sig =22 0,4 Ref =off ( OPEN_ACC\06080856.D) mAU 50 HPLC 40 30 20 10 0 0 1 2 3 4 5 6 7 8 min 0.060 0.050 0.040 UPLC AU 0.030 0. 020 0.010 0.000 0.50 1.00 1.50 2. 00 2. 50 3.00 3.50 4.00 4.50 5.00 Mnutes i 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 Monolithic Stationary Phases    Limitations... discussed here!  For more about UHPLC, see: – J R Mazzeo, U W Neue, M Kele, and R S Plumb, Anal Chem 77 (20 05) 460A-467A For more about monolithic materials in LC, see: – F Svec, C G Huber, Anal Chem 78, 21 00 -21 07 (20 06) For more about SMB, see: – F Charton, R M Nicoud, J Chrom A 7 02, 97-1 12 (1995)   23 ... biological mixture) Figures from Dionex, Inc application note, www.dionex.com 12 Two-Dimensional Liquid Chromatography (2D-LC) ● 2D LC: two LC experiments run back-to-back, with the effluent from the first LC column broken up and injected on a second LC column Fast RP LC dimension P Dugo et al., Anal Chem 78, 7743-7750 (20 06) Slower NP LC dimension Micro-LC  Micro (and nano) LC refers to precision... SP): Simulated Moving Bed Chromatography   Simulated moving bed (SMB) – a more practical way to “move” the stationary phase, compatible with modern columns and pumps Step 1 - inject inject Flow Drawings courtesy Dr G Terfloth, GSK 21 Simulated Moving Bed Chromatography  Step 2 – move injector, inject again inject Flow Drawings courtesy Dr G Terfloth, GSK Simulated Moving Bed Chromatography   Step... chemical analysis 17 Preparative Chromatography Slide courtesy of Novasep The Langmuir Isotherm Slide courtesy of Novasep 18 Non-Linear Chromatography Slide courtesy of Novasep Batch Preparative Chromatography  Inject and collect – delay between injections! Inject mobile phase mobile phase Inject again mobile phase Drawings courtesy Dr G Terfloth, GSK Collect 19 True Moving Bed Chromatography  What if we... could move the SP backwards too? Column 1 Column 2 Column 3 Column 4 mobile phase mobile phase stationary phase Drawings courtesy Dr G Terfloth, GSK True Moving Bed Chromatography  What if we move the stationary phase backwards too? Column 1 Column 2 inject Column 3 collect Column 4 collect mobile phase stationary phase Drawings courtesy Dr G Terfloth, GSK 20 SMB – Martin and Kuhn  Original Patent from... e.g with UHPLC What is a monolith? – A continuous porous stationary phase or SP support How are they made? – Polymerization reactions that yield voids Image from F Svec, C G Huber, Anal Chem 78, 21 00 -21 07 (20 06) 11 Monolithic Stationary Phases   Typical monoliths (SEM images of the support for the stationary phase) Both mesopores and micropores are apparent http://www.iristechnologies.net/CIM/monolith_structure.gif... Moving Bed Chromatography   Step 3 – collect, then move injector again, inject again Continuous chromatography – keep moving, injecting, collecting as needed Because it can go on for so long, it can separate closely-eluting compounds collect Flow inject collect Drawings courtesy Dr G Terfloth, GSK 22 Further Reading  Please note that many other new LC technologies are being developed that are not... courtesy of Nanostream Inc 16 Nanostream PLC  Features of the Nanostream system include: – 24 UV absorbance detectors – A 8-head Autosampler – Stationary phase – 10 mm (Van deemter plot!) – Column Length – 80 mm – Equivalent i.d – 0.5 mm – Injection volumes 0.4-1.0 mL Preparative Chromatography  Preparative chromatography (and preparative separations sciences): the use of a separation method to isolate . 153 AU 0.00 0.05 0.10 Minutes 0.00 5.00 10.00 15.00 20 .00 25 .00 30.00 2. 1 x 50 mm, 5 µm Peak Capacity = 123 AU 0.00 0.05 0.10 Minutes 0.00 5.00 10.00 15.00 20 .00 25 .00 30.00 2. 1 x 50 mm, 1.7 µm 6x Faster 3x Sensitivity AU 0.00 0.05 0.10 Minutes 0.00. real data: min 0 1 2 3 4 5 6 7 8 mAU 0 10 20 30 40 50 DAD1 A, Sig =22 0,4 Ref=off (OPEN_ACC6080856.D) AU 0.000 0.010 0. 020 0.030 0.040 0.050 0.060 Minutes 0.50 1.00 1.50 2. 00 2. 50 3.00 3.50 4.00. 1 Liquid Chromatography 2 Lecture Date: April 14 th , 20 08 Outline of Topics  UHPLC – ultra-high pressure liquid chromatography (also referred to as UPLC TM ,