Liquid Chromatography 2

Liquid Chromatography 2 Lecture Date: April 14th, 2008 Outline of Topics  UHPLC – ultra-high pressure liquidchromatography (also referred to as UPLCTM, as sold by Waters) –Smaller particle packed columns  Monolithic stationary phases: –Dionex ProSwiftTM –Phenomenex OnyxTM  2D LC  Micro-HPLC –Eksigent Technologies 8-channel HPLC –NanoStream 24 column HPLC –Other examples  Preparative and Simulated Moving Bed (SMB) LC 1 Particle Size Evolution Late 1960’s 40µm pellicularnon-porous coated 100-500psi (7.1-35.5 bar) 1000plates/meter 1m columns 10min Diagrams from Waters Inc. Early1970’s 10µm Irregular micro-porous 1000-2500psi (71-177 bar) 25,000plates/meter 3.9 x 300mm 10min 1980’sto present day 3.5 - 5µm sphericalmicro-porous 1500-4000psi (106.4-283.7 bar) 50,000- 80,000 plates/meter 3.9 x 300mm 10min 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 The evolutionof the van Deemter plot  Smaller particles provide increased efficiency  Withsmaller particlesthis efficiency increase extendsover a wider linear velocity  Thisprovidesthe ability for both added resolution and increased speed of separation  Particlesare central to the quality of the separation Diagram from Waters Inc. 2 Faster Chromatography Can Reduce Resolution “Compressed Chromatography” 5umReversed Phase Column 1 2 * 50 mm column * Higher Flow Rates 2.0 mL/min 1 2 Peak Rs 1 --2 3.3 Peak Rs RT Area %RSD %RSD 0.4 0.1 0.3 0.3 RT Area %RSD %RSD 1 3.0 mL/min. 2 -- 0.8 0.3 2.3 0.6 0.4 0.0 Time in Minutes 3.0 Fails Rs Goal of 3 Limitation Run time is reduced, but resolution is lost! Diagram from Waters Inc. UPLCSeparations Diagram from Waters Inc. 3 Achieving Speed without Compression 2.1x 50 mm, 5 µm 0.10 0.05 Peak Capacity = 153 0.00 0.00 5.00 10.00 15.00 20.00 Minutes 25.00 30.00 2.1x 50 mm,1.7 µm 0.10 Peak Capacity = 123 0.05 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 0.10 0.05 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Minutes 6x Faster 3x Sensitivity 3.50 4.00 4.50 5.00 Diagram from Waters Inc. HPLC and UPLCTM 8 Diuretics + impurity 0.30 Rs= 2.30 Rs= 1.86 2.1x100mm4.8µm HPLC 0.30 Rs= 9.15 10.0 2.1x100mm1.7µmACQUITYUPLC Rs= 4.71 More Resolution ACQUITY UPLCTM 0.0 Timein Minutes 10.0 Diagram from Waters Inc. 4 HPLC and UPLCTM 0.30 Rs= 9.15 2.1x100mm1.7µmACQUITYUPLC Rs= 4.71 ACQUITY UPLCTM 0.33 Rs= 3.52 10.0 Rs= 1.82 2.1x30mm1.7µmACQUITYUPLC Scaled Gradient SameResolution as HPLC, Less Time ACQUITY UPLCTM 0.0 Timein Minutes 3.5 Diagram from Waters Inc. ... - tailieumienphi.vn