High Temperature Liquid Chromatography
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1 High Temperature Liquid Chromatography C.V. Mceff 1*, B. Yan 1, D. R. Stoll 2, R.A. Henry 3 1 ZirChrom Separations, Inc., 617 Pierce Street, Anoka, M Department of Chemistry, University of Minnesota, 207 Pleasant St. Minneapolis, M Independent Consultant, 983 Greenbriar Drive, State College, PA ZirChrom Separations, Inc.
2 OUTLIE Advantages of High Temperature HPLC Theoretical Effects of High Temperature HPLC Practical Analytical Advantages of Using High Temperature HPLC Development of ew Stationary Phases Selectivity Comparison of Zirconia Based Stationary Phases with C18 Silica and Other Columns High Temperature Separations Using Temperature to Control Selectivity Importance of Selectivity in HPLC Optimization 2
3 Theoretical Advantages to High Temperature LC van Deemter Plot h A B 23 / 3D m = + + Cν + Dν + 2 ν ν 8kd R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988). d p Practical Limit Temperature Dependence Guiochon, Georges, Anal. Chem., 52, (1980). t (1 + k') L P 2/3 2/3 max η 1/3 T Three ways that temperature increases efficiency and speed Increased temperature increases diffusivity, thus decreasing the reduced velocity Increased temperature accelerates sorption kinetics Increased temperature decreases mobile phase viscosity 3
4 Theoretical Effect of Temperature on Column Efficiency o C 8 75 o C H/d p o C o C ud p /D m,25 R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988). 4
5 Estimated Effect of Temperature on Viscosity* 1.0 η ( cp) t η Water 50 % AC MeOH 0.4 AC T ( o C) *H. Chen and Cs. Horvath, "Rapid Separation of Proteins by RP-HPLC at Elevated Temperatures," Anal. Methods Instrum., 1, (1993). 5
6 Effect of Temperature on Theoretical Analysis Time at Constant Pressure and Plate Count* t Analysis,T /t Analysis, 25 o C fold improvement! Temperature ( o C) *R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988). 6
7 Practical Advantages of Column Stability Extraordinary Chemical Stability ph Stability Thermal Stability ph < 1 ph > 13 Lower Pressure Drop Less Organic Solvent Thermally Optimize Selectivity Cleaning with Conc. Acid Ion Supression for Acids Ion Supression for Amines Sanitation/ Depyrogenation Less Wear and Tear Higher Flow Rate: Fast Analysis More Robust Analysis Easier Method Development 7
8 List of HTLC compatible reversed-phase columns Manufacturer Column ame Stationary Phase Type Temperature Limit ( o C) Selectivity versus C18 Polymer Laboratories PLRP Polymer 200 Different Selerity Blaze Silica 200 Different Supelco DiscoveryZR-Carbon Carbon Clad Zirconia 200 Different Supelco DiscoveryZR-CarbonC18 Modified Carbon on Zirconia 200 Different Thermo-Electron Hypercarb Carbon 200 Similar ZirChrom Separations, Inc. ZirChrom-CARB Carbon Clad Zirconia 200 Different ZirChrom Separations, Inc. Diamondbond-C18 Modified Carbon on Zirconia 200 Different Jordi Jordi DVB Polymer 150 Different Sachtleben Sachtopore-RP Polymer coated Titania 150 Different Supelco DiscoveryZR-PBD Polymer Coated Zirconia 150 Different Supelco DiscoveryZR-PS Polymer Coated Zirconia 150 Different ZirChrom Separations, Inc. ZirChrom-PS Polymer Coated Zirconia 150 Different ZirChrom Separations, Inc. ZirChrom-PBD Polymer Coated Zirconia 150 Different Agilent SB Extend-C18 Silica 90 Similar Waters X-Bridge Silica 80 Similar 8
9 Stationary Phase Comparison Average Scatter of κ κ Plots for Two Kinds of Stationary Phases Using 22 Solutes Standard Deviation Carbon-ZrO 2 PBD-ZrO 2 C18-SiO 2 (ODS) Phenyl-SiO 2 C-SiO 2 PRP C-ZrO 2 C PRP Phenyl ODS PBD-ZrO 2 ODS Phenyl PRP C For non-electrolytes, C-ZrO 2 and aliphatic phases have the most different selectivities. 9
10 orm Fast Separations on-steroidal Anti-Inflammatories Column Temperature = 150 o C Separation in 1 minute! LC Conditions: Column, 50 x 4.6 DiamondBond TM -C18; Mobile phase, 25/75 AC/40mM phosphoric acid, ph 2.3; Flow rate, 5.5 ml/min.; Temperature, 150 o C; Injection volume, 1ul; Detection at 254nm; Solute concentration, 0.15 mg/ml.; Solutes, 1= Acetaminophen, 2=Ketoprofen, 3=aproxen, 4=Ibuprofen, 5=Oxaprofen. min 10
11 Fast β-blockers Separation mau Column Temperature = 150 o C, ph = H 2 OC 1000 HO OH CHCH 2 HCH CH 3 Labetalol CH 2 CH CH 3 OCH 2 CH 2 OCH 2 CHCH 2 HCH(CH 3 ) OH Metoprolol CH 2 CH CH OCH 2 CHCH 2 HCH(CH 3 ) 2 Alprenolol OH Separation in 0.4 minute! LC Conditions: Column, 50 x 4.6 Diamondbond-C18, OD A; Mobile phase, 45/55 AC/20mM Ammonium Phosphate ph11.0; Flow rate, 3.0 ml/min; Temperature, 150 o C; Injection volume, 1.0 ul; Detection at 210 nm; Solutes, 1=Labetalol, 2=Metoprolol, 3=Alprenolol min 11
12 Resolution: The Importance of Selectivity α Efficiency R= 4 Retention k k +1 α= k j k i Selectivity α-1 α Selectivity (α) has the greatest impact on improving resolution. Resolution (R) k α k 12
13 Comparison of Variables Affecting Selectivity 30% AC vs. 50% AC R 2 =0.989 SD=0.05 Stationary Phase Type Carbon-ZrO 2 vs. PBD-ZrO 2 MeOH vs. THF 2.00 R 2 =0.896 SD= logk' (50/50 AC/H 2O) 80 o C vs. 30 o C R 2 =0.995 SD= R 2 =0.385 SD= R 2 =0.973 SD=0.09 logk' (THF/H 2 O) C18-SiO 2 vs. PBD-ZrO logk' (PBD-ZrO 2 ) logk' (C18, 30 o C) Stationary phase type has a very large effect on selectivity logk' (PBD-ZrO 2) 13
14 Thermally Tuned Tandem Columns (T 3 C) A Mechanism to Continuously Adjust the Stationary Phase Temperature 1 Temperature 2 Pump Injector Column 1 Column 2 Detector e.g. C18-SiO 2 e.g. C-ZrO 2 Column 1 1,2 3 4 Optimized T 3 C Column ,4 14
15 Solutes: 1. Simazine 2. Cyanazine 3. Simetryn 4. Atrazine 5. Prometon Separation of Ten Triazine Herbicides by T 3 C CH 3 S, CH 3 O, Cl =R C18-SiO 2 C-ZrO 2 30 o C 125 o C H R 1 H 6. Ametryn 7. Propazine 8. Terbutylazine 9. Prometryn 10. Terbutryn Other conditions: 30/70 AC/water 1ml/min; 254 nm detection R 2 Absorbance (mau) T 3 C can improve separation without increasing analysis time Time (min) 9 C18-SiO 2 30 o C C-ZrO 2 60 o C 3 T 3 C 10 15
16 Conclusions (1) Zirconia Based Stationary Phases are ultra-durable and efficient, stable at the extremes of ph and at column temperatures as high as 200 o C. (2) ZirChrom -CARB has the most different selectivity relative to conventional ODS phases for the 22 selected non-ionizable compounds. (3) High Temperature Liquid Chromatography (HTLC) is a powerful technique that can be used as a routine analytical tool in the development of separation methods. (4) HTLC is a unique tool in altering chromatographic selectivity (T 3 C method), increasing analysis speed. (5) HTLC capability will become an important part of HPLC system design in order to fully utilize the benefits of columns prepared with ultra-small particles and utrafast analyses. 16
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