Retention and Selectivity Variables

Applications of Ultra-Stable Phases for HPLC: High Temperature Ultra-Fast Liquid Chromatography and Thermally Tuned Tandem Column (T 3 C) Liquid Chromatography Yun Mao, Merck & Co. Jon Thompson, Peter W. Carr, University of Minnesota Clayton V. McNeff, ZirChrom Separations, Inc. Richard Henry, Angelos Kyrlidis, Greg Gaudet, Cabot Corporation HPLC Columns TM

Outline Development of Ultra-Stable Stationary Phases Theoretical and Practical Benefits of High Temperature HPLC Stability of Zirconia-based HPLC Columns (Z-phases) Examples of Ultra-Fast High Temperature Separations Using Temperature to Control Selectivity Importance of Selectivity in HPLC Optimization Selectivity of Zirconia-based HPLC Columns (Z-phases) Thermally Tuned Tandem Columns (T 3 C) Concept Examples of T 3 C Applications Conclusions

Retention and Selectivity Variables Once an HPLC column is selected, there are four main variables to adjust. Temperature is the least often used, but may be the most convenient to try. Temperature affects both retention and selectivity. ph Solvent Composition (Both Type and %) HPLC Column (Stationary Phase) Temperature (ambient- o C) Additive (Both Type and Concentration)

Effect of Temperature on Retention Time * *R. D. Antia and Cs. Horvath, J. Chromatogr., 3, - (988). t Analysis,T / t Analysis, o C..8.6.. -fold improvement!..8.6.. Temperature ( o C) Keeping Pressure and Plate Count Constant

Effect of Temperature on Column Efficiency H/d p 8 6 o C 7 o C o C 7 o C 3 6 7 8 9 ud p /D m, Ways that temperature increases efficiency and speed: Increased temperature increases diffusivity, thus decreasing the reduced velocity Increased temperature accelerates sorption kinetics The result is speed without loss of resolution and sensitivity, even at high flow rates van Deemter Plot h A B 3 / 3D m = + + Cν+ Dν + ν ν 8kd R. D. Antia and Cs. Horvath, J. Chromatogr., 3, - (988). d p

Estimated Effect of Temperature on Viscosity* η ( cp) * H. Chen and Cs. Horvath, "Rapid Separation of Proteins by RP-HPLC at Elevated Temperatures," Anal. Methods Instrum.,, 3- (993)...8.6... t N η 6 8 6 8 T ( o C) Water % ACN MeOH ACN Ways that increased temperature increases efficiency and speed:. Increased temperature increases diffusivity, thus decreasing the reduced velocity. Increased temperature accelerates sorption kinetics 3. Increased temperature reduces k values. Increased temperature decreases mobile phase viscosity which enables higher flow rates

Why Stable Phases? Advantages of Extraordinary Chemical Stability ph Stability Thermal Stability ph < ph > 3 Lower Pressure Drop Less Organic Solvent Thermally Optimize Selectivity Clean with conc. Acids Suppress Ions for Acids Suppress Ions for Amines Sanitation Depyrogenation Less Wear and Tear Higher Flow Fast Analysis More Robust Analysis Easier Method Development Stable stationary phases have advantages in terms of analysis time, selectivity and column lifetime. If LC/MS is employed, stable phases exhibit low bleed background.

ZirChrom Particle Properties ZirChrom -Carb and DiamondBond particles are prepared by coating base particles with a thin layer of carbon using a chemical vapor deposition process ZirChrom -PBD and -PS particles are prepared by coating with a layer of highly crosslinked polymer Characteristic Property Surface Area (m /g) Pore Volume (cc/g).3 Pore Diameter (Å) -3 Porosity. Density (g/cc).8 (.x silica) Particle Diameters (µ) 3.,.,. NH Y + HA + NaNO = AN N Y + H O + NaA + N N + Y -C8 Bonding Reaction on Carbon Clad Zirconia Carbon Clad Zirconia Diazonium Salt Modified Carbon Clad Zirconia

DiamondBond-C8 Stability 8 7 6 k' 3 k' Butylbenzene (.M Nitric Acid) k' Butylbenzene (.M NaOH) k' Butylbenzene ( o C) 7 Column Volumes Stable phases allow retention and selectivity variables to be fully explored and exploited. LC Conditions: Base Stability DiamondBond TM Phase A, 3 x.6 mm id; Mobile phase, / ACN/Water; Flow rate,. ml/min.; Temperature, 3 o C; Injection volume, ul; Detection at nm. Acid Stability DiamondBond TM Phase A, x.6 mm id; Mobile phase, / ACN/Water; Flow rate,. ml/min.; Temperature, 3 o C; Injection volume, ul; Detection at nm. Temperature Stability-- DiamondBond TM Phase B, x.6 mm id; Mobile phase, / ACN/Water; Flow rate,. ml/min.; Temperature, 3 o C; Injection volume, ul; Detection at nm.

Increasing Separation Speed Column Temperature vs. Column Length vs. Flow Rate T increases o C k decreases 3-fold Retention Time Retention Time Cold Hot Long Short Temperature Column Length Increasing only flow rate to reduce retention time is the least desirable option because pressure increases and performance drops. Increasing temperature and decreasing column length are better ways to decrease retention time and speed up analysis; however, decreasing length reduces efficiency and resolution, while increasing temperature doesn t.

Antihistamines- Fast Isocratic ma U 6 3 VWD A, Wavelength= nm (P\TEST6.D) 3 o C 3 Temperature: o C Flow rate:.3 ml/min. Pressure drop: 9 bar Resolution (,3):. Analysis time:. minutes..6.8 3 3. min ma U 6 8 VWD A, Wavelength= nm (P\TEST.D) 3 min 8 6 3 8 o C Temperature: 8 o C Flow rate: 3. ml/min. Pressure drop: 9 bar Resolution (,3):. Analysis time:. minutes.9.9..... min 6 8 min LC Conditions: (A) Mobile Phase, 9/7 ACN/mM Tetramethylammonium hydroxide, ph.; Flow Rate,.3 ml/min.; Injectionl,. ul; nm detection; Column Temperature, C; Pressure drop = 9 bar; Solutes: =Doxylamine, =Methapyrilene, 3=Chlorpheniramine, =Triprolidine, =Meclizine=Triprolidine, =Meclizine, x.6 ZirChrom-PBD (B) same as A, except Mobile Phase, 6./73. ACN/mM Tetramethylammonium hydroxide, ph.; Flow Rate, 3. ml/min.; Column Temperature, 8 C; Pressure drop = 9 bar.

Beta-Blockers- Fast Isocratic mau Column Temperature = o C, ph = H NOC HO OH CHCH NHCH CH 3 Labetalol CH CH 8 CH 3 OCH CH OCH CHCH NHCH(CH 3 ) 6 OH Metoprolol CH CH CH OCH CHCH NHCH(CH 3 ) Alprenolol OH Separation in. minutes!..6 LC Conditions: Column, x.6 Diamondbond-C8, OD6A; Mobile phase, / ACN/mM Ammonium Phosphate ph.; Flow rate, 3. ml/min; Temperature, o C; Injection volume,. ul; Detection at nm; min

Nitrosamines- Fast Gradient mau ph 9., 7 ºC... 3 min Column: -C8,.6mm Mobile Phase:.-9%B from -3 minutes A: mm Ammonium hydroxide, ph 9. B: % Acetonitrile Flow rate:. ml/min. Temperature: 7 ºC Injection volume:. µl Detection: 3 nm Back Pressure: bar

Non-Steroidal Anti-inflammatories Norm. 7 Column Temperature = 8 o C 7 3 6 3 6 7 8 min LC Conditions: Column, x.6 DiamondBond TM -C8; Mobile phase, -8 A over minutes, A=ACN, B=mM ammonium phosphate, mm ammonium fluoride, ph.; Flow rate,. ml/min.; Temperature, 8 o C; Injection volume, ul; Detection at nm; Solute concentration,. mg/ml.; Solutes, =Acetaminophen, =Ketoprofen, 3=Ibuprofen, =Naproxen, =Oxaprofen, 6=Indemethacin.

Non-Steroidal Anti-inflammatories (FAST) Norm. 3 3 Column Temperature = o C Separation in minute! 3...6.8...6.8 min LC Conditions: Column, x.6 DiamondBond TM -C8; Mobile phase, /7 ACN/mM phosphoric acid, ph.3; Flow rate,. ml/min.; Temperature, o C; Injection volume, ul; Detection at nm; Solute concentration,. mg/ml.; Solutes, = Acetaminophen, =Ketoprofen, 3=Naproxen, =Ibuprofen, =Oxaprofen.

PTH-Amino Acids mau 8 6 3 6 Column Temperature = 8 o C 7 8 3 6 7 8 9 min LC Conditions: Column, x.6 DiamondBond TM -C8; Mobile phase, /8 ACN/.% TFA, ph.; Flow rate,. ml/min.; Temperature, 8 o C; Injection volume, ul; Detection at nm; Solute concentration,. mg/ml.; Solutes, =PTH-Arginine, =PTH-Serine, 3=PTH-Glycine, =PTH-Alanine, =PTH-Isoaminobutyric acid, 6=PTH-Aminobutyric acid, 7=PTH-Valine, 8=PTH-Norvaline.

The Impact of Selectivity on Resolution 3. Efficiency Retention Selectivity. α R= N α = k k + k j k i α- α Resolution (R).... N k Selectivity (α) has the greatest impact on improving resolution........ α N k

Comparing and Adjusting Selectivity in HPLC Mobile Phase Composition (B%) ** Mobile Phase Type (ACN, MeOH, THF) Stationary Phase Type (C8-SiO, C-ZrO, PBD-ZrO ) Temperature ** Only works in mixed-mode log k (condition ) 3 α =.3 α =.3 3 log k' (condition ) κ κ plots r =., s.d. =. r =.66, s.d. =.7 log k (condition ). Horvath, Snyder and Carr 3 Poor correlations in the κ κ plot indicate changes in selectivity. α =. α =. 3 log k' (condition )

Non-Electrolyte Solutes Nonpolar Benzene Toluene Ethylbenzene p-xylene Propylbenzene Butylbenzene Cl Polar Br Cl O O Bromobenzene p-dichlorobenzene Anisole O O Methylbenzoate Napthalene Acetophenone O NO CN NO NO Cl Benzonitrile Nitrobenzene p-nitrotoluene p-nitrobenzyl Chloride Benzophenone HB Donor Cl OH OH NH H OH OH O Benzylalcohol 3-Phenyl Propanol N-Benzyl Formamide Phenol p-chlorophenol The selectivity of the stationary phase becomes paramount for non-ionic solutes. Temperature and mobile phase variables become less important.

Selectivity Comparison.. -. - -. Diamondbond-C8 ODS- Unmodified Carbon coated zirconia Mobile phase, /6 Acetonitrile/Water; Flow rate,. ml/min.; Temperature, 3 o C; Detection at nm; ml Injection volume.

Comparison of Variables Affecting Selectivity 3% ACN vs. % ACN.. R =.989 SD=. Stationary Phase Type Carbon-ZrO vs. PBD-ZrO.. MeOH vs. THF R =.896 SD=.7... logk' (/ ACN/H O) 8 o C vs. 3 o C R =.99 SD=.3.. R =.38 SD=..... logk' (THF/H O) C8-SiO vs. PBD-ZrO R =.98 SD=.9 -.. logk' (PBD-ZrO ). 3 logk' (C8, 3 o C) -. -... logk' (PBD-ZrO ) Stationary phase type has a large effect on selectivity.

Carbon Surface Has Unique Selectivity Mobile phase: 3/6 A/B A: ACN B: Water LC Conditions Column: ZirChrom -PBD, x.6 mm Flow rate:. ml/min. Temperature: 3 o C Injection volume: µl Detection: nm mau 8 6 PBD or ODS α=.3, Analytes - Ethylbenzene - p-xylene 3 6 7 8 9 min Mobile phase: 37.//7. A/B/C A: ACN B: THF C: Water LC Conditions Column: DiamondBond-C8, x.6 mm Mobile phase: 3./67. A/B A: ACN B: Water LC Conditions Flow rate:. ml/min. Temperature: 6 o C Injection volume: µl Detection: nm Column: ZirChrom -CARB, x.6 mm Flow rate:. ml/min. Temperature: 6 o C Injection volume: µl Detection: nm mau 3 mau 8 6 DB-C8 α=. 3 6 7 8 9 CARB α=.8 Bonded Carbon min Carbon 3 6 7 8 9 min

Selectivity Difference vs ODS (Orthogonality) Column R Selectivity Difference * ZirChrom -PBD.98 DiamondBond -C8.889 33 ZirChrom -Carb.9 67 * S=(-R )., as described by U. Neue at FACSS For non-ionizable solutes: ZirChrom-Carb and Diamondbond-C8 columns have very different selectivity from traditional C8-silica columns, exceeding that of embedded polar phases. ZirChrom-PBD has selectivity similar to C8-silica. For ionizable solutes: The selectivity difference is even greater on zirconia phases due to mixed mode possibilities.

Zirconia Has Unique Surface Chemistry Lewis acid O O - P O - A + OH A + Bronsted base and acid A + A + O - OH O H O OH OH H O H O Hydrophobic Coating (PBD, Carbon) O Zr O Zr O Zr O Zr O Zr O Zr O Zr O Zr O Zirconia by itself has very rich surface chemistry Coated zirconia phases (Carbon and PBD) have mixed surface properties The retention of various basic and acidic analytes can be fine tuned by changing ph, buffer and salt concentration, in addition to mobile phase modifier concentration and type

Zirconia Features Tunable Surface Properties Net Zirconia Surface Charge State with weak Lewis Base Buffers + + - - + + + + - - - - + + + + + + + - - - - - - - ph 3 9 Net Zirconia Surface Charge State with strong Lewis Base Buffers - - - - - - - - - - - - - - - - - - - - - - - - - - - - The choice of buffer and ph on zirconia columns affects the surface charge and the elution properties of ionizable analytes. Mixed-mode (RP plus CEX) selectivity can be created. ph 3 9 Silica Surface Charge State - - - - - - - - - - - - - - - - - - - - ph 3 9

Antihistamine Drug Selectivity Comparison Mobile Phase: /6 Acetonitrile/ mm Phosphate, ph=7 3 +3 C8-SiO 3 o C Absorbance (mau) Absorbance (mau) +7 6 3 8 6 9 8 7 log k' (ODS at o C) ZirChrom-PBD 3 o C 9... R =.7 S=9 9 8 3 6 7.... log k'(pbd-zro at o C) The selectivity of zirconia based columns towards ionizable compounds becomes very different from that of traditional silica columns. Buffer type and ph have an effect on mixedmode retention (RP and CEX) by Z-phases.

Thermally Tuned Tandem Columns What if you could continuously adjust the selectivity of your HPLC column? Think of temperature as a replacement for solvent strength in adjusting retention time.

Thermally Tuned Tandem Columns (T 3 C) Pump Injector Temperature Column Temperature Column Detector e.g. C8-SiO e.g. C-ZrO Column, 3 Optimized T 3 C 3 Column 3, T 3 C is a method to continuously adjust HPLC selectivity using tandem, orthogonal columns and dual, independent temperature control.

Requirements for T 3 C Two columns with different (ideally orthogonal) selectivity At least one thermally stable column (e.g. Zirconia-based) Thermally stable compounds Temperature control for at least one column (both preferred) Easy method development Theory and practice of T 3 C (references -3) Guidelines for method development

Effect of Temperature on T 3 C Selectivity Pump Injector Temperature Column Temperature Column Detector f = k ' α = fα f α net ' k + k ' + Low T Low T f = k ' ' k + k ' Low T High T High T Low T α net α αnet α Temperature continuously changes the T 3 C selectivity between α and α.

Method Development for T 3 C Pump Injector T T Column Column Detector t Rnet (T,T ) = t R (T )+ t R (T ) t R = t + ( k ') t R = t + ( k ') log k =A +B /T log k =A +B /T trial runs trial runs

Guidelines for Optimizing T 3 C Choose Two Stationary Phases Window Diagram Optimization Choose Mobile Phase (<k <) Calculate T 3 C Retentions ln k =A+B/T t rnet =t r +t r No Different Selectivity? Yes Two More Runs at Higher Temperatures

Separation of Ten Triazine Herbicides by T 3 C Absorbance (mau) 3 3 6 7 8 9 +7 8 3 9 7 6 8 9 3 6 Time (min) C8-SiO 3 o C C-ZrO 6 o C C8-SiO C-ZrO 3 o C o C 3 T 3 C CH 3 S, CH 3 O, Cl =R Solutes:. Simazine. Cyanazine 3. Simetryn. Atrazine. Prometon N N N N H R Other conditions: 3/7 ACN/water ml/min; nm detection H N R 6. Ametryn 7. Propazine 8. Terbutylazine 9. Prometryn. Terbutryn T 3 C can improve separation without increasing analysis time. Extra length is offset by shorter k values.

Comparison of T 3 C with Solvent Optimization T 3 C is more powerful than mobile phase optimization on ODS 6 + 3 %ACN 6 7 8 9..8. ACN R<. 3 6 3+ 6 8 6 3 6+7 %MeOH 8 9 6 7+8 3% THF 9+ Minimum Rs..3.3.......6.8.6.. MeOH R<.3 THF R<. 6 8 Time (min)....3 Percentage of organic modifier

More T 3 C Separations Absorbance (mau) 7 8 8 6 8 6 3 6 8 3 3 6 7 8 * Urea and Carbamate Pesticides +9 + T 3 C 39 o C+89 o C 3 6 7 8 Time (min) * 76 8 9 * C8-SiO 3o C C-ZrO 9 o C 9 + 8 Absorbance (mau) 6 8 6 8 8 6 6 8 +3 3 6 7 8 9 3 Barbiturates +7 C8-SiO 3o C 6 7 8 9 3 Time (min) C-ZrO 3 o C 6 8 9 T 3 C 8 o C+ o C

Basic Drugs: C8-Silica and PBD-Zirconia orthogonal for nonionics Temperature Temperature C8-SiO Carbon-ZrO Partition Mechanism Adsorption Mechanism Even more orthogonal for ionics Phosphate Buffer ph=7 Temperature Temperature C8-SiO Reversed Phase Mode + Cation-Exchange Mode PBD-ZrO Cation-Exchange Mode + Reversed Phase Mode

Separation of Antihistamine Drugs by T3C Mobile Phase: /6 Acetonitrile/ mm Phosphate, ph=7 +3 C8-SiO 3 oc 3 log k' (ODS at oc) 9 8 7 6 Absorbance (mau) Absorbance (mau). 9 R=.7 S=9!. 8 3 6 +7 PBD-ZrO 3 oc. 6 T3 C 3.. Resolution....6.8....6.8.. 9. log k'(pbd-zro at oc) 8 3. 8 6 oc +3 oc 7 9 Tempearure on ODS column 7 (a) 3 3 8 Time (min) 3 7 6 Temperature on PBD-ZrO column 3

Advantages and Disadvantages of T 3 C Advantages: Provides much better separation Can improve selectivity without big change in analysis time Relatively easy method development Good selection of orthogonal stationary phases available Disadvantages: Stationary phases and sample must be thermally stable Potential for higher pressure drop and longer run time Need to carefully select stationary and mobile phase Need well-designed column oven; two ovens or dual-zone oven provide more selectivity

Conclusions Zirconia-based phases are ultra-stable to extreme conditions in both temperature and ph The durability of Zirconia-based phases allows for ultrafast separations at elevated temperatures ZirChrom zirconia-based phases offer selectivity that can be very different from traditional silica-based stationary phases The combination of unique (orthogonal) selectivity and exceptional thermal stability allows the development of novel separations using the Thermally Tuned Tandem Column (T 3 C) approach For a given analysis, T 3 C requires two stationary phases with orthogonal selectivity and different critical pairs.

References. Jun Mao and Peter W. Carr, Anal Chem., 7, -8.. Jun Mao and Peter W. Carr, Anal. Chem., 73, 78-8. 3. Jonathan D. Thompson and Peter W. Carr, Anal. Chem., 7, 7-3.

Acknowledgements The author wishes to thank the following: the technical support group at Zirchrom Separations, Inc. the research group at Cabot Corporation the research group under Peter Carr at University of Minnesota Richard A. Henry rhenry@psualum.com