Method Development on Next Generation RPLC Supports

Method Development on Next Generation RPLC Supports Dr. Peter W. Carr President Dr. Clayton McNeff Vice President 1-866-STABLE-1 www.zirchrom.com Specialists in High Efficiency, Ultra-Stable Phases for HPLC

ZirChrom Separations, Inc. ZirChrom Separations, Inc. is a company formed in 1995 located in Anoka, Minnesota, USA. ZirChrom manufactures a full line of zirconia-based high performance chromatographic materials for the analytical analysis of compounds primarily by high performance liquid chromatography (HPLC). The extreme stability of zirconia and its unique chromatographic selectivity allows for the optimization of separation conditions, which are totally incompatible with other types of supports. ZirChrom columns will give you full access to all chromatography tools that have traditionally been limited when using silica (I.e. ph, high temperature, and buffers). Zirconia-based supports promise to revolutionize the way that liquid chromatographic analyses are done. These supports will last longer, and allow for faster more selective separations resulting in significant cost savings per analysis

Outline RPLC Columns Sold By ZirChrom Surface Chemistry Considerations Method Development on ZirChrom Columns Where Do I Start Column Choice Mobile Phase ph Column Temperature Example Method Development of A Real Pharmaceutical Samples

Why Use ZirChrom Phases? Selectivity - similar, different, or very different to ODS-silica Stability - run the operating conditions needed to get the separation, don t worry about the column Speed - get 3x - 5x faster analysis by modest increases in temperature. Cost - Longer column life, greater productivity

Analytical Diameter Porous Zirconia Particles 2.9 µ 2.8 µ

Properties of Porous Analytical Zirconia Characteristic Property Surface area (m²/g) 22 Pore volume (cc/g) 0.13 Pore diameter (Å) 250-300 Porosity 0.45 (silica 0.48) Density (gm/cc) 2.6 (2.5x silica) Particle size (µm) 3.0 (130,000 p/m) Prep-scale particles also available

4 RPLC Stationary Phases Column Name ZirChrom-PBD ZirChrom-PS ZirChrom-CARB DiamondBond-C18 Stationary Phase Polybutadiene Polystyrene Carbon Octadecylphenyl-Carbon

Typical Silica C18 CH 3 CH 2 CH 2 CH 2 ODS CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 10 CH 2 10 CH 2 CH 2 CH 2 CH 3 CH 2 CH 3 CH 3 CH 2 CH 3 CH 3 CH 2 CH Si CHCH 2 CH 3 CH 3 CH 2 CH Si CHCH 2 CH 3 O OH OH OH O Silica Substrate

ZirChrom -PBD CH 2 CH CH 2 CH CH CH 2 CH 2 CH CH CH 2 CH 2 CH CH 2 CH CH CH 2 CH 2 CH CH CH 2 ~20 Zirconia Substrate

ZirChrom -PS Zirconia Substrate

ZirChrom -CARB C C C C C C C C C C C C C C Zirconia Substrate

DiamondBond -C18 CH 3 CH 3 CH 3 (CH 2 ) 10 (CH 2 ) 10 (CH 2 ) 10 16 16 16 This bond is chemically and thermally stable -- great for LC/MS CH 2 CH 2 CH 2 C C C C C C C C C C C C C C Zirconia Substrate

How do I Start? Sample size, matrix and solubility? Column(s) Selection Mobile Phase Optimization Organic modifier ph Buffer choice Temperature Can I speed it up?

General Starting Point Separation Variable Preferred Initial Choice Dimension 10 x 0.46 cm Particle Size 3 µm Stationary Phase ZirChrom-PBD or DBC18 Mobile Phase Water/Acetonitrile %B Variable Buffer 25 mm phosphate, ph 7.0 Additives Variable Flow Rate 1-2 ml/min Temperature 30-80 o C Sample Volume 5 microliters Sample Mass 100 µg

Which Column Do I Use? Are the analytes acidic, basic or neutral? Are the analytes very polar? Are the analytes closely related? Chiral? Are the analytes highly aromatic? Are the analytes thermally stable? How complex is the sample?

Reversed-Phase Column Selection Guide CURRENT PROBLEM/CONCERN COLUMN SUGGESTED CONDITIONS Improve Selectivity Need improved selectivity for nonelectrolytes, isomers, disastereomers. Currently using carbon, cyano, phenyl or fluoro phases Need improved selectivity for bases. DiamondBond TM C18, ZirChrom -CARB ZirChrom -PBD Temp >50 o C, with acetonitrile or preferably THF eluent. Use with Lewis base buffer (e.g. phosphate, citrate, fluoride) at ph 7.0, 5-50 mm. If not effective, increase ph to 11.0 or higher - See Technical Bulletin #241. Need improved selectivity for acids. Change Retention Need more retention for very polar (hydrophilic) nonelectrolytes. Currently using nearly 100% water eluent or polar embeded phase Need more retention for very polar bases. Currently using nearly 100% water eluent or polar embeded phase or sulfonic acid paired ion reagent Need more retention for very polar acids. Currently using nearly 100% water eluent or polar embeded phase or quaternary amine paired ion reagent Need less retention with any solute type. Improve Efficiency / Productivity Inadequate stability and selectivity. Having trouble with silica-based phases, changed to alumina or polymer column and problems were still not sufficiently resolved Poor column stability. Experiencing retention drift at low or high ph, at above ambient temperature or when using phosphate or carbonate buffer. Separations taking too long. DiamondBond TM C18 DiamondBond TM C18, ZirChrom -CARB ZirChrom -PBD DiamondBond TM C18 ZirChrom -PS DiamondBond TM C18, ZirChrom -PBD DiamondBond TM C18, ZirChrom -PBD DiamondBond TM C18, ZirChrom -PBD Use with 10 mm phosphate and 2 mm fluoride at ph 7.0. If not effective, drop ph to 2.0 or lower. Can use in high water mobile phase. Temperature can be lowered to room temperature if needed to increase retention. Use at ph 7.0 with < 5mM phosphate. Vary ph as needed. Can use in high water mobile phase. Use low ph to proton ate acid. Choose a ph << pka, even ph 0.5 is no problem. Least hydrophobic ZirChrom phase. Can easily achieve use of 100% water eluent. ph range: 0.5-13, Temp < 200 o C. Any of the listed reversed phases will give higher efficiency and better peak shape than alumina or polymer columns. Our phases are the Most Durable phases on the market Increase temperature up to max. operating range for LC &/or analyte. Increase flow rate. Easily improves speed 2-3 fold. Column overloaded too easily with basic solutes. ZirChrom -PBD Use phosphate buffer. Improve Detection Sensitivity Need to go to shorter wavelength to enhance sensitivity in UV. ZirChrom -PS Use a high water or pure water eluent and go deep into UV. Solute does not have long wavelength absorption or is very dilute Need to decrease bleed in LC/MS. DiamondBond TM Recommend THF or acetonitrile as eluent modifier; can also C18 enhance sensitivity. See www zirchrom com literature articles numbers 1 3 4 18 27 28 30 35 38 39 41 44 46 50 51 55 59 Technical Bulletin #240

Part II. Zirconia the Un-Silica. The difference is the surface chemistry.

Surface Chemistry of Zirconia O OH O Zr O Zr O O H 2 O HO O H 2 O OH 2 OH OH OH2OH2 O Zr O Zr O Zr O Zr O Zr O O O O O O O Brönsted Acid: Brönsted Base: ZrOH + - OH ZrO - + H 2 O + Zr O H + Zr H + Zr O Zr Lewis Acid: Zr(OH 2 ) + ZrOOC R + H 2 O - OOC R

Interaction Strength of Lewis Bases with Lewis Acid Sites on Zirconia Interaction Strength Strongest Weakest Lewis Base (A) Phosphate Fluoride Citric acid Sulfate Acetic acid Formic acid Nitrate Chloride Lewis bases with higher electron density and lower polarizability interact more strongly with zirconia.

Resolution: The Importance of Selectivity 3.0 2.5 α Efficiency R= N 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) 2.0 1.5 1.0 0.5 N k 0.0 1.00 1.05 1.10 1.15 1.20 1.25 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 α N k

Effect of α and N on Resolution In general it is better to optimize selectivity rather than column efficiency as resolution is directly related to selectivity changes, but only varies with the square root of N. α Column N Run Time 1.10 4 cm, 3 µm 6,000 2-5 min 1.05 30 cm, 5 µm 25,000 30-60 min 1.02 5 m, 10 µm 160,000 8-15 h

Method Development Knobs on Zirconia RP Phases Stationary Phase 4 Types ph (1-14) Organic Modifier Type (many) Temperature up to 200 o C Percent Organic Modifier Buffer - Type and Concentration

Method Development Knobs on Silica RP Phases Stationary Phase Many Types ph (2-9) Organic Modifier Type (many) Temperature up to 50 o C Percent Organic Modifier

Why Choose Zirconia? Silica Zirconia Which tool set would you rather have to keep your method development vehicle going?

When Do We Use the Stationary Phase Knob? Before we try anything else... Stationary Phase To change selectivity To change retention

22 Non-electrolyte Solutes Nonpolar Benzene Toluene Ethylbenzene p-xylene Propylbenzene Butylbenzene Cl Polar Br Cl O O Bromobenzene p-dichlorobenzene Anisole O O Methylbenzoate Naphathalene Acetonphenone O NO 2 CN NO 2 NO 2 Cl Benzonitrile Nitrobenzene p-nitrotoluene p-nitrobenzyl Chloride Benzophenone Cl HB Donor OH OH NH H OH OH O Benzylalcohol 3-Phenyl Propanol N-Benzyl Formamide Phenol p-chlorophenol

Butylbenzene Propylbenzene 2.00 1.50 1.00 0.50 0.00-0.50-1.00-1.50 Selectivity Comparison ZirChrom-PBD ZirChrom-CARB DiamondBond-C18 ZirChrom-PS Phenol 3-phenylpropanol p-chlorophenol Acetophenone ** Benzonitrile Nitrobenzene ** Methylbenzoate ** Anisole ** p-chlorotoluene ** p-nitrobenzyl chloride Benzophenone ** Bromobenzene ** Napthalene ** p-xylene ** p-dichlorobenzene ** Benzene ** Toluene Ethylbenzene Solute N-benzylformamide Benzylalcohol Normalized Log k' (k' solute/k' benzene)

Selectivity Matrix* CARB DB-C18 PBD RP18 C18 (2) PLRP-S RP-1 ZirChrom-CARB 1 DiamondBond-C18 0.80 1 ZirChrom-PBD 0.51 0.90 1 Xterra RP18 0.53 0.85 0.90 1 Luna C18 (2) 0.53 0.86 0.93 0.97 1 PLRP-S 0.60 0.90 0.93 0.92 0.96 1 Gammabond RP-1 0.52 0.88 0.96 0.97 0.98 0.95 1 * Column names are the trademarks of their respective manufacturers. ZirChrom-PBD is the most similar to ODS for non-ionic analytes ZirChrom-CARB is the most different to ODS DiamondBond-C18 is ODS-like but with some CARB selectivity (generally better peak shapes than CARB)

Selectivity and Shape: Isomeric Analytes Compound ODS Carbon di(phenethyl)amide 1.19 1.20 cis-/trans-stilbene 1.02 22 O CH 3 CH 3 N H di( phenethyl)amide cis-/trans-stilbene

Dinitroaniline Herbicide Separation (EPA Method 627) ODS NO 2 R Carbon N R' X NO 2 4 6 8 10 12 14 16 Minutes 2 3 4 5 6 7 8 Minutes

DiamondBond-C18 Selectivity* a) 150 x 4.6 mm ODS b) 100 x 4.6 mm DiamondBond-C18 1,2 1 2 Ethylbenzene p-xylene 0 1 2 3 4 5 6 7 8 9 min 0 1 2 3 4 5 6 7 8 9 min LC Conditions: a) Column, 150 x 4.6 Zorbax Eclipse XDB-C8 S/N: USRK010769; Mobile phase, 65/35 ACN/Water; Temperature, 30 o C; Flow rate, 1.0 ml/min.; Injection volume, 5 ul; Detection at 254 nm; Solutes: 1=Ethylbenzene, 2=p-xylene. b) Column, 100 x 4.6 DiamondBond-C18, OD082401A; Mobile phase, 37.5/5/57.5 ACN/THF/Water; Temperature, 60 o C; all other conditions the same as a). * Column names are the trademarks of their respective manufacturers.

When Do We Use the ph Knob? ph (1-14) For ionizable compounds To improve peak shape of acidic or basic compounds To change selectivity of acidic or basic compounds To change retention of acidic or basic compounds

Silica after aging with Base* Extend C18 before aging Extend C18 after aging Aging conditions: 0.1g of Extend C18 was placed in 50mls of 40:60 - Methanol:1M Potassium hydroxide solution. It was aged for 24 hours at room temperature in a shaking bath. * Column names are the trademarks of their respective manufacturers.

Why Use ph Extremes? ph Stability ph < 1 ph > 13 Cleaning with Conc. Acid Ion Supression for Acids Ion Supression for Amines Sanitation/ Depyrogenation

ZrO 2 / SiO 2 Surface Chemistry O O O OH Zr O Zr O O O R O Lewis base: PO 4 3-, F -, RCOO - O C OH H 2 O O OH2 OH OH O 2 OH 2 Zr Zr Zr Zr Zr O O O O O O O O O Basic Compound H N H + R _ O OH Si Si Si Si O O O O O O O Si Si Si Si Lewis Acid Site Active Silanol Correct buffering can eliminate unwanted residual surface interactions (or enhance desirable residual surface interactions)

High ph Stability Comparison* k' Propranolol 9.00 8.00 7.00 6.00 5.00 4.00 3.00 ZirChrom-PBD ph12.0 Gammabond RP-1 ph 12.0 Luna C18 (2) ph 10.0 Extend C18 Rapid Res. ph 11.5 Xterra RP18 ph 12.0 DiamondBond-C18 ph 12.5 2.00 1.00 0.00 0 2000 4000 6000 8000 10000 12000 14000 16000 Column Volumes of Mobile Phase Exposure Conditions: Mobile phase, ACN/50mM Potassium phosphate buffer at indicated ph; Temperature, 30 o C. LC Conditions: Mobile phase, ACN (or THF)/50mM Potassium phosphate buffer at indicated ph; Flow Rate, 1.0 ml/min.; Temperature, 30 o C; Injection Volume, 5 ul; Detection, 254nm. * Column names are the trademarks of their respective manufacturers.

Effect of ph on Bases ph 4 1 2 3 4 5 6 RNH 2 + H + <---> RNH 3 + RP RP + CEX ph 7.5 1 2 3 4 5 6 ph 9.5 1 2 4 3 5 6 ph 12 2 3 4 1 6 5 5 and 6 switch elution order 0 10 20 30 40 (min)

Fast Separations of NSAIDs on DiamondBond TM -C18 mau 80 70 1 ph = 1.75 60 50 2 40 3 4 30 20 10 0 0 1 2 3 4 5 6 min LC Conditions: Column, 100 x 4.6 DiamondBond TM -C18; LC Conditions: Mobile phase, 50/50 ACN/50mM Phosphoric acid, ph 1.75; Flow rate, 1.0 ml/min.; Temperature, 65 o C; Injection volume, 1.0 ul; Detection at 254nm; Solutes: 1=Acetominaphen, 2=Ketoprofen, 3=Ibuprofen, 4=Naproxen

Why Work at High ph? To adjust retention To improve peak shape by deprotonating amines To adjust selectivity Why tie your hands with columns with limited lifetime, limited buffer types and limited ph range? ZirChrom RPLC phases are stable: 1< ph < 14

When Do We Use the Mobile Phase Knobs? Percent Organic Modifier Organic Modifier Type (many) To change selectivity To change retention

Molecular Interactions in LC Dispersion Dielectric Interactions Dipole Hydrogen Bonding

Overview of Mobile Phase Optimization Define optimum solvent strength so that 1 < k < 20 a. Do stepwise isocratic study in 20% steps starting at 100% organic. b. Do gradient determination of % organic. Define the best type of modifier; MeOH, ACN or THF ---TRIANGLE OPTIMIZATION. Keep in mind that the relative strengths of MeOH, ACN and THF are related.

EASY SAMPLES Separation Optimization Initial run with ACN/water Define k range vs % ACN Best % ACN for k range and band spacing AVERAGE SAMPLES Initial run with MeOH/water Initial run with THF/water Define k range vs % MeOH Define k range vs % THF Best % MeOH for k range and band spacing Best % THF for k range and band spacing

Triangle Optimization HARD SAMPLES MeOH Use k range from earlier runs 1 H 2 O ACN 4 6 7 2 5 3 THF

Snyder Scheme for RPLC Optimization Define % solvent for 1 < k < 20 in MeOH(Solvent 1). If band spacing is not OK then use equieluotropic mixtures for ACN and THF. (Solvents 2 and 3) If 2 or 3 are not OK then do mid-points as shown. (Solvents 4,5,6). If a significant improvement is seen then look for intermediate ternary mixture. If no significant improvement is seen then do mixture for all three solvents (Solvent 7).

Solvent Properties Triangle

When Do We Use the Buffer Knob? Buffer - Type and Concentration To improve peak shape of acidic or basic compounds To change selectivity of acidic or basic compounds To change retention of acidic or basic compounds To modify band spacing or elution order

Effect of Different Lewis Base Additives Acetate 4 2,3 1 5 6 N NH (5) pk a 9.4 (6) pk a 9.7 Fluoride Phosphate 1 4 1 2 3 2 3 4 5 6 5 6 1. Lidocaine 2. Norpseudo ephedrine 3. Tryptamine 4. Quinidine 5. Amitriptyline 6. Nortriptyline 0 10 20 30 40 (min) 20% ACN, 20 mm Lewis base additive, ph 7.5; 0.8 ml/min; 30 C

Effect of Additive Concentration on Bases 1 2 3 4 5 6 20 mm NH 4 H 2 PO 4 ph 7.5 1,2 3 4 5 6 0 10 20 30 40 100 mm NH 4 H 2 PO 4 ph 7.5 (min) CEX is adjustable by ionic strength of mobile phase

Effect of Buffer Concentration on Retention 2.0 (A) ZirChrom-PBD 1.5 (B) ODS 1.5 1.0 Log k 1.0 Log k 0.5 0.5 0.0 0.0 0.5 1.0 1.5 log [Phosphate (mm)] -0.5 0.5 1.0 1.5 log [Phosphate (mm)] Plot of log k for antihistamines versus logarithm of phosphate buffer concentration in milimole on (A), ZirChrom-PBD column and (B), ODS column. Experimental conditions: mobile phase, 40/60 acetonitrile/potassium phosphate buffer at ph 7.0; temperature, 30 C; solutes: ( ), pheniramine; ( ), thenyldiamine; ( ), chlorpheniramine; ( ), brompheniramine; ( ), cyclizine; ( ), thonzylamine; ( ), meclizine; ( ), chlorcyclizine; ( ), pyrrobutamine.

Acetate Fluoride 3 1 4 Separation of Antiarrythmic Drugs 5 1 2 6 7 2 3 5 4 6 7 1, 2 1. Chlorpropamide 2. Tolbutamide 3. Procainamide 4. Acetylprocainamide 5. Propionylprocainamide 6. Lidocaine 7. Quinidine Cl (1) pk a = 4.9 O S O NH O C NH Phosphate 3 5 6 4 0 2 4 6 8 7 min H 3 C O O S NH C NH O (2) pk a = 4.9 Gradient elution; 30mM Additive, 15 mm TRIZMA, ph 7.5; 0.8 ml/min; 40 C

Separation of Alkoxybenzoic Acids on ZirChrom -PBD 40 mm Acetate ph 4 Problem 50 x 4.6 mm N = 260 A s = 3.35 0 2 4 6 8 10 12 14 No Problem 40 mm Phosphate ph 2.15 50 x 4.6 mm N = 3250 A s = 1.07 0 2 4 6 8 10 12 14 25% ACN, 40 mm above additive, 5 mm NH 4 F; 0.6 ml/min; 30 C; 254 nm.

Carboxylate Problem - Solved! Phosphate as buffer ([PO 4 ] > 20-30 mm). Small amount of fluoride (5mM). Low ph (< 3). High temperature (40 o C). ACN as organic modifier.

Separation of Carboxylic Acid NSAIDs on ZirChrom -PBD Absorbance (mau) 150 100 50 0 1 2 3 4 5 6 1. Acetaminophen 2. Aspirin 3. Ketoprofen 4. Fenoprofen 5. Ibuprofen 6. Indomethacin No Problem 0 2 4 6 8 Time (min) A, 40 mm H 3 PO 4, 5 mm NH 4 F, ph 2.1; B, 50% ACN + A; 10-60% B, 0-2 min; 60% B, 2-10 min; 0.8 ml/min; 40 C.

Solve Any Tailing Problem Use High ph on Zirconia Phases

Combination of ODS and PBD-ZrO 2 for the separation of basic drugs Temperature 1 Temperature 2 C18-SiO 2 Carbon-ZrO 2 Partition Mechanism Adsorption Mechanism Phosphate Buffer ph=7 Temperature 1 Temperature 2 C18-SiO 2 Reversed Phase Mode + Cation-Exchange Mode PBD-ZrO 2 Cation-Exchange Mode + Reversed Phase Mode

Separation of Anti-Histamine Drugs by T3C C18-SiO2 30 oc 2+3 25 1 20 4 15 9 5 10 8 7 6 5 0 1.5 log k' (ODS at 40oC) 30 9 R2=0.147 8 4 1.0 5 3 2 6 0 5 10 1+7 20 PBD-ZrO2 30 oc 15 10 0.5 62 5 5 10 15 20 25 15 T3 C 12 10 3 4 1.0 1.5 50 Resolution 45 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 9 0 0 0.5 log k'(pbd-zro2 at 40oC) 8 324 7 1 0.0 40 oc +35 oc 8 5 6 7 9 5 0 Tempearure on ODS column Absorbance (mau) Absorbance (mau) 25 (a) 40 35 30 25 20 80 0 5 10 15 Time (min) 20 25 30 70 60 50 40 Temperature on PBD-ZrO2 column 30

When Do We Use the Temperature Knob? Temperature up to 200 o C To speed up analyses To modify band spacing

Temperature The Third Dimension in HPLC Temperature Mobile Phase Stationary Phase

Why Use Temperature? Thermal Stability Lower Pressure Drop Less Organic Solvent Thermally Optimize Selectivity Less Wear and Tear Higher Flow Rate: Fast Analysis More Robust Analysis Easier Method Development

Antihistamine Example: Speed it up! ma U 60 50 40 30 VWD1 A, Wavelength=254 nm (P102501\TEST0146.D) 1 3 2 4 2 3 Temperature: 21 o C Flow rate: 1.35 ml/min. Pressure drop: 195 bar Resolution (2,3): 2.1 Analysis time: 12.5 minutes 20 10 2.4 2.6 2.8 3 3.2 min 5 0 ma U 0 2 4 6 8 10 12 14 VWD1 A, Wavelength=254 nm (P102501\TEST0151.D) 3 min 80 60 40 1 2 3 4 2 Temperature: 80 o C Flow rate: 3.00 ml/min. Pressure drop: 195 bar Resolution (2,3): 2.1 Analysis time: 2.5 minutes 20 5 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 min 0 0 2 4 6 8 10 12 14 LC Conditions: (A) Mobile Phase, 29/71 ACN/50mM Tetramethylammonium hydroxide, ph 12.2; Flow Rate, 1.35 ml/min.; Injection volume, 0.5 ul; 254 nm detection; Column Temperature, 21 C; Pressure drop = 195 bar; Solutes: 1=Doxylamine, 2=Methapyrilene, 3=Chlorpheniramine, 4=Triprolidine, 5=Meclizine4=Triprolidine, 5=Meclizine, 100 x 4.6 ZirChrom-PBD (B) same as A, except Mobile Phase, 26.5/73.5 ACN/50mM Tetramethylammonium hydroxide, ph 12.2; Flow Rate, 3.00 ml/min.; Column Temperature, 80 C; Pressure drop = 195 bar. min

Fast β-blockers Separation mau Column Temperature = 150 o C, ph = 11 1200 H 2 NOC 1000 HO OH CHCH 2 NHCH CH 3 Labetalol CH 2 CH 2 800 CH 3 OCH 2 CH 2 OCH 2 CHCH 2 NHCH(CH 3 ) 2 600 OH Metoprolol CH 2 CH CH 2 400 OCH 2 CHCH 2 NHCH(CH 3 ) 2 Alprenolol 200 0 OH Separation in 0.4 minute! 0 0.4 0.6 LC Conditions: Column, 50 x 4.6 Diamondbond-C18, OD0121601A; Mobile phase, 45/55 ACN/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

Summary

Causes of Bad Peak Shape Bad Column (poorly packed). Build up of GARBAGE on the column. Too much sample per injection. Wrong solvent for sample. Extra-column effects. Chemical or secondary effects (Lewis acid site interactions). Poor buffering.

Operational Similarities for SiO 2 and ZrO 2 RP Media k increases with molecular hydrophobicity (CH 3, CH 2, phenyl, etc.). Elution sequence of non-electrolytes similar. k decreases 2 fold per 10% increase in volume % modifier. log k linear with % organic modifier. k decreases as temperature is increased (3 fold/50 o C). Solvent strength: THF > ACN > MeOH.

Operational Differences in SiO 2 and ZrO 2 RP Media Lewis base modifier at mid-range ph creates mixed-mode RPC/IEC possibilities. Cations are typically more retained and sometimes much more retained on ZrO 2 in buffered (PO 4 ) eluents than on SiO 2 phases. Elution sequence of anions and cations can be very different on ZrO 2 & SiO 2 at neutral ph. Must use Lewis base eluent for carboxylic analytes. Strongly advise use of 5mM or more phosphate (or TMAH) for all electrolytes.

Conclusions Silica-based HPLC columns work well between ph 3 and 8; outside these limits, these columns can have short lifetimes Zirconia is stable at ph 1-14 and at elevated temperature Zirconia can be functionalized by coating / cross-linking polymers, for very stable supports. ZirChrom s zirconia columns reproducibly combine silica s efficiency with polymer stability

Thanks very much for listening! ZirChrom Separations & Cabot Corporation Partners in Chromatography 1-866-STABLE-1 www.zirchrom.com For more information and web access to the free Buffer Wizard: www.zirchrom.com

Additional Information

Efficiency Comparison of Leading HPLC Columns* average plates per meter 140,000 120,000 100,000 80,000 60,000 40,000 20,000 LC Conditions: Mobile Phase, 65/35 Acetonitrile/50mM Potassium phosphate buffer, ph 3.2; Flow Rate, 1.0 ml/min.; Injection volume, 1 ul; 254 nm detection; Column Temperature, 21 C. Solutes: uracil, phenol, pyridine, 4-butylbenzoic acid, N,N dimethylaniline, toluene. 0 ZirChrom -PBD Gammabond RP-1 Luna C18(2) Symmetry C18 Hypurity Elite C18 Supelcosil ABZ+Plus Zorbax 300SB-C18 Hamilton PRP -1 Polymer Labs PLRP -S Jordi Gel DVB C18 Jordi Gel DVB SM -500 DiamondBond -C18 ZirChrom -CARB Hypercarb Mobile Phase: 45/5/50 Acetonitrile/THF/50mM Potassium phosphate buffer, ph 7.0; Column Temperature: 30 C; all other conditions are identical. * Column names are the trademarks of their respective manufacturers.

Reproducibility Data for 50 ZirChrom -PBD Columns Parameter Average %RSD k' (Toluene) 2.32 2.4% α (Methylbenzoate/ Benzonitrile) 1.80 1.1% N (plates/m) 151,000 6.8% Symmetry 0.93 7.2% LC Conditions: Mobile phase, 35/65 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 o C; Injection volume, 5 µl; Detection at 254 nm

Testing of DiamondBond Stability 8 7 6 k' 5 4 3 2 1 0 k' Butylbenzene (0.5M Nitric Acid) k' Butylbenzene (1.0M NaOH) k' Butylbenzene (200 Degrees Celsius) 0 250 500 750 1000 1250 2000 Column Volumes LC Conditions: Base Stability DiamondBond TM Phase A, 30 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 o C; Injection volume, 5ul; Detection at 254nm. Acid Stability DiamondBond TM Phase A, 50 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 o C; Injection volume, 5ul; Detection at 254nm. Temperature Stability-- DiamondBond TM Phase B, 50 x 4.6 mm id; Mobile phase, 50/50 ACN/Water; Flow rate, 1.0 ml/min.; Temperature, 30 o C; Injection volume, 5ul; Detection at 254nm.

Synthesis of ZirChrom-PBD (Polybutadiene) CH 2 CH CH 2 CH CH CH 2 CH 2 CH CH CH 2 CH 2 CH CH 2 CH CH CH 2 CH 2 CH CH CH 2 ~20 CH 3 O O CH 3 C C O O C C CH 3 CH 3 Dicumyl Peroxide Vacuum 160 ºC CH 2 CH CH 2 CH CH CH 2 CH 2 CH CH CH 2 CH 2 CH CH 2 CH CH CH 2 CH 2 CH CH CH 2 ~20

ZirChrom-PS Synthesis CH CH 2 CH CH 2 Step 1. Synthesis of Copolymer (CMS/VMS) Step 2. Adsorption of Copolymer Si (OC 2 H 5 ) 2 CH 3 CH 2 Cl n Polymer chain m Si Si Step 3. Thermal Crosslinking O O Zr Zr Zirconia's surface CMS/VMS-ZrO 2 PS-ZrO 2 CH 2 Cl + CH 2 Cl 0 160 C H C H ClCH 2

Synthesis of ZirChrom-CARB Lewis Acid Site Zr Zr Zr Zr Zr Zr O O O H O H O O O O O O O O O O O O O Bulk Zirconia

Synthesis of DiamondBond-C18 General approach - Cabot Corporation (Billerica, MA): functionalizing agent X-R-Y X reacts with surface Y = functional group X is typically a diazonium salt NH 2 Y + 2 HA + NaNO 2 = AN N Y + 2 H 2 O + NaA + N N + Y Carbon Clad Zirconia Diazonium Salt Modified Carbon Clad Zirconia

ZirChrom-CARB ZirChrom-CARB has RPLC like properties but has radically different selectivity than ODS or ZirChrom -PBD -- great for steroids, and for geometric isomers. Much more hydrophobic & retentive than ODS -- great for polar analytes ZirChrom -CARB is very stable. Can be used at low and high ph. Can be used at high temperatures. Lewis acid-base properties are still important on all zirconia-based phases. Peak shapes best with THF (10%) and/or elevated temperature.

Role of Temperature in LC High-Performance Liquid Chromatography at Elevated Temperatures: Examination of Condition for the Rapid Separation of Large Molecules, R. D. Antia and Cs. Horvath, J. Chromatogr., 435, 1-15 (1988). Temperature as a Variable in Reversed Phase High- Performance Liquid Chromatographic Separations of Peptide and Protein Samples, W. S. Hancock, R. C. Chloupek, J. J. Kirkland and L. R. Snyder, J. Chromatogr. A, 686, 31-43 (1994) Superheated Water: A New Look at a Chromatographic Eluent for Reversed-Phase Liquid Chromatogrpahy, R. M. Smith and R. J. Burgess, LC-GC, 17, 938-945 (1999)

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 1 2 3 4 Column 2 1 2 3,4

Analogy between Column Temperature and Column Length T increases 50 o C k decreases 3-fold Retention Retention Cold Hot Long Short Temperature Column Length Increasing temperature and decreasing column length both decrease retention time

Pump Effect of Temperature on T 3 C Selectivity Temperature 1 Temperature 2 Injector Column 1 Column 2 Detector f 1 = k ' 1 α = f1α f α net 1 ' 2 2 k1 + k ' 2 + Low T Low T f 2 = k ' 1 ' k2 + k ' 2 Low T High T High T Low T Temperature continuously changes the T 3 C selectivity between α 1 and α 2. α net α 1 αnet α2

Requirements for T 3 C 1. Two columns with different (ideally orthogonal) selectivities 2. One column must be thermally stable Zirconia based phase are thermally stable up to 200 o C Polybutadiene-coated zirconia (PBD-ZrO 2 ) Carbon-coated zirconia (C-ZrO 2 ) 3. Method development must be easy Theory for T 3 C Guideline for method development

Method Development for T 3 C Pump Injector T 1 T 2 Column 1 Column 2 Detector t Rnet (T 1,T 2 ) = t R1 (T 1 )+ t R2 (T 2 ) t R = t 1+ 1 01( k1 ') t R = t 1+ 2 02( k2 ') log k 1 =A 1 +B 1 /T log k 2 =A 2 +B 2 /T 2 trial runs 2 trial runs

Guidelines for Optimizing T 3 C Choose Two Stationary Phases Window Diagram Optimization Choose Mobile Phase (1<k <20) Calculating T 3 C Retentions ln k =A+B/T t rnet =t r1 +t r2 No Different Selectivity? Yes Two More Runs at Higher Temperatures

Thermally Tuned Tandem Column (T 3 C) Concept Pump Injector Temperature 1 C18-SiO 2 Temperature 2 C-ZrO 2 Detector Absorbance (mau) 25 20 15 10 5 0 30 20 10 0 Optimized Single Column 12 3 4 0 5 10 15 20 25 30 2+7 5 4 5 8 1 6 7 9 8 0 5 10 15 20 25 30 Time (min) 6 9 10 C18-SiO 2 10 C-ZrO 2 3 Absorbance (mau) 20 10 0 Optimized T 3 C 2 1 4 5 3 7 6 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (min) T 3 C Works!

Absorbance (mau) 25 20 15 10 5 0 30 20 10 0 20 10 0 12 Separation of Ten Triazine Herbicides by T 3 C 3 2+7 2 1 4 6 7 8 5 4 5 8 4 1 5 3 9 7 6 8 9 10 0 5 10 15 20 25 30 6 10 Time (min) 9 C18-SiO 2 30 o C C-ZrO 2 60 o C 3 T 3 C 10 CH 3 S, CH 3 O, Cl =R Solutes: 1. Simazine 2. Cyanazine 3. Simetryn 4. Atrazine 5. Prometon C18-SiO 2 C-ZrO 2 30 o C 125 o C N N N N H R 1 H N R 2 Other conditions: 30/70 ACN/water 1ml/min; 254 nm detection 6. Ametryn 7. Propazine 8. Terbutylazine 9. Prometryn 10. Terbutryn T 3 C can improve separation without increasing analysis time.

Steps in T 3 C Optimization of Triazine Herbicides Log k (C-ZrO 2, 60 o C) 1.6 1.2 0.8 2 0 1 0 0 R 2 =0.107, sd=0.342 3 6 10 9 1 8 2 4 5 7 0.8 1.2 1.6 log k (ODS, 30 o C) 3 4 6 2 7 1 5 Minimum Resolution 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 140 120 T 3 C, 3ml/min Temperature on C-Zr Column 100 T ODS =30 o C T C-ZrO2 =125 o C Rs=3.30 80 R=2.02 8 9 10 60 30 40 50 60 70 80 Temperature on ODS Column 0 1 2 3 4 5 6 7 8 9 1 0 Time (min)

Compare T 3 C with Mobile Phase Optimization 60 40 1+2 3 4 40%ACN 6 7 8 9 10 1.2 0.8 ACN R<1.0 20 5 0.4 0 30 20 10 0 40 20 0 2 4 6 2 1 0 2 4 6 8 10 12 14 16 53 1 2 3+4 4 6+7 5 6 50%MeOH 8 9 7+8 30% THF 9+10 10 Minimum Rs 0.0 0.30 0.35 0.40 0.4 0.2 0.0 0.4 0.5 0.6 0.8 0.6 0.4 0.2 MeOH R<0.3 THF R<0.5 0 2 4 6 8 10 Time (min) 0.0 0.20 0.25 0.30 Percentage of organic modifier T 3 C is more powerful than mobile phase optimization on ODS

More Applications of T 3 C Concept Urea and Carbamate Pesticides 7 20 5+9 C18-SiO 2 30 o C 10 11+12 10 0 6 123 4 * 8 Absorbance (mau) 8 4 0 0 2 4 6 8 4 12 5 3 76 8 9 * C-ZrO 2 90 o C 10+11 12 0 4 8 12 16 8 4 T 3 C 39 o C+89 o C 12 5 9 3 4 6 7 8 10 11 * 12 0 0 1 2 3 4 5 6 7 8 Time (min)

Introducing the Metalox TM 200-C High Temperature Column Heater Designed from the ground up for chromatographers. Overcomes issues such as thermal mismatch and inaccurate column temperature reporting.

Metalox TM 200-C Diagram Patent Pending

Metalox TM 200-C Four Zone Temperature Performance 250 System Temperatures vs. Flow Rate @ 200 o C Prototype High Temperature Column Control System Column Inlet Column Outlet Column Exchanger Outlet Detector Inlet Temp (Post Heat Exchanger) System: Waters 2690 + 2487 detector Column: 4.6 mm x 3.3 cm Diamondbond C18 Column Heating system- prototype. 200 o Set-Point Temperat C 150 100 Reducing thermal mismatch to a minimum at any flow rate solves broadening and peak splitting! "Influence of Thermal Conditions on the Efficiency of High-Performance Liquid Chromatography." H. Poppe and J.C. Kraak, J. Chromatogr., 282, 399-412 (1983). 50 0 0 1 2 3 4 5 6 Flow Rate, Water (ml/min) Conclusion: Regardless of flow rate, the column inlet and outlet temperature are within 3 C of each other.

Metalox TM 200-C Features Advantages, and Benefits Feature Adiabatic type oven (Adiabatic: Occuring without loss or gain of heat), Patent Pending Advanced oven design includes reduced internal dead volume by using laser welded components in the heat exchanger Close coupled heater element and temperature sensors Patent Pending Advantage All critical components (column insulation and mobile phase) are at a stable and defined temperature Reduces band broadening normally associated with adding a "modular column heater" Provides for more efficient heat transfer to mobile phase over air bath only type Benefit Greatly reduces retention time shifts and band broadening commonly found in other column heater designs and thus improves overall data quality Allows use of narrow bore columns and/or gradient elution without excessive increase in run time or loss of column efficiency Allows for faster and more responsive heat transfer with smaller (less expensive) instrument design.

Metalox TM 200-C Features Advantages, and Benefits Feature Heat exchanger at mobile phase inlet Four monitored temperature zones Column resides in defined location (max length 15 cm, max id 4.6 mm) Advantage Pre-heats incoming mobile phase closer to desired temperature as well as reducing exiting mobile phase temperature as it enters the detector Monitors temperature at critical points in flow path When column is changed the positioning geometry will be the same even with different operators Benefit Reduces needed heater size which lowers instument size and cost. Lower detector temp. means less thermal noise and drift from detector cell. Ensures that column is at or near uniform temp., reducing thermal band broadening. Provides built in capabilities for instrument validation. Column is at constant temp. during gradient elution and/or flow programming. Eliminates the problem caused by temp. variability inside large air bath ovens and thus removes another point of instrumental error and lowered chromatographic performance.

Metalox TM 200-C Features Advantages, and Benefits Feature Small footprint and stand alone operation. Front panel displays all four temperature controlled zones as well as column inlet and outlet differential temperature RS-232 and analog output of all four temperature zones Advantage Easy to attach to existing systems and/or move between systems for added flexibility Operator can easily tell when oven and column are truly ready for use - instead of just a single readout of an air temperature or block sensor Provides two convenient monitoring modes to log data to external devices Benefit User get top chromatographic performance in modular format Avoids making reruns because column was not truly at desired operating temperature Very useful when setting up validation protocols, no need to attach extra sensor and recording devices

Metalox TM 200-C Features Advantages, and Benefits Feature Bonded heaters are powered by isolated 24V DC Back pressure regulator down stream of detector Two thermal cutout sensors inside of oven Advantage No line voltage in close proximity to chromatographic flow path or operator Keep constant back pressure on entire chromatographic system Shuts off heater power in case of thermal runaway Benefit Provides extra safety to both instrument and personnel in case of shorted heater component Prevents mobile phase form boiling in detector and prevents mobile phase boiling in column upon loss of mobile phase supply Provides extra measure of safety for unattended operation

The Metalox TM 200-C High Temperature Column Heater Introductory Offer The new Metalox 200-C High Temperature Column Heater Back Pressure Regulator A set of three ZirChrom revolutionary RP HPLC Columns. ZirChrom-PBD ZirChrom-CARB DiamondBond C18

Metalox TM 200-C Preliminary Specifications Operational Capabilities Max. Column Operating Temp: 200 C (6 ml/min with water) Min. Temperature: 7 C above ambient Max. Flow Rate: 6ml/min (200 C, water mobile phase) Max. Cal./s: 17.9 Temperature Reproducibility: ± 0.5 C Accuracy of Temp. Reading: ± 1% Display Resolution: 1 C Physical Specifications Weight: 15 lbs Footprint: 6 x10 x14 Power Requirements: 115-230V 47-440 Hz Internal Transfer Volumes: Pre-Column: 10.5 ul Post-Column 4.0 ul Note: Metalox 200-C design and specifications are pending patent

Quantitative Value Assessment Steroids Separation Customer: Contract Lab XYZ Date: 1/31/02 Leading ODS ZirChrom-PBD Column Cost $395.00 $595.00 Analysis Time (min) 10.0 5.0 Average Time per Successful Cycle (min) 12.3 7.2 Possible Cycles per Instrument per Year 42,705 73,474 Cost Components Column Cost per Successful Cycle $1.01 $1.30 Solvent Cost per Successful Cycle $0.02 $0.04 Waste Disposal Cost per Successful Cycle $0.01 $0.01 Total Fixed Cost per Successful Cycle $2.37 $1.38 Total Operator Cost per Successful Cycle $0.26 $0.15 Total Cost per Analysis* $3.67 $2.88