Ronald E. Majors, Ph.D. Product Marketing Specialist Consumables and Accessories Business Unit May 31, PDF Free Download

Ronald E. Majors, Ph.D. Product Marketing Specialist Consumables and Accessories Business Unit May 31, 2000 Logical Method Development for Small Molecular Weight Ionizable Compounds using RP-HPLC 11:00 a.m. EST Telephone Number: 816-650-0621 Chair Person: Tim Spaeder

Logical Method Development for Small Molecular Weight Ionizable Compounds using RP-HPLC Ronald E. Majors Agilent Technologies, a subsidiary of Hewlett-Packard Company, Little Falls Analytical Division, Wilmington, DE USA Slide 4

Outline of Reversed Phase HPLC Method Development Talk 1. The silica base material and bonded phase chemistry, especially with respect to degradation chemistry 2. The chemistry of ionizable compounds as we change ph of mobile phase during method development 3. Optimize conditions, bonded phase chemistries and conditions for method development 4. Summarize the results of the stationary phase selection and method development approach Slide 5

Experimental Parameters for Optimizing Separations and Column Stability by Reversed- Phase Chromatography For neutral, non-ionizable compounds, use water-acetonitrile or water-methanol mixtures. For ionizable compounds (e.g. amines, carboxylic acids, phenols, etc.), above conditions but must optimize ph, buffer, temperature for best separations. Effect of ph on the Separation of Ionizable Compounds - Low ph (ph 1-3) - Intermediate ph (ph 3-8) - High ph (ph 7-12) Column Stability as a Function of ph - Variables that Affect Column Stability especially at Low and High ph Phase Design for Optimal Separations and Column Stability Slide 6

Silica Support Surface OH OH OH OH-----OH ---Si-- ----Si---- --Si Si--- Free Geminal Associated Silanol Silanols Silanols Decreasing Acidity OH + + ---M--- ---M---Si--- Surface Metal Internal Metal (most acidic) Slide 7

Preparation of Silica Bonded Phases l l l l l Silica Surface Organochlorosilane Reactants End-capping Reactants Slide 8

Mechanisms of Degradation of Silica-Based HPLC Packings Low ph (ph <3) Hydronium catalyzed hydrolysis of bonded phase siloxane - Loss of Bonded Phase - Change in retention times (usually decrease) Intermediate-to-High ph (ph >6-7) Dissolution of the underlying silica by the hydroxide ion - Loss of silica, void development - Loss of resolution Slide 9

Best to Begin Method Development at Low ph Both silica support silanol groups and solutes (basic and acidic) are protonated Generally produces narrow bands, best peak shape and separation reproducibility Mobile phase modifiers (also called competing bases) (e.g., triethylamine, TEA) rarely are required for good peak shape Many conventional short-chain phases are unstable Slide 10 001275P1.PPT

Retention of a Basic Compound as a Function of ph and pka Weakly Basic Compound Retention Time A B pk a ± 1.5 pk a C 2 3 4 5 6 7 ph Analyte charge: Silica surface charge: + + / neutral neutral neutral - / neutral - Slide 11 001281P1.PPT

Minimizing Silanol-Base Interactions at Low ph Silica Particle SiO - + H + Lower ph Higher ph Silica Particle SiOH Slide 12 000302P2.PPT

Initial Experimental Conditions For Separating Ionizable Compounds Separation Variable Column Dimensions Particle Size Bonded Phase Mobile Phase Solvents A and B % B Buffer Additives/competing base Flow Rate Temperature Sample Size Volume Mass Initial Choice 15 (or 25) x 0.46 cm 5 or 3.5 µm C18 or C8 Aqueous buffer / ACN Variable (k = 0.5-20) 25 mm potassium phosphate, ph 3 25-50 mm TEA or TEA acetate, if needed 1-2 ml / min 40 C 50 µl < 25 µg Slide 13

Packings Designed for Maximum Stability in Specific ph Ranges StableBond, ph 1-5 1. Uses bulky silanes 2. Non-endcapped Eclipse XDB, ph 3-8 1) extra Densely Bonded dimethylalkylsilanes 2) proprietary double-endcapping Bonus-RP, ph 3-8 1) polar alkyl phase 2) triple endcapped 3) uses bulky silanes Extend-C18, ph 2-11.5 1) unique bidentate structure 2) double endcapped * C18 C18 O Si Si O O Si NH O Zorbax Rx-SIL Silica Support Surface At low ph, silica breaks down by hydrolyzing the siloxane* bond. This breakdown can be almost entirely eliminated by using bulky silanes, found in StableBond columns (left). At mid-range and high ph, breakdown occurs from silica dissolution, and is minimized by using endcapping procedures such as those in Eclipse XDB, Bonus-RP and Extend. Slide 14 001241P2.PPT

Comparitive Performance of C18 Packings At Low ph and High Temperatures (ph 0.8, 90 C) Accelerated Degradation Test Purge Solvent: 50% methanol/water with 1.0% TFA Solute: Toluene Slide 15 Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) 473-480.

Stability of ZORBAX SB-CN at ph 2.0, 50 C % k REMAINING 100 80 60 40 20 0 0 1000 2000 3000 4000 5000 6000 COLUMN VOLUMES Si-(iPr) 2 PrCN Sterically Protected Silane Si-(Me) 2 PrCN Traditional Silane Kirkland, J.J., J.L. Glajch, and R.D. Farlee, Analytical Chemistry (1989), 61, 2. Slide 16 000948P1.PPT

Ambient 35 C New Selectivities Using Elevated Temperature and Stable, Short-Chain Bonded Phases Zorbax 300SB-C3 45 C 1. Leucine Enkephalin 2. Angiotensin II 3. RNase A 4. Insulin (BOV) 5. Cytochrome C 6. Lysozyme 7. Myoglobin 8. Carbonic Anhydrase 60 C Mobile Phase: A:5:95 ACN : Water with 0.10 % TFA (v/v%); B: 95:5 ACN : Water with 0.085% TFA (v/v%) Gradient: 15-53 % B in 20 minutes, postime: 12 min. Flow: 1.0ml/min, Protein concentration: 2-6µg., Inj. vol.: 10µL, UV/VIS=215 nm, Temp.: 35 C Slide 17 001084P1.PPT

The Move from Conventional HPLC Columns to Fast LC Columns Slide 18

Separation of Aspartame and Metabolites With Columns of 5 µm and 3.5 µm Particles 1 2 5 µm Detector Response A 3 2 1 3 4 4 3.5 µm Mobile Phase: 15% ACN / 85% 0.1% trifluoroacetic acid Flow rate: 1.0 ml/min.; Temp: Ambient A. 4.6 x 15 cm Zorbax SB-C18, 5 µm particles B. 4.6 x 7.5 cm Zorbax SB-C18, 3.5 µm particles 1. Phenylalanine 2. 5-Benzyl-3,6-dioxo-2- piperazine-acetic acid 3. Asp-phe 4. Aspartame 0 5 10 Retention Time, (min) Slide 19

Review of Suggested Conditions For Basic Compounds With Silica-Based Columns Less acidic silica support Mobile phase ph 3 Buffer concentration in mobile phase 10 mm Mobile phase modifier (I.e. competing base), 20-50 mm triethylamine for older column types Sample size 1 µg/peak Slide 20

Reasons for Reversed-Phase Separations at Intermediate ph Compounds of interest are unstable at low ph Unable to obtain desired selectivity at low ph Belief that bonded-phase columns are unstable at low ph Protonated (hydrophilic) basic compounds too poorly retained at low ph (maybe ion pair RPC) Slide 22

Potential Problems at Intermediate ph Unreacted silanol groups and basic solutes often partially ionized Difficulties maintaining good peak shape, high efficiency Rugged separations problematic must closely control ph and temperature ± 0.1 ph, ± 1 C greatly effect separation Column degradation by silica support dissolution strongly dependent on: - ph - mobile phase buffer type, concentration - temperature - silica support type Slide 23

Silica Comparisons Rx-SIL (Sol type) Xerogel (sil-type) STRUCTURE: UNIFORM SUB PARTICLES SPONGE-LIKE, POLYMERIC NETWORK POROSITY (%): 50 70 PARTICLE SIZE/ SURFACE AREA (M 2 /G) STRENGTH: HIGH ph RESISTANCE: PURITY: PORE SIZE, PARTICLE SIZE DISTRIBUTION 80A/180 HIGH GOOD HIGH NARROW 100A/300 MODERATE POOR LOW - HIGH BROAD Slide 25

Characteristics for Typical Silica Supports Used for Bonded-Phase Packings Column Designation Silica Support b a Surface Type Silica Type b Surface Area, m 2 /g Pore Diameter, Å Porosity, cm 3 /ml Hypersil BDS-C8 A SolGel 170 130 0.65 Inertsil-C8 B SilGel 320 150 NA c Supelcosil ABZ+ A SolGel 175 120 0.60 Symmetry-C8 B SilGel 340 100 0.84 YMC-Basic B SilGel 325 120 1.0 Zorbax XDB-C8 B SolGel 180 80 0.50 b a Data taken from commercial literature or manufacturer s sources b NA = not available Slide 26

Effect of Column Type on Stability 1 2 3 1. URACIL 2. NORTRIPTYLINE 3. DOXEPIN 4. AMITRIPTYLINE 5. TRIMIPRAMINE 4 5 COLUMN A (SilGel Silica; Single Endcap) Initial Detector Response 1 2 3 4 5 COLUMN A After 1826 Column Volumes COLUMN B (SolGel Silica; Double Endcap) Initial Columns: 4.6 x 150 mm Column B: ZORBAX Eclipse XDB-C8 Initial Mobile phase: 60% ACN / 40% sodium phosphate buffer, ph 7.0 Flow rate: 1.5 ml/min. Temp: 40 C After- after purging with >1800 column volumes of 20% ACN / 80% 0.25M sodium phosphate buffer, ph 7 Flow rate: 1.0 ml/min. Temp: 60 C 1 2 3 5 COLUMN B After 1843 Column Volumes Sample: tricyclic anti-depressants 4 0 2 4 6 8 10 12 14 min. Slide 27

Buffers for Reversed-Phase HPLC Buffer pk a Buffer Range Buffer pk a Buffer Range Phosphate Formate 3.8 2.8-4.8 pk 1 2.1 1.1-3.1 Acetate 4.8 3.8-5.8 pk 2 7.2 6.2-8.2 Tris(hydroxymethyl) pk 3 12.3 11.3-13.3 aminomethane 8.3 7.3-9.3 Ammonia 9.2 8.2-10.2 Citrate Borate 9.2 8.2-10.2 pk 1 3.1 2.1-4.1 Pyrrolidine 10.5 9.5-11.5 pk 2 4.7 3.7-5.7 pk 3 5.4 4.4-6.4 ph Stabilizers at ph < 2.5 Phosphoric acid, 0.1% TFA, 0.1% Slide 28

Preferred Sequence of Developing Reversed- Phase Methods Buffered intermediate ph 4-8 Retention should not be ph dependent Buffer must be carefully chosen (buffering capacity, wavelength dependency, etc.) Often must use a basic mobile phase modifier (i.e.,competing base) Endcapped packing can be useful Slide 29

Effect of Buffer Type on Column Stability Columns: 4.6 x 150 mm Zorbax XDB-C8; Purge: 20% ACN/80% 0.25 M buffer, ph 7, 1.0 ml/min; Test: 60% ACN/40% 0.01 M sodium phosphate buffer, ph 7.0, 1.5 ml / min; 22 C Toluene Plate Height, cm 0.010 0.008 0.006 0.004 0.002 A Phosphate Citrate TRIS Amitriptyline Plate Height, cm 0.010 0.008 0.006 0.004 0.002 B Phosphate Citrate TRIS 0.000 0 5000 10000 15000 20000 25000 30000 0.000 0 5000 10000 15000 20000 25000 30000 Column Volumes of Purge Column Volumes of Purge Slide 30

Effect of Buffer Type and Concentration at ph 7 500 Columns: Zorbax Rx-C18, 4.6 x 150 mm; purge: 20% acetonitrile / 80% sodium phosphate and TRIS buffers; 1.0 ml / min; 60 C Amount of Silica Dissolved, mg 400 300 200 100 Sodium Phosphate Buffer 0.25 M TRIS Buffer 0.05 M 0.25 M 0 0.05 M 0 2 4 6 8 10 12 14 16 Volume of Eluent, Liters Slide 31

Improving Column Life by Reducing Operating Temperature Amount of Silica Dissolved, mg 500 400 300 200 100 0 Column: Zorbax Rx-C18, 15 x 0.46 cm; 20% ACN/ 80% 0.25 M buffer; 1.0 ml/min 60 C 40 C ph 7.0 0 2 4 6 8 10 12 14 16 Volume of Eluent, Liters Silica solubility in ph 7 phosphate buffer increases dramatically with temperature increase Solubility increase is also more significant as ph is increased above 3 to 4 Slide 32 001076P1.PPT

Alternative: Develop Methods on Silica-Based Bonded Phase Packings at ph > 9 Basic compounds often in free base form - Advantage: additional retention and selectivity Unreacted silanol groups totally ionized Thus, ionic interactions with packing eliminated - Good peak shape for basic compounds Zirconia, alumina, porous polymers and carbon have been proposed, but not widely used Limitations: - column efficiency - separation reproducibility - peak shape - mobile phase restrictions Alternative methods for LC/MS with volatile mobile phases Slide 34

Effect of Buffer Type on Column Stability at ph 11 Plate Height, cm 0.012 0.010 0.008 0.006 0.004 0.002 0.000 3 Peak Asymmetry Value 2 1 A B Propranolol; phosphate Propranolol; piperidine Toluene; phosphate Toluene; piperidine Propranolol; phosphate Propranolol; piperidine Toluene; phosphate Toluene; piperidine Column: Zorbax XDB-C8, 4.6 x 150 mm Aging: 50/50 ACN/0.014 M K Phosphate. 1.5 L/min; 24 C test: same, but 1.0 ml/min; 40 C Aging: 55/45 MeOH/0.05 M 1-methyl-piperidine 1.0 ml/min; 24 C; test, same. 0 0 5000 10000 15000 20000 25000 30000 35000 Column Volumes of Purge Slide 36

Conditions for Routine Use of Silica-Based Columns at High ph Use columns based on sol gel silica supports Use densely-reacted, endcapped, long chain (C18, C8) phases - short-chain phases less stable - phases with polar groups less stable - bidentate structure is optimum Use organic (Tris, glycine, pyrrolidone,etc.) or borate buffers - avoid phosphate and carbonate where possible - organic buffer stability: Li > Na > K > NH 4 Utilize column temperatures of 40 C Do not exceed buffer concentrations of 50 mm Slide 37

Silica-Based Column Aging at ph 11 Column: Zorbax XDB-C8, 4.6 x 150 mm; Aging: 55% MeOH / 45% 1-Methyl-piperidine-HCL buffer, ph 11 Flow: 1.0 ml/min; 24 C; UV, 215nm; Sample: b-blocker drugs; Injection: 5 µl; Chromatographic Test: Same Conditions A Pindolol Metoprolol Oxprenolol XDB-C8 Initial Propranolol N=7500 AS=0.99 t 0 B XDB-C8 After 31,284 Column Volumes N=6600 AS=1.37 0 2 4 6 8 10 12 14 16 18 20 22 24 Retention Time, (min) Slide 38

Using pka Differences to Move Peaks Band Spacing Changes Between ph 3 and 11 For Diuretic Drugs 0.12 0.10 0.08 0.06 0.04 0.02 1 2 1 Amiloride 2 Caffeine 3 Benzthiazide (pka=4.5) 3 ph 3 A 0.00 0.10 0.08 0.06 0.04 0.02 1 3 2 ph 11 Column: 4.6 x 150 mm, Zorbax XDB-C8; Flow: 1.0 ml/min; 24 C Mobile Phase: A: 55% MeOH / 45% 0.025M sodium phosphate buffer, ph 3.0 B: 55% MeOH / 45% 0.05M 1-methyl-pyridine-HCL buffer, ph 11.0 B 0.00 0 2 4 6 8 10 12 14 16 18 20 Minutes Slide 39 001200P1.PPT

A Bidentate C18 Bonded Phase Designed for Use at High ph Zorbax Extend-C18 C18 C18 O Si Si O Silica Support Slide 40

Plate Heights, cm Bidentate C18 Phase Stability at High ph Aging of ZORBAX Extend-C18 Column in Ammonium Hydroxide Mobile Phase, ph 10.5 10 9 8 1-Chloro-1-nitrobenzene Trimipramine k Values 7 6 5 4 3 2 1 Column: 4.6 x 150 mm Extend-C18 Mobile phase: 80% Methanol/20% 20 mm NH4OH, ph 10.5; Flow rate: 1.5 ml/min; Aging at 24 C, tests at 40 C 0 0.005 1-Chloro-1-nitrobenzene Trimipramine 0.004 0.003 0.002 0.001 0 10000 20000 30000 40000 50000 Column Volumes of Mobile Phase Slide 41

Effect of Bonded Phase Type on Silica Support Dissolution at ph 11 180 Amount of Silica Dissolved, mg 160 140 120 100 80 60 40 20 Zorbax XDB-C8 Zorbax XDB-C18 Zorbax Extend-C18 Columns: 4.6 x 150 mm Purge: 50% ACN / 50% 0.02 M potassium phosphate buffer ph 11; F= 1.5 ml / min; 25 C silicate concentration by silicomolybdate color reaction Bidentate bonded-phase chemistry provides additional high-ph stability over current materials 0 0 2 4 6 8 10 12 14 16 Volume of Eluent, Liters Slide 42 001369P1.PPT

Bidentate-C18 Stability at High ph Buffer, ph 80% Methanol / 20% 20 mm NH4OH, ph 10.5 55% methanol / 45% 50 mm 1-methylpiperidine buffer, ph 11.0 55% methanol / 45% 0.05 M pyrrolidine buffer ph 11.5 4 working months (10,000 Column Volumes (CV) is approximately 1 working month of 8- hour days) Lifetime No change in retention factor (k) or plate number after 40,000 CV 3-4 working months No change in k after 40,00 CV. Minor loss in plate number after 30,000-40,000 CV. 2 working months No change in k or plate height after 20,000 CV. After 20,000 CV, k and plate number slowly decreases. Column is still useable after 40,000 CV. Slide 43

Separate Basic Compounds in Their Free Base Form at High ph Separation of ß-Blocker Drugs with Zorbax Extend- C18 at ph 11.5 Separation of Antidepressants Using Zorbax Extend-C18 at ph 11.5 A 1 1. Pindolol 2. Metoprolol 3. Oxyprenolol 3 4. Toluene 5 5. Propranolol % Nortriptyline 2 4 N = 14,060 A s = 1.03 N = 11,500 A s = 0.99 Doxepin I Imipramine Amitriptyline Trimipramine 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 2 4 6 8 10 12 14 16 min. Column: ZORBAX Extend-C18 4.6 x 150 mm Flow Rate: 0.5mL/min. Mobile Phase: Panel A: 55% Methanol / 45% 50 mm Pyrrolidine Buffer, ph 11.5 Panel B: 75% Methanol / 25% 50 mm Pyrrolidine Buffer, ph 11.5 Flow Rate: Panel A: 1.5 ml / min.; Panel B: 1.0 ml/min. Temperature: 40 C Detectoion: UV at 215 nm Slide 44

Separate Peptides/Proteins at High ph Comparison of Angiotensins: Separation With TFA and NH4OH TIC (200-1500 m/z) 5.0E6 4.0E6 3.0E6 2.0E6 1.0E6 0 5.0E7 4.0E7 3.0E7 2.0E7 Acidic Conditions A- 0.1% TFA in water B- 0.085% TFA in 80%AcN 0 2.5 5 7.5 10 12.5 Basic Conditions A- 10 mm NH 4 OH in water B- 10 mm NH 4 OH in 80%AcN 5.499 AII 8.477 AI 9.621 AII+AIII AIII 12.050 13.261 AI min Conditions: 2.5 µl sample (50 pmol each); ZORBAX Extend- C18, 2.1 x 150 mm; 0.2 ml/min; 35 C; 15-50% B in 15 min.; Pos. Ion ESI- Vf 70V, Vcap 4.5 kv, N 2-35 psi, 12 L/min., 325 C Angiotensin I and II separate at high ph due to their different selectivity (in this case different charges). 1.0E7 0 0 2.5 5 7.5 10 12.5 min Slide 45

Separate Peptides/Proteins at High ph with Low Noise LC/MS Comparison of Angiotensin I Mass Spectra With TFA and NH4OH 100 80 433.0 649.5 +3 +2 Max: 10889 Acidic Conditions A- 0.1% TFA in water B- 0.085% TFA in 80%AcN 60 40 20 0 227.1 250.9 340.3 398.0 458.0 659.7 720.3 767.3 844.8 1025.2 1179.1 1277.9 1326.3 1373.3 1468.8 Conditions: 2.5 µl sample (50 pmol); ZORBAX Extend-C18, 2.1 x 150 mm; 0.2 ml/min; 35 C; 15-50% B in 15 min.; Pos. Ion ESI- Vf 70V, Vcap 4.5 kv, N 2-35 psi, 12 L/min., 325 C 500 1000 m/z 100 80 60 40 20 0 433.0 649.0 +2 659.8 500 1000 Max: 367225 1296.6 m/z Basic Conditions A- 10 mm NH 4 OH in water B- 10 mm NH 4 OH in 80%AcN +3 Note that the +1 ion is visible with the high ph mobile phase, but buried in the +1 noise with the low ph mobile phase. Slide 46

Effect of Stationary Phase Type on Separation Selectivity Selectivity Separation of Triazine Pesticides on Polar- Embedded Reversed Phase and Alkyl C8 Reversed Phase Columns mau 800 400 0 5 1 2 3 4 5 6 Bonus-RP 7 Columns: 4.6 x 150 mm Instrument: HP 1100 Mobile phase: MeOH:0.1% TFA (70:30)* Flow rate: 1 ml/min. Temperature: Ambient Inj.: 2 µl UV: 254 nm Sample: 1. Prometryne 5. Diuron 2. Tebuthion 6. Propanil 3. Atrazine 7. Dacthal 4. Propazine mau 1 Alkyl-C8 800 400 2 5 6 3 4 0 0 2 4 6 8 min *For low ph work, a TFA mobile phase is often preferred over phosphate, and is compatible with LC/MS. Si NH O Zorbax Rx-SIL Silica Support Surface O Slide 47

Overview of Method Development: Start at low ph, work toward higher ph A METHOD-DEVELOPMENT STRATEGY THAT MAXIMIZES COLUMN LIFE: REVERSED-PHASE HPLC OF IONIZABLE COMPOUNDS ZORBAX StableBond B asic com poun ds havin g low-p H instability [STEP 1a] A dd 2 0 mm TE A o r TEA acet ate Re-adjust p H [STEP 2a] Vary co lumn t emp., up to 80 C; up to 90 C for Zo rbax SB-C18 Ta il in g pe aks Band spacing p roble ms SAMPLE Chan ge b onde d-ph ase fu nctiona lity to Zorbax SB-CN, SB-Ph eny l, or SB-C3 Resta rt at STEP [1] Initia l separation Zorbax SB-C 18 or SB -C8 ph 2 (1-3), 2 0-50 mm b uffer T= 30 C (am bient to 80 C, SB-C18 to 90 C) Adjust % A CN for 0.5 < k < 20 Band spacing problems Incr ease or de crea se % of organic modifier by 5% (v/v) Band spacing p roble ms Chan ge o rgan ic modifie r (MeOH or THF) Adjust % organic fo r 0.5 < k < 20 Resta rt at STEP [2] Band spacing problems [START] [STEP 1] [STEP 2] [STEP 3] [STEP 4] Poor ly retain ed ba sic compo unds Band spacing p roble ms [STEP 1d] Incr ease or de crea se % of or ganic m odifier by 5 %( v/v) Band spacing p roble ms OR BAS IC COMPOUNDS ( Ion Pai ring) Use M eoh or ganic m odifier, 25-50 mm hexane sulfonic acid, 10 mm p H 3 bu ffer Adjust % Me OH for 0.5 < k < 20 Band spacing pr oblem s [STEP 1c] Vary te mpe ratur e within re comm ende d ran ge for bon ded p hase Band spa cin g pr oblem s [STEP 1b] [STEP 1e] Low ph ph 3 Band spacing pr oblem s Chan ge o rgan ic modifie r ( ACN or THF) Adjust fo r 0.5 < k < 20 OR [STEP 5] Zorbax Eclipse XDB- C8 or XDB-C18 ph 7 (6-9), 2 0-50 mm b uffer T= 30 C (am bient to 40 C) Adjust % Me OH for 0.5 < k < 20 Poor retention or ba nd spa cing pr oblem s Poor ly retain ed acidic co mpo unds ZORBAX Eclipse XDB/Bonus RP [STEP 6a] Vary te mpe ratur e within re comm ende d ran ge fo r bo nded phase Band spacing p roble ms Chan ge o rgan ic modifie r (A CN or THF) Adjust fo r 0.5 < k < 20 Resta rt at ST EP [6] [STEP 7] Band spacing pr oblem s [STEP 8] Chan ge b onde d-ph ase fu nctiona lity to Zorbax Eclipse XDB-Phenyl or Bonus RP Resta rt at ST EP [5] Band spacing pr oblem s Incr ease or de crea se % of organic modifier by 5% (v/v) Band spacing pr oblem s [STEP 6] [STEP 5d] Increase or decrease % of or ganic m odifier by 5 %( v/v) Band spacing p roble ms Band spacing pr oblem s [STEP 5c] [S TEP 5b] ACIDIC COMPOUNDS (Ion Pairi ng) Use M eoh or ganic m odifier, 25-50 m M te trabu tylamm onium ph ospha te, 10 mm p H 7 bu ffer Adjust % Me OH for 0.5 < k < 20 Vary te mpe ratur e within re comm ende d ran ge for bonded phase Band spa cin g pr oblem s [STEP 5e] Chan ge o rgan ic modifie r ( ACN or THF ) Mid ph ph 7 Band spacing pr oblem s Adjust for 0.5 < k < 20 [STEP 9] Adapted and updated by R.D. Ricker, B.A. Bidlingmeyer and J.J. Kirkland, from J.J. Kirkland, LC/GC, 14 (1996) 486. ZORBAX Extend-C18 [STEP 10a] Vary te mpe ratur e within re comm ende d ran ge fo r bo nded phase Band spacing pr oblem s Band spacing p roble ms Zorbax Extend-C18 ph 1 0.5 (9-12 ); 5 m M am mon ia, or TEA, or 10-50 m M or gan ic buffer, or bora te bu ffer T= 25 C (am bient to 40 C) Adjust % Me OH for 0.5 < k < 20 Band spacing pr oblem s Chan ge o rgan ic modifie r (ACN or THF) A djust fo r 0.5 < k < 20 [STEP 10] Try different HPLC mode. Call Agilent Technologies Technical Support (800) 227-9770. Band spacing pr oblem s High ph ph 9 Adapted and updated by R.D. Ricker, B.A. Bidling meyer and J.J. Kirklan d, from J.J. Kirkland, LC/ GC, 14 (1996) 486. Reco mm en ded Colu mn 1 2 3 4 5 6 7 8 9 10 11 12 13 ZO RBAX Stab le Bon d ZO RBAX Eclip se XDB ZO RBAX Bon us RP ZO RBAX Exte nd - C18 ph- Rang e for O ptimal Colum n lif e Unique Selectiv ity ABBREVIATIO NS AC N = ace tonitr ile MeOH = methanol TEA = triethylamine TFA = trifluoroacetic acid THF = tet rahydrofuran Slide 48

Summary of Phase Selection Presentation 1. Choose reversed-phase stationary phase based on separation requirements of your analytes and optimum column lifetime. - Low ph, 1-4 (siloxane protecting phase, no end capping, ZORBAX StableBond) - Intermediate ph, 3-8 (high coverage phase, fully end capped, ZORBAX Eclipse XDB) - High ph, 8-12 (Bidentate or polymer-coated phase, fully end-capped, C8 or C18, no short chain phases, ZORBAX Extend C18) - Low % organic and basic compounds that tail with above phases, ph 3-8 (polar-embedded alkyl phase, ZORBAX Bonus) 2. If possible, always use low ph values since best chromatography for ionizable compounds; if not, use intermediate ph and doubly end capped packing; if insufficient retention, then choose high ph with bidentate packing; if peak shape for basic compounds poor, then try alkyl polar-embedded phase. 3. Choose smallest particle size consistent with efficiency needs; use Type B solgel silica 4. Optimize solvent selection and composition, buffer type, buffer concentration, and temperature; add competing base, if needed Slide 49