Reversed Phase Chromatography: Mobile phase Considerations

Reversed Phase: Mobile Phase Considerations Dr. www.forumsci.co.il/hplc Example of a Reversed Phase Method with UV Detection 1

Chromatographic Process B+A Mobile phase Elution through the Column-movie B A Stationary Phase Distribution: K = C s /C m.34.3.3.8.6.4...18.16 AU.14.1.1.8.6.4.. -. k' = Area A = 3, Area B = 1, Time t R - t t k = Chromatogram t R(A) A - 93.448 9.5 91. 91.5 9. 9.5 93. 93.5 94. 94.5 95. 95.5 96. Minutes C s φ Cm t R(B) B - 94.776 Chromatographic Process in Reversed Phase Mode: Polar Mobile Phase and A-polar Stationary Phase B+A Mobile phase polar: Water, Buffers MeOH, Acetonitril, IPA Hydrophobic Stationary Phase A B Distribution: K = C s/c m B (More Hydrophobic) A t= t Migration through the Column: Hydrophobic Components are Retained Longer Chromatogram

Reversed Phase Elution Order: Propyl>Methyl.3 OH.5 OH. O O AU.15.1 O H 3 C O H 3 C.5 Methylparabene Propylparabene...4.6.8 1. 1. 1.4 1.6 1.8...4.6.8 3. 3. 3.4 3.6 3.8 4. 4. 4.4 4.6 4.8 5. Minutes MOBILE PHASE Parameters that Affect Separation * TYPE OF MODIFIER (MeOH, ACN) * MOBILE PHASE COMPOSITION (% modifier) * ph * TYPE OF BUFFER (phosphate, acetate) * IONIC STRENGTH (Salts, buffer concentration) * ION-PAIRING REAGENTS (alkyl-amines, -sulfonates) 3

OPTIMIZATION: CHOICE OF SOLVENTS % MeOH % ACN % EtOH % THF main solvent: H O MOBILE PHASE Parameters that Affect Separation * TYPE OF MODIFIER (MeOH, ACN) * MOBILE PHASE COMPOSITION (% modifier) * ph * TYPE OF BUFFER (phosphate, acetate) * IONIC STRENGTH (Salts, buffer concentration) * ION-PAIRING REAGENTS (alkyl-amines, -sulfonates) 5

Analyte Retention as a Function of % Modifier: Negative Slope k (retention) for each analyte changes independently as % Modifier changes. Thus, the resolution between peaks changes. SOLVENT STRENGTH k' = t R - t t M. Gilar et al. / J. Chromatogr. A 958 () 167 18 OPTIMIZATION: % SOLVENTS % MODIFIER 4% MODIFIER 6% MODIFIER 8% MODIFIER The higher the % of modifier The sorter are all RT 6

k can be Very Sensitive to Compositional Changes of the Mobile Phase k' = t R - t t.6.5.4 AU.8.6.4...18.16.14.1.1.8.6 69.8% A 69.9% A 7.% A 7.1% A 7.% A AU.3.4....86.88.9.9.94.96.98 1. 1. 1.4 1.6 1.8 1.1 Minutes.1..1..3.4.5.6.7.8.9 1. 1.1 Minutes MOBILE PHASE * TYPE OF MODIFIER (MeOH, ACN) * MOBILE PHASE COMPOSITION (% modifier) * ph * TYPE OF BUFFER (phosphate, acetate) * IONIC STRENGTH (Salts, buffer concentration) * ION-PAIRING REAGENTS (alkyl-amines, -sulfonates) 7

.36.7.18.9..48.3.16. -.16.48.36.4.1..5 1. 1.5..5 3. 3.5 4. 4.5 5. Minutes.5 1. 1.5..5 3. 3.5 4. 4.5 5. Minutes.5 1. 1.5..5 3. 3.5 4. 4.5 5. Minutes Reversed Phase Chromatography: Mobile phase Considerations ph as a Selectivity Tool For Impurities.36.7 1 ph 9 Ammonium Bicarbonate AU.18.9..5 1. 1.5..5 3. 3.5 4. 4.5 5. Minutes 1 ph 7 Sodium Phosphate AU 1 ph 5 Ammonium Acetate 1 ph Sodium Phosphate AU Ionization of Acids and Bases Dissociation of Molecule Acid HA H + + A - (Un-ionized) (Ionized) 5% ph= pka 5% 1% ph<<pka % % ph>>pka 1% 8

Ionization of Acids and Bases Dissociation of Molecule Base B BH + + OH - (Un-ionized) (Ionized) 5% ph= pka 5% % ph<<pka 1% 1% ph>>pka % Retention Factor versus ph for Acids, Bases and Neutrals Retention Factor -Linear Acids (HA) ( pka =4.8 ) Un-Ionized Bases (BH + ) ( pka = 9. ) - Ionized k k = HA K a + k A + [ H ] K a 1 + + [ H ] Apparent pka B Un-Ionized Neutral A - Ionized 1 3 4 5 6 7 8 9 1 11 1 13 14 ph (mobile phase) 9

Behavior of Acidic, Basic and Neutral Compounds in Reversed-phase Chromatography 4 35 3 Note: Retention of neutral analytes not affected by ph Acid Neutral Capacity Factor (k) 5 15 1 Robust ph zone Robust ph zone 5 Base 4 6 8 1 1 ph Impact of Organic Concentration on the ph of the Mobile Phase Aqueous buffers prepared from an acid and mixed with an organic solvent pk a of the acid increases ph of the buffers increases Acid Aqueous pka pka in 5% methanol Phosphoric.11 3.1 Phosphoric 7.19 8.4 Formic 3.73 4.35 Acetic 4.77 5.54 Appendix: Methanol References and 3 1

Impact of Organic Concentration on the ph of the Mobile Phase Aqueous buffer prepared from a base and mixed with an organic solvent pk a of the base decreases ph of the buffers decreases Acid Aqueous pka pka in 5% methanol Ammonium 9.4 8.76 Appendix: Methanol References and 3 Impact of Organic Concentration on the pk a of the Analyte In general: Basic Compounds: pk a will decrease with the addition of an organic solvent Acidic Compounds: pk a will increase with the addition of an organic solvent The specific change in pk a will be compound dependent 11

Shift of pk a With Organic Solvent Composition: Amitriptyline (Basic Compound) Capacity Factor (k) 4 35 3 5 15 1 Aqueous pk a = 9.4 4% Acetonitrile Apparent pk a = 8. Compound un-ionized 5 Compound ionized Phoebe, Tran 1 3 4 5 6 7 8 9 1 11 1 Aqueous ph Amitriptyline + NH CH 3 N CH 3 CH 3 CH 3 Ionized Un-ionized pka (H ) 9.4 apparent pk a (% ACN) 8.5 apparent pk a (3% ACN) 8.3 apparent pk a (4% ACN) 8. Note: you cannot determine the degree or the direction of the pk a shift with out experimentally running each organic concentration 1

Shift of pk a With Organic Solvent Composition: Naproxen (Acidic Compound) 7 Capacity Factor (k) Phoebe, Tran 6 5 4 3 1 compoundu n-ionized Apparent pk a = 4.6 Aqueous pk a = 4. % Acetonitrile 1 3 4 5 6 7 8 9 1 11 1 Aqueous ph compound ionized Naproxen CH 3 COOH CH 3 COO - H 3 CO Un-ionized H 3 CO Ionized pka (H ) 4. apparent pk a (% ACN) 4.6 apparent pk a (3% ACN) 4.8 Note: you cannot determine the degree or the direction of the pk a shift without experimentally running each organic concentration 13

Shift of the ± ph unit rule with the Apparent pk a : Basic compound 4 Aqueous pk a = 9.4 compound un-ionized Capacity Factor (k) 3 1 4% Acetonitrile Apparent pk a = 8. compound ionized 1 3 4 5 6 7 8 9 1 11 1 ± ph Shift of the ± ph unit rule with the Apparent pk a : Acidic compounds 7 6 un-ionized Aqueous pk a = 4. Capacity Factor (k) 5 4 3 % Acetonitrile Apparent pk a = 4.6 1 ionized 1 3 4 5 6 7 8 9 1 11 1 ± ph 14

General Retention and Mobile Phase ph Rules Retention of ionizable analytes is most affected within the ± ph unit envelope surrounding the pk a Strong selectivity changes are observed at ph s close to the pka Requires tight control of mobile phase ph for reproducible chromatographic retention Most stable chromatographic retention times observed outside of the ± ph unit envelope Remember always measure ph before adding the organic solvent Resolution of Two Acidic Compounds at Different Mobile Phase ph s Capacity Factor (k) 1 9 8 7 6 5 4 3 Best retention Indoprofen 1 Silanols Un-ionized 1 3 4 5 6 7 8 9 1 11 1 ph Silica gel high ph limit Decreasing retention Critical ph control necessary Ketoprofen Silanols ionized ph ph 8 1 Minutes 1 Minutes 15

Enhanced Resolution of Basic Compounds at High ph 4 k versus ph Amitriptyline ph 3 k N CH 3 CH 3 Amitriptyline 1 1 3 Minutes Nortriptyline Amitriptyline ph 8 ph control critical N H CH 3 Nortriptyline 1 Nortriptyline 1 3 4 5 6 7 8 9 1 11 1 Phoebe, Tran ph 8 Nortriptyline 1 3 Minutes Amitriptyline ph 1 1 Minut 3 es Dependence of Selectivity on ph AU AU.115.75.35 -.5.135.11.17.93.79.65.51.37.3.9 -.5 3 1 5 4 ph.5 4 8 1 16 4 8 1 6 Time (min) 4 8 1 16 Time (min) ph 8. 5 3 6 4 AU AU.115.75.35 -.5.5.15.5 -.5 1 3 4 5 ph 5. 4 8 1 1 6 Time (min) 1 3 4 5 Time (min) 6 ph 1.6 5 3 4 Conditions: Column: XTerra RP 18, 3.9 X 15 mm, 5 µm Mobile Phase: 65% mm Buffers, 35% ACN Column Temp.: 3 C Flow Rate: 1. ml/min Detector: 1nm for ph.5, ph 5., and ph 7.; 3nm for ph 1.6 1:acetaminophen :lidocaine 3:doxepin 4:imipramine 5:nortriptyline 6:ibuprofen ph the most powerful selectivity tool Lu, Cheng; Waters 16

Impact of ph on the Retention of a Zwitterionic Compound 1 1 Aqueous pk a = 4.3 Aqueous pk a = 9.5 k 8 6 4 Positive Charge Dual Charge Negative Charge 1 3 4 5 6 7 8 9 1 11 1 O H N H + ph H O Fexofenadine (Antihistamine) C H 3 C H 3 C O O - ph Dependent Loadability: Equal Load 1 Load: 1.4 mg.51 Column: 4.6 mm x 15mm XTerra MS C18 Samples: Oxybutynin, Diphenhydramine, Terfenadine Flow rate: 1.75 ml/min Detection: UV at 58 nm % Mobile phase: 4% acetonitrile/5% water/1% 1%TFA.33 1 5.37 % Mobile phase: 6% acetonitrile/8% water/ 1%.1 M (NH 4 ) CO 3, ph 1. 3.5. 4. 6. 8. 1. 1. 14. Time Wheat, Zheng 17

UV/Vis Spectral Change Between Ionized and Non-ionized Forms 1. 1. 1.. A U 8. 6. 4.. 1 6 4 54 nm 5 6 7 7 8 Astemizol e 8 6 3 n m ph. ph 1. 3 3 4 3 6 3 8.8.6.4.. Ketoconazole 54 nm ph. ph 1. 93 4 6 8 3 3 34 36 38 nm Phoebe, Tran Typical Chromatograms for ph Failure of an Ordinary C 18 -Silica Column 1 3 4 Initial 5 1. Uracil. Propranolol 3. Naphthalene 4. Acenaphthene 5. Amitriptyline 1 After failure 3 4 5 4 minutes 18

Recommended Buffers for ph s -7 (Silica and Hybrid Packing Materials) Additive or Buffer pk a ph range (± 1 ph unit) Volatile or Non-Volatile Recommended Concentration Ranges and Common Counter- Ions TFA.3 Volatile (.-.1%) Acetic Acid 4.76 Volatile (.1-1.%) Formic Acid 3.75 Volatile (.1-1.%) Acetate 4.76 3.76 5.76 Volatile/Nonvolatile Formate 3.75.75 4.75 Volatile/Nonvolatile Phosphate.15 1.15 3.15 Non-volatile (1-1mM) NH 4, Na, K (1-1mM) NH 4, Na, K 7. 6. 8. Non-volatile Not for ph s >7. (reduce the temperature & conc. for longer column lifetime) Non-Recommended Buffers for ph s >7 Buffer pk a ph range (± 1 ph unit) Volatile or Non-Volatile Effect if buffer used (Dissolution) Phosphate 1.3 11.3 13.3 Non-Volatile Short Column Lifetime Borate 9. 8. 1. Non-Volatile Short Column Lifetime Not recommended for either hybrid (XTerra) or silica gel based columns.

Recommended Buffers at ph s >7 (Hybrid & Polymeric HPLC Packings) Buffer pk a ph range (± 1 ph unit) Volatile or Non-Volatile Recommended Concentration Ranges 4-Methyl- Morpholine ~8.4 7.4 9.4 Volatile 1mM Ammonia 9. 8. 1. Volatile <1 mm and <3º C Bicarbonate 1-Methyl- Piperidine 1.3 1.3 9.3 11.3 9.3 11.3 Volatile Volatile 5mM (do not use carbonate) 1mM Triethylamine 1.7 9.7 11.7 Volatile (.1-1%) also DEA Pyrrolidine 11.3 1.3 1.3 Volatile 1mM Recommended Organic Buffers for ph s -7 Additive or Buffer pk a ph range (± 1 ph unit) Volatile or Non-Volatile Recommended for use with Extended ph Packings TFA.3 Volatile Yes (.-.1%) Acetic Acid 4.76 Volatile Yes (.1-1.%) Formic Acid 3.75 Volatile Yes (.1-1.%) Acetate 4.76 3.76 5.76 Volatile/Nonvolatile Formate 3.75.75 4.75 Volatile/Nonvolatile Yes (1-1mM) NH 4, Na, K Yes (1-1mM) NH 4, Na, K Phosphate.15 1.15 3.15 Non-volatile Yes 7. 6. 8. Non-volatile No for ph s >7. (lower the temperature the longer the column lifetime) 1

Some Considerations in the Selection of Mobile Phase Buffers for Reversed Phase HPLC Phosphate is more soluble in CH3OH/water than in CH3CN/ water or THF/water. NH 4 > K > Na soluble in organic/water mobile phases. TFA and TEA degrade with time and increase their UV absorbance. Mobile phases containing these buffers should be made fresh often. Citrate buffers attack stainless steel. When using these buffers, be sure to flush them out of the system as soon as you complete your assay. Microbial growth can quickly occur in buffered mobile phases that contain little or no organic modifier. This growth will accumulate on column inlets and damage chromatographic performance. These mobile phases should be made fresh daily and pre-column filters should be used to protect columns. Using boiled water to prepare buffered mobile phase and storing it under refrigeration will help reduce the problem of microbial growth. At ph greater than 7, phosphate buffers accelerate the dissolution of silica and severely shorten the lifetime of silica-based HPLC columns. Organic buffers should be used at ph higher than 8. MOBILE PHASE Parameters that Affect Separation * TYPE OF MODIFIER (MeOH, ACN) * MOBILE PHASE COMPOSITION (% modifier) * ph * TYPE OF BUFFER (phosphate, acetate) * IONIC STRENGTH (Salts, buffer concentration) * ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

k' Does not Change Significantly as Function of Buffer Concentration 4 K' vs Buffer Concentration 3 K' 1 5N-,4,6T 4A-6C-1,3B HCT TMT 1 3 5 7 4 6 mm NaHPO4 ph 5.5 8 9 1 Types of Buffers and Recommended Ionic Strength ph 1: Borate mm H 3 BO 3 ph 7: Phosphate mm K HPO 4 ph 4-5: Acetate 1 mm CH 3 COONH 4 1 mm CH 3COOH ph -3.5: Phosphate mm H 3PO 4 - KH PO 4 3

MOBILE PHASE Parameters that Affect Separation * TYPE OF MODIFIER (MeOH, ACN) * MOBILE PHASE COMPOSITION (% modifier) * ph * TYPE OF BUFFER (phosphate, acetate) * IONIC STRENGTH (Salts, buffer concentration) * ION-PAIRING REAGENTS (alkyl-amines, amines, -sulfonates) Ion Pair Reagent Alkylamines + Alkylsulfonates Pentanesulfonate - SO 3 Na+ CH 3CH CH3CH CH CH CH 3CH + CH 3 CH Triethylamine (TEA) N OH- OH- N CH CH CH CH 3 CH 3CH CH CH CH CH CH CH 3 Hexanesulfonate Heptanesulfonate - SO 3 Na+ - SO 3 Na+ Tetrabutylamine (TBA) H 3 C OH- N+ CH CH 3CH CH CH CH CH CH 3 CH 3 Octanesulfonate Dodecylsulfonate SO - 3 Na+ - SO 3 Na+ Dibutyl-dimethylamine 4

Alternative Mobile Phase Modifiers N N N N Triethylamine (TEA) Dimethylbutylamine (DMBA) Tributylamine (TBA) diisopropylethylamnie (DIEA) N ( ) 5 dimethyloctylamine (OA) N Tripropylamine (TPA) NH Dihexylamine (DHA) NH Octylamine (OA) NH Hexylamine (HA) Choice of Ion-Pairing Reagent Ion-Pairing Agent Acid Abbreviation Triethylamine acetic acid TEAA Triethylamine bicarbonic acid TEAB Dimetylbutylamine acetic acid DMBAA Tetrabutylamine acetic acid TBAA Triethylamine hexafluoroisopropanol TEA-HFIP 5

TCC CTA GCG TTG AAT TGT CCC TTA GCG GGT (truncated from 3 ) 1 11 5 3 5 UV 6 nm Choice of Ion-Pairing Reagent Triethylammonium Acetate Reagent 11 1 13 14 15 16+17 18+19 1 3 9 4 8 3 1 Minutes 3 UV 6 nm Triethylammoniu m HFIP Reagent T G 11 1 A A T T 13 14 15 16 G 17 C 19 T 18 C T T 1 T G 5 3 G A 4 9 G T G 8 3 C 7 6 Minutes 3 Optimizing Ion-Pairing Reagent (5mer PS) 5 4 TEA-HFIP 16.3mM-4mM 3 19 1 N+x 15 TEAA 1 mm minutes 3 6

PS Diastereomeric Separations: C 18, 135Å,.5 µm (.1x5mm) TEAA TEA-HFIP CCT CAT GsCC GTG TCC CTG TsT Ion Pair Chromatography S. Levin 7

Dependence on Chain Length of Ion-Pair Reagent S. Levin Concentration of Ion-Pair Reagent in the Mobile Phase The larger the alkyl, the longer are retention times The larger alkyls saturate the stationary phase at lower concentrations k ' C8 C7 C6 C5 5 mm Conc. of Ion Pair Reagent in the Mobile Phase 8