ACE C18-AR and C18-PFP - Hplc.eu

Exploiting Selectivity in HPLCand UHPLC With RationalStationary Phase DesignThomas J. Waeghe, Ph.D.twaeghe@mac-mod.comMAC-MOD Analytical, Inc.800-441-7508www.mac-mod.com

®ACEOutlineHPLC / UHPLC Columns2Chromatographic selectivityStationary phase design conceptsThe unique ACE ® C18-AR and ACE ® C18-PFP phasesIntroducing the NEW ACE ® Excel UHPLC productsExamplesConclusions

®ACEHPLC / UHPLC ColumnsChromatographic Peak Resolution33.02.5EfficiencyR s =√ N4Selectivity-1Retentionk1+kResolution (R s)2.01.51.0Nk0.50.01.00 1.05 1.10 1.15 1.20 1.250 5000 10000 15000 20000 250000 5 10 15 20 25NkZhao, J.H. and P.W. Carr. Analytical Chemistry, (1999) 71, 2623-2632

®ACEHPLC / UHPLC ColumnsThe Importance of N, k and For Resolution4Typical separation:N = 10,000 platesk = 3.8 / 4.2 (4.0 mean) = 1.1R SR S14 1.81.11410,000 1.1 14R s =√ N4-1k1+kWhich looks like

®ACEHPLC / UHPLC ColumnsThe Importance of N, k and For Resolution5Double Efficiency (e.g., 5 m 2.5 m):N = 10,000 20,000 platesR s =√ N4-1k1+kR SR S14 2.61.11420,000 1.1 14R s = 1.8~40% IncreaseR s = 2.6Opportunity to optimize further, e.g. reduce column length to speed up

®ACEHPLC / UHPLC ColumnsThe Importance of N, k and For Resolution6Double Retention Factor (e.g., decrease solvent strength):k = 4 8√ NR s =4-1k1+kR SR S14 2.01.11810,000 1.1 18R s = 1.8~10% IncreaseR s = 2.0Slight improvement in resolution has led to increased analysis time

®ACEHPLC / UHPLC ColumnsThe Importance of N, k and For Resolution7Increase Selectivity (e.g., change column): = 1.1 1.2R s =√ N4-1k1+kR SR S1 1.214 10,0004 1.2 14 3.3R s = 1.8Can now shorten run time with shorter column and/orfaster flow rate~80% IncreaseR s = 3.3Significant opportunity to speed up for modest change in selectivity

®ACEHPLC / UHPLC ColumnsSelectivity: The Key to Chromatographic Peak Resolution8EfficiencySelectivity Retention3.02.5R s =√ N4-1k1+k~40% ~80% ~10%Resolution (R s)2.01.51.0Nk0.5From the examples, selectivity hasthe greatest impact on increasingpeak resolution0.01.00 1.05 1.10 1.15 1.20 1.250 5000 10000 15000 20000 250000 5 10 15 20 25NkZhao, J.H. and P.W. Carr. Analytical Chemistry, (1999) 71, 2623-2632

®ACEHPLC / UHPLC ColumnsWhich Factors a Affect Selectivity?9MOSTInfluenceIsocratic SeparationsColumn stationary phaseGradient SeparationsAll parameters for isocraticOrganic modifierGradient steepnesspH (ionised analytes only) k*% Organic modifierDwell volumeBuffer selectionColumn dimensionsColumn temperatureBuffer concentrationLEASTInfluencek*tG F 85 V Sma Adapted from ‘Introduction to Modern Liquid Chromatography”, 3 rd Edition, Snyder, Kirkland, Dolan, 2010, p.29, Wiley & Sons

®ACEHPLC / UHPLC ColumnsInfluencing Selectivity – Bonded Phase Effects / Basic Analytes10ACE C18 – Increase RetentionACE CN – Elution OrderACE C8 (start point)ACE C4 – Decrease RetentionACE Phenyl – Elution OrderUse ultra high purity silica for good chromatography and reproducibilityColumn: 250 x 4.6mm, 5m Mobile phase: 80:20 MeOH/25mM KH 2 PO 4 (pH 6.0) Flow: 1.00ml/minComponents; 1: Norephedrine, 2: Nortriptyline, 3: Toluene, 4: Imipramine, 5: Amitriptyline Wavelength: 215nm

®ACEHPLC / UHPLC ColumnsHPLC End User Surveys a ...Listening To The Analyst11Column reproducibility and column lifetime are majorfactors for analysts− Have been the top 2 feedback points since 2007− Critical in pharmaceutical and other major industries for methodtransfers / consistency and long term performanceReversed-phase is the dominant separation mode− C18 & C8 = 60%; Phenyl = 16%; CN = 9.5%; Fluorinated = 5.9%− 92% analysts use C18 at some time in their work...they typically meetthe above criteria− BUT limited selectivitya Current Trends in HPLC Column Usage, R.E. Majors, 1Jan12, Modern Medicine website, accessed on 15Feb12: http://www.modernmedicine.com/modernmedicine/article/articleDetail.jsp?id=755108

®ACEHPLC / UHPLC Columns16 Pharmaceutically Relevant Analytes – C18 Columns12ACE Excel 2 C18P max: 309 barWatersAcquity 1.7 BEH C18P max: 478 barPhenomenaKinetex 1.7 C18P max: 446 bar1, 2121Agilent ZorbaxEclipse 1.8 XBD C18P max: 396 bar 1 23, 4253334445 65 67675 6787898810910, 119101211109111213111612 13 14 1513 14 15 161614 1514 1512161350x2.1mmA: 20 mM KH 2 PO 4 , pH 2.7B: 20 mM KH 2 PO 4 , pH 2.7 inMeOH/H 2 O (65:35 v/v)Gradient: 3 – 100 %B in 5 minFlow rate: 0.6 ml/minTemperature: 60CDetection: 214 nm1. N‐Acetylprocainamide2. 3‐Hydroxybenzoic acid3. Pindolol4. Methylphenylsulfoxide5. Benzyl alcohol6. Quinoxaline7. 1,4‐Dinitrobenzene8. Phenacetin9. 1,2‐Dimethoxybenzene10. Furosemide11. Anisole12. Methyl benzoate13. Remacemide14. Nimesulide15. Ethyl benzoate16. Diflunisal1 2 3 4 5C18 phases show ‘similar’ selectivity...minAll trademarks are recognised...comparative separations may not be representative of all applications

®ACEHPLC / UHPLC ColumnsThe Challenge...13To engineer new phases with alternative selectivity butwith the robust properties of the C18 ligand− Reproducible (column-to-column & batch-to-batch)− Excellent column lifetime− Superb efficiency provided by ultra-inert, ultra-pure silica particle− Low MS bleed− Usable in 100% aqueous eluentsAvailable for HPLC & UHPLC separationsAvailable as a ‘Phase III Ready’ product familyGlobally available, supply chain, reproducible, multiple batches etc

®ACEHPLC / UHPLC ColumnsAromatic Functionality – Engineering New Stationary Phases14Phases with aromatic functionality include phenyl andpentafluorophenyl (PFP) based ligands−−AdvantagesAromatic functionality potentially offer unique interactions withanalytes (c.f. C18) giving alternative selectivityProvides enhanced retention of polar compounds− Many aromatic functionality-based phases can be used in 100%aqueous eluents−−DisadvantagesPhenyl / PFP phases may suffer phase bleedBatch-to-batch reproducibility & robustness may be weak

®ACEHPLC / UHPLC ColumnsAromatic Functionality: π – π Interactions15A type of electron donor-acceptor interactionOriginates from π systems in unsaturated functionalgroups on analytes and the stationary phaseTypes of π-π interaction can be manipulated formaximum effect (orthogonality) in phase design− eg phenyl: electron rich ring on the stationary phase also actsas π-base and interacts well with electron poor analytes− eg PFP: electron poor ring on the stationary phase also actsas π-acid and interacts well with electron rich analytes

®ACEHPLC / UHPLC ColumnsThe Power of π...Scientific Led Stationary Phase Design16Electron Donating Groupseg NH 2 , NR 2 , alkyl, OCH 3OR, CH 3 , Ar etcSi e.g.δ+δ-Electron Rich RingSiC 6 H 6Classic π-πinteractionElectron Withdrawing Groupseg NO 2 , halides, NR 3+ , CO 2 H,CN, CO 2 R, SO 3 H, COH etcSie.g.Fδ-δ-Fδ+Fδ-δ-FFδ-Electron Deficient RingActivity: π-donor (π-base)Activity: π-acceptor (π-acid)How do we exploit these properties for new stationary phases?C18+Phenyl = ACE ® C18-ARC18+PFP = ACE ® C18-PFP

®ACEHPLC / UHPLC ColumnsUniquely Designed Stationary Phases17 ACE ® C18-AR (USP L1)− Ligand has C18 hydrophobic element PLUS phenyl character ACE ® C18-PFP (USP L1)− Ligand has C18 hydrophobic element PLUS PFP characterUltra-inert, ultra-pure silica particle technology as usedin all ACE ® products for high peak efficiency Available in 3, 5 & 10m, (ACE ® ) and 2m (ACE ® Excel TM )Multi-mode interaction mechanisms result in enhancedchromatographic selectivity giving the analyst newoptions for method development

®ACEHPLC / UHPLC ColumnsACE ® C18-AR: Multi-Mode Separation Mechanisms18Combining the character of C18+phenyl into a single individualphase harnesses the best of both phases for unique selectivitySeparationmechanismTypicalC18TypicalPhenylACE ®C18-ARHydrophobicity ++++ + / ++ ++++- Interaction - +++ +++Dipole - Dipole - + +Hydrogen Bonding - ++ ++Shape Selectivity ++ ++ ++ / +++The predominance of each retention mechanism will bedictated by the analyte’s physicochemical properties, itsstructure and the chromatographic conditions appliedMulti-Mode Interactions Offer the Chromatographer More

®ACEHPLC / UHPLC ColumnsACE ® C18-AR Aromatic Selectivity19Illustrating hydrophobicity and π-base character / aromaticselectivity with a simple example using substituted aromatics1. TNB 2. DNB 3. NB 4. TolNO 2NO 2NO 2NO 2NO 2NO 2NO 2NO 2Log P*π-acidity (order)O 2NO 2N1.2 1.6 1.9 2.71 > 2 > 3 -O 2NO 2NmAU123 4 ACE 3 C183421ACE 3 Phenyl150x4.6 mm id1 mL/min40C, 210nm1:1 v/v MeOH:H2O23140 2 4 6 8 10 12 14 16 18ACE 3 C18-ARmin* Predicted data from ACDLabs software, 30May12

®ACEHPLC / UHPLC ColumnsACE ® C18-PFP: Multi-Mode Separation Mechanism20Combining the character of C18+PFP into a single individualphase harnesses the best of both phases for unique selectivitySeparationmechanismTypicalC18TypicalPFPACE ®C18-PFPHydrophobicity ++++ + / ++ ++++- Interaction - +++ +++Dipole - Dipole - ++++ ++++Hydrogen Bonding - +++ +++Shape Selectivity ++ +++ ++++The predominance of each retention mechanism will bedictated by the analyte’s physicochemical properties, itsstructure and the chromatographic conditions appliedMulti-Mode Interactions Offer the Chromatographer More

®ACEHPLC / UHPLC ColumnsACE ® C18-PFP Selectivity*21Peak Number:1 2 3 4 5 6 7 8Log P:π-basicity (order):1,2,3- 1,2,4- 1,2- 1,4- 1,3- 1,3,5TMB TMB DMB DMB MB DMB TMB Tol1.7 1.6 1.7 2.1 2.2 2.2 1.6 2.71 1 2 2 3 2 1 -Elution / retention not simply a function of π-basicity and Log PRetention mechanism for C18-PFP multi-modal*Structures fromwww.chemspider.comPredicted data from ACDLabs software, 30May12

®ACEHPLC / UHPLC ColumnsACE ® C18-PFP Selectivity1,2345,67ACE 3 C18Hypersil GOLD 3 μm PFPHydrophobicreference8C18 or PFP mechanisms alonenot enough to fully resolve themethoxybenzene isomers2213425186 732456 7 8ReducedHydrophobicityACE 3 C18-PFPACE C18-PFP mechanismcombines hydrophobicity,shape selectivity, dipole-dipoleand π-π interactionsElution order, retention andselectivity all seen to differ5 10 15 20 25Minutes1) 1,2,3‐trimethoxybenzene, 2) 1,2,4‐trimethoxybenzene, 3) 1,2‐dimethoxybenzene, 4) 1,4‐dimethoxybenzene 5) methoxybenzene, 6) 1,3‐dimethoxybenzene,7) 1,3,5‐trimethoxybenzene, 8) toluene (ref) Mobile phase 50:50 v/v MeOH / H 2 O; Column= 150 x 4.6 mm id; 1.00 ml/min; 40C; 254 nmAll trademarks are recognised...comparative separations may not be representative of all applicationsPowerful positional isomer andshape selectivity

®ACEHPLC / UHPLC ColumnsACE ® Phase Comparisons With The Selectivity Descriptor*23Selectivity = 100 x √ (1 – R 2 )t rcolumn 1xxxxR 2 = 0.9987S = 19S = 15C18C18MeCNt rcolumn 2MeOHS = 7 S = 8>100 acidic, basic, neutral analytesS = 11S = 12S = 18S = 18S = 8Selectivity = 100 x √ (1 – R 2 )= 100 x √ (1 – 0.9888)= 10.6C18-PFPC18-PFPS = 10C18-ARS = 15C18-AR* Neue, O’Gara, Méndez “Selectivity in Reversed-Phase Separations: Influence of the Stationary Phase”, J. Chromatogr. A 1127 (2006), 161-174

®ACEHPLC / UHPLC ColumnsRanking ACE ® Phase Orthogonality With MeOH and MeCN24For the 102 acidic, basic and neutral analytes assessedMeOHMeCNColumn 1 Column 2 Selectivity ‘S’Column 1 Column 2 Selectivity ‘S’C18 C18-AR 12C18 C18-AR 8C18 C18-PFP 11C18-AR C18-PFP 8C18-AR C18-PFP 10C18 C18-PFP 7MeOH MeCN Selectivity ValueC18-PFP C18 19C18-AR C18 18C18-AR C18-PFP 18C18-PFP C18-AR 18C18-PFP C18-PFP 18C18 C18-AR 17C18 C18-PFP 17C18 C18 15C18-AR C18-AR 15Shows value of usingthe 3 phases in a 2solvent screen formethod developmentwork

®ACEHPLC / UHPLC ColumnsWhat Do I Use These Novel Phases For: ACE C18-PFP?25Useful for analytes that contain electron donating moietieseg -NH 2 , -NR 2 , -OCH3, -OH, -alkyl, -Ar etc eg nucleotides, nucleosides, nucleobases, halogenated aryl /aromatics, catecholamines, tetracyclines, beta blockers,structural isomers, coumarins etcExcellent shape and positional isomer selectivity

®ACEHPLC / UHPLC ColumnsWhat Do I Use These Novel Phases For: ACE C18-PFP?26C18-PFP: chloroacetophenone halogenated isomers separation

®ACEHPLC / UHPLC ColumnsWhat Do I Use These Novel Phases For: ACE C18-AR?27Useful for analytes that contain electron withdrawing moietieseg -NO 2 , -halides, -NR 3+ , -SO 2, -CO 2 H, -SO 3 H, -CO 2 R, -CHO etceg aromatic compounds, anthocyanins, steroids, analgesics,phenolics, water soluble vitamins, sulphur containingcompounds, quinolones, positional isomers etcModerate shape selectivity

®ACEHPLC / UHPLC ColumnsWhat Do I Use These Novel Phases For: ACE C18-AR?mAUACE C18921112310/7 1/4 568Steroid MixtureColumns: 4.6 x 150 mm, 3 mA: WaterB: ACNTemp. 20CFlow Rate: 1 mL/minGradient: 25 to 46%B, 24 min9ACE C18‐AR21112310 16 57 4 8ACE Phenyl92/1112 310 1 5/867 40 5 10 15 20 25min1) 17α Estradiol, 2)Prednisolone, 3) Corticosterone, 4) 19‐Norethindrone, 5) Estrone, 6) 17α Ethynylestradiol,7) Cortisone 21‐acetate, 8) 21‐Hydroxyprogesterone, 9) Estriol, 10) 17β Estradiol, 11) Hydrocortisone,12) Cortisone

®ACEHPLC / UHPLC ColumnsCombining Selectivity With The NEW ACE ® Excel Format30NEW high efficiency, ultra-inert 2m silica particles suitable forUHPLC at 1000bar (15,000psi)Nine selectivities – including the unique C18-AR and C18-PFPHigh reproducibility: column-to-column and batch-to-batchUltra-robust phases: NEW low dispersion column hardware andNEW High Stability Column (HSC) packing technologyEngineered with lower back pressures (~30%) compared to other< 2m phases, due to 2m particle size and frit technologyFully scalable to ACE® 3m, 5m and 10m phasesFully compatible with all commercial HPLCs and UHPLCs

®ACEHPLC / UHPLC ColumnsACE ® Excel UHPLC Column Robustness311000 bar for ~2000 gradient runsIsocratic efficiency assessments every ~100 runs more demanding!ACE ® Excel 2 C18(with HSC Packing Technology)Prototype 2 m C18(without HSC Packing Technology)Efficiency virtuallyunchanged across2000 injections at1000 bar100x2.1mm; MPA 0.1% FA (aq); MPB: 0.1% FA in MeOH; 0.73mL/min; gradient: 20‐90%B in 6 mins.NEW High Stability Column (HSC) Packing TechnologySignificantly Improves UHPLC Column Robustness

®ACEHPLC / UHPLC ColumnsACE ® Excel UHPLC Columns – Scalability & Reproducibility32ACE Excel 2 C18150 x 2.1mm0.21mL/min1 2 34562 µmUHPLCACE 3 C18150 x 3.0mm0.40mL/min3 µmHPLCACE 5 C18150 x 4.6mm1.00mL/min5 µmACE 10 C18150 x 21.2mm21.2mL/min10 µm0 1 2 3 4 5 6 7 8PrepLCMP: 35:65 v/v MeCN:0.1% TFA (aq); 22C; 254nm; 1. uracil; 2. 4‐hydroxybenzoic acid; 3. acetylsalicyclic acid; 4. benzoic acid; 5. 2‐hydroxybenzoic acid 6. ethyl paraben

®ACEHPLC / UHPLC ColumnsACE ® Excel Has Typically Lower Back Pressure For UHPLCSpecifically engineered for lower UHPLC backpressuresACE Excel 2 C18‐AR10.9521.22 31.69 41.7952.2462.5182.8172.9193.55103.81114.19124.47134.53P max: 364 bar33Waters Acquity 1.7um BEH C18120.900.87 31.48 41.5152.0672.1762.2982.50103.3093.34124.02114.09134.32P max: 581 barPhenomenex Kinetex 1.7um C181, 20.8031.34 451.36 1.9172.0362.1582.3810123.91 133.18 911 4.193.223.94P max: 540 barAgilent ZORBAX Eclipse 1.8 um XDB C1820.9010.933, 41.5152.1362.37 82.63103.3993.51124.13114.18134.41P max: 540 bar0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 minConditions: A = 5mM formic acid (aq); B = 5mM formic acid in MeOH; tg= 3 to 100%B in 5 min; 0.6 ml/min; 40C; 254nm1. Paracetamol 2. Hydrochlorothiazide 3. Methylphenylsulphoxide 4. Methylphenylsulphone 5. Aspirin 6. Phenacetin 7. 1,3-dinitrobenzene 8. 1,2,4-Trimethoxybenzene9. Ethyl benzoate 10. Nimesulide 11. Ibuprofen 12. Indomethacin 13. Mefenamic acidAll trademarks are recognised...comparative separations may not be representative of all applications

Selectivity Changes by Changing Organic Modifier10,12mAU0 100 2005768,11916ACE C18‐AR13 1415MeOH/0.1% HCOOHmAU0 100 2004,569ACE C18‐PFP10,12,13157,8 11 1614MeOH/0.1% HCOOH3.0 4.0 5.0 6.05) benzoic acid, 6) myrecetin, 7) p‐cresol, 8) propranolol, 9) ethylparaben, 10) furosemide, 11) anisole,12) 1,3,5‐trinitrobenzene, 13) toluene, 14) nimesulide, 15) mefenamic acid, 16) 1,2,3‐trichlorobenzene10ACE C18‐AR4.0 5.04) phenacetin, 5) p‐cresol, 6) acetophenone, 7) quercetin, 8) carvedilol, 9) 1,3‐dinitrobenzene,10) tolmetin 11) 3,4‐dichlorobenzoic acid, 12) ethyl benzoate 13) toluene 14) ibuprofen 15)indomethacin, 16) 1,2‐dichlorobenzene4ACE C18‐PFP0 100 2005678 9 111213141516ACN/0.1% HCOOHmAU0 100 2005678910 1113 141215162.1 x 50 mm, 3µm columnsACN/0.1% HCOOH3.0 4.0 5.03.0 4.0 5.0

Gradient Column Phase Screening:Optimum selectivity + UHPLC efficiencyAmbien Tablet, H 2O 2degradedTrazodone Tablet, H 2O 2degradedmAU0 2 4ACE Excel C18mAU0 2 418 8ACE Excel C18# Peaks4.0 5.0 6.0 7.0 8.0Time (min)4.0 5.0 6.0 7.0 8.0Time (min)mAU0 2 418ACE Excel C18‐ARmAU0 2 414ACE Excel C18‐AR4.0 5.0 6.0 7.0 8.0Time (min)mAU0 2 414ACE Excel C18‐PFPmAU0 2 44.0 5.0 6.0 7.0 8.0Time (min)Agilent 1200SL 600 bar systemoptimized ECV, 2 µL flow cell8ACE Excel C18‐PFP4.0 5.0 6.0 7.0 8.0Time (min)4.0 5.0 6.0 7.0 8.0Time (min)0–80% ACN/0.1% HCOOH, 10 mM NH 4 COO, 12‐minutes

Selectivity Changes by Changing Organic Modifier10,12mAU0 100 2005768,11916ACE C18‐AR13 1415MeOH/0.1% HCOOHmAU0 100 2004,569ACE C18‐PFP10,12,13157,8 11 1614MeOH/0.1% HCOOH3.0 4.0 5.0 6.05) benzoic acid, 6) myrecetin, 7) p‐cresol, 8) propranolol, 9) ethylparaben, 10) furosemide, 11) anisole,12) 1,3,5‐trinitrobenzene, 13) toluene, 14) nimesulide, 15) mefenamic acid, 16) 1,2,3‐trichlorobenzene10ACE C18‐AR4.0 5.04) phenacetin, 5) p‐cresol, 6) acetophenone, 7) quercetin, 8) carvedilol, 9) 1,3‐dinitrobenzene,10) tolmetin 11) 3,4‐dichlorobenzoic acid, 12) ethyl benzoate 13) toluene 14) ibuprofen 15)indomethacin, 16) 1,2‐dichlorobenzene4ACE C18‐PFP0 100 2005678 9 111213141516ACN/0.1% HCOOHmAU0 100 2005678910 1113 141215162.1 x 50 mm, 3µm columnsACN/0.1% HCOOH3.0 4.0 5.03.0 4.0 5.0

®ACEHPLC / UHPLC ColumnsACE ® Excel C18-PFP Selectivity & Throughput (Isocratic)38Aim: obtain R s≥ 1.7 in shortest possible time for mixtureWaters XBridge 5µm C18150 x 4.6 mm1.00 ml/min163 barR s= 1.71 23HPLC: 5µm C1813.6 mins(L/d p= 3)413.60 minWaters Acquity 1.7µm BEH C1850 x 2.1 mm0.21 ml/min246 bar3R s= 1.11 24.69 min4UPLC: < 2µm C184.75 mins(L/d p= 2.9)To maintain R s andreduce run time,keep L / dp ratioconstant~ x23QuickerACE Excel 2μm C18-PFP30 x 2.1 mm1.30 ml/min492 barUHPLC: ACE Excel 2µm C18‐PFP< 1 min2 4 6 8 10 12 14 16min1R s = 3.4230.65 min4R s= 1.9Using UHPLC and selectivity, itis possible to dramatically improveresolution allowing shorter columns& increased flow ratesSample: 1) 1,2,dimethoxybenzene, 2) 1,3‐dimethoxybenzene, 3) 1,3,5‐trimethoxybenzene, 4) toluene (reference).Mobile phase 50:50 MeOH / H 2 O; Temperature 40°C; 254 nmAll trademarks are recognised...comparative separations may not be representative of all applications

®ACEHPLC / UHPLC ColumnsACE ® Excel C18-PFP Selectivity & Throughput (Gradient)39Aim: obtain R s≥ 1.7 in shortest possible time for mixtureACE 5µm C18100 x 4.6 mm1 ml/min, t G= 29 minmax pressure: 92 bar40 min cycle timeR s= 1.81325476, 22.49 minHPLC: 5µm C1822.49 minsACE Excel 2µm C1850 x 2.1 mm0.6 ml/min, t G= 5 minmax pressure: 367 bar9 min cycle timeACE Excel 2 μm C18-PFP30 x 2.1 mm2.5 ml/min, tG = 0.7 minmax pressure: 914 bar1 min cycle time00R s = 1.80R s= 1.51327, 0.61 minR s = 1.912345R s= 1.760.5 15 42.5 57R s = 1.7UHPLC: 2µm C18‐PFP0.61 mins6, 4.17 minUHPLC: 2µm C184.17 mins1.5 2 2.5 3 3.5 425~ x25QuickerminR s ≥ 1.705 10 15 20 251, aspirin; 2, phenacetin; 3, 1,3‐dinitrobenzene ; 4, ethyl benzoate; 5, nimesulide; 6, ibuprofen; 7, indomethacin.min

®ACEHPLC / UHPLC Columns40Pressure EffectsHPLC ↔ UHPLC

®ACEBackgroundHPLC / UHPLC Columns41Pressure is a complex physical parameter that affectsmany elements of a chromatography systemChromatographic selectivity and retention changes atelevated pressures have been investigated and reported aObservations are highly dependent upon the analytesand may be seen with any manufacturer phases operatedunder UHPLC conditionsChanges are typically not helpful for HPLC ↔ UHPLCactivitiesa Fallas et al. / J. Chromatogr. A 1209 (2008) 195–205

®ACEHPLC / UHPLC ColumnsEffect of Pressure on Selectivity and Retention Factor42Initial 2µm and 3µm data aresimilar (A, B)Scalability looks goodAgilent 1290, 50 x 2.1 mm (constant flow and restrictor capillary used)Mobile phase: A=0.1% FA in water: B=0.1% FA in MeOH (51:49 v/v)Flow Rate: 0.21 ml/min, Temperature: 40 °CK= Ketoprofen; S= Sulindac; N=Naproxena Fallas et al. / J. Chromatogr. A 1209 (2008) 195–205

®ACEHPLC / UHPLC ColumnsEffect of Pressure on Selectivity and Retention Factor43Initial 2µm and 3µm data aresimilar (A, B)Scalability looks goodRetention and selectivity seento change with pressure (BE)Agilent 1290, 50 x 2.1 mm (constant flow and restrictor capillary used)Mobile phase: A=0.1% FA in water: B=0.1% FA in MeOH (51:49 v/v)Flow Rate: 0.21 ml/min, Temperature: 40 °CK= Ketoprofen; S= Sulindac; N=Naproxena Fallas et al. / J. Chromatogr. A 1209 (2008) 195–205

®ACEHPLC / UHPLC ColumnsSummary: Unwanted Selectivity Changes44Pressure induced k and changes may be seen forany manufacturer phases under UHPLC conditionsChanges in selectivity and retention may be significantwith ionised analytes and large MW analytes a , but theimpact on neutral molecules is typically smallerCurrent discussions / theory focus on changes inanalyte molar volume as the principle cause forchanges in k and observedSuccessful HPLC ↔ UHPLC possible...the analyst justneeds to be vigilanta Fallas et al. / J. Chromatogr. A 1209 (2008) 195–205

®ACEHPLC / UHPLC Columns45Connections : Losses in N and A sPeak Dispersion

®ACEBackgroundHPLC / UHPLC Columns46UHPLC / optimised HPLC instruments are very sensitiveto the introduction of extra column volumeAny time you install a column (from any manufacturer) itis vital to ensure good connectionsAim for a ‘fresh connection’ every time to ensure a snugfit between tubing and column and reduce the likelihoodof an unwanted gap and / or tubing slippageFree movement of the ferrule and nut when installing thecolumn gives you a fresh connection

®ACEHPLC / UHPLC ColumnsLosses in Performance Due to Incorrect Column Fitting47mAUCorrectly fitted column300 1.5363.147 5.6658.22625020015010050N 5%h= 12,800 platesPW 5%h= 0.291 minA s= 0.94Correctly fittedcolumns make themost of your columnand system02 4 6 8 10minIncorrectly fitted columnmAU300 1.5373.160 5.7132502001501005008.3132 4 6 8 10< ~ 0.5mm gapN 5%h= 9,850 platesPW 5%h= 0.335 minA s= 0.84min Incorrectly connectedcolumns lead toreduced efficiency,reduced peaksymmetry, andpossibly, leaksLoss of ~23% for NLoss of ~11% for A s

®ACEHPLC / UHPLC ColumnsSummary: Column Connections48Extra column volume reduces peakefficiency and increases asymmetryMake a fresh connection every timeyou install any columnACE recommends reusable fittingsfor a fresh connection every timeAll ACE ® Excel TM columns have aFREE ‘Making Great UHPLCConnections’ leaflet in every box

®ACEHPLC / UHPLC ColumnsMaking Good UHPLC Column ConnectionsAlso downloadable from the ACE website:www.ace-hplc.com

®ACEHPLC / UHPLC ColumnsOverall Summary & Conclusions50Understanding the properties of building blocks in stationaryphase design led to these unique ACE ® productsACE ® C18-AR and ACE ® C18-PFP are powerful tools for methoddevelopment due to unique but complementary selectivitiesThese unique phases are available for HPLC as the ACE ® rangeand also for UHPLC as the NEW ACE ® Excel 2 µm formatThese phases meet analyst demands of reproducibility,robustness & low phase bleed with excellent peak efficiencyOperating at high pressures can deliver excellent results butremain vigilant - selectivity and retention may be affected...andeven column connections become critical!

®ACEHPLC / UHPLC ColumnsFull Information On All ACE Products Available51Unique SelectivitiesFree GuidesMACMOD Analytical = http://www.mac-mod.com/ACT = http://www.ace-hplc.com

Thank You For Your Attentiontwaeghe@mac-mod.cominfo@mac-mod.comwww.mac-mod.comACE ® is a registered trademark of Advanced Chromatography Technologies Ltd. ACE Excel and HSC are trademarks of Advanced Chromatography Technologies Ltd.UPLC, Xbridge, ACQUITY are trademarks of Waters Corporation; ZORBAX, Eclipse are trademarks of Agilent Technologies Inc.; Kinetex is a trademark of Phenomenex Inc.; GOLD is a trademark of Thermo Fisher Scientific.