Trends in Method Development for HPLC and UHPLC... the movement toward using solid-core particles Richard A. Henry, Separation Technology Consultant On behalf of: Supelco/Sigma-Aldrich, Bellefonte, USA www.sigma-aldrich.com rhenry@psualum.com
Fused-Core Milestones- Pioneering the Particles (2007) First 2.7µm particles- achieve efficiencies >250,000 N/m (1) Ruggedness proven to hold up extremely well in use Efficiencies comparable to sub-2µm particles Pressure drop (flow resistance) comparable to 3µm particle columns Allows use of traditional 400 bar instruments Narrow UHPLC peaks reveal limitations of instrument dispersion Both 90Å for small molecules and 160Å for larger molecules available (2012) First 5µm particles- achieve efficiencies >150,000 N/m (2) Operate at low pressures with unsurpassed ruggedness. Efficiencies exceed most 3µm particles (150,000 N/m observed routinely at low pressure) Pressure drop of 5µm particle columns Designed for traditional instruments & routine methods. C18 & F5 available now; all standard phases available by Nov 2012 Trademark: Fused-Core is a registered trademark of Advanced Materials Technology, Inc. 2
FASTER HPLC ON ANY SYSTEM Fused-Core Particles More plates with less backpressure 2.7µm replaces 1.7-3µm 5µm replaces 3-5µm 2.7 µm Fused Core 5 µm Fused Core Fused-core 1.7 µm 2.5 µm 3.0 µm 5.0 µm Higher Backpressure 3
Narrower Particle Distribution of Fused-Core 16 2.7um core shell 1.7um fully porous N u m b e r C o u n t 14 12 10 8 6 Particle samples taken from columns 3.0um fully porous 5.0um fully porous 5.0um core shell Fused-Core forms efficient, uniform beds Stable bed resists voiding ( % ) 4 2 0 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 Particle Size (um) 4
More Efficiency with Ascentis Express Columns 20.00 18.00 16.00 Fused Core 2.7um Porous 3 um Fused Core 5um Porous 5 um Column: C18 150 x 4.6mm Mobile Phase: 60% Acetonitrile Temperature: 35ºC Sample: 10 LToluene 14.00 12.00 5µm porous H 10.00 8.00 3µm porous 5µm Fused-Core N max = 25,000 (167K/m) 6.00 4.00 2.00 N = L/H 2.7µm Fused-Core N max = 37,500 (250K/m) 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 flow (ml/min) Fused-Core Outperforms Porous Particles Trademark: Ascentis is a registered trademark of Sigma-Aldrich Co. LLC 5
Pressure Drop* with Ascentis Express Compares to Same Size Porous Columns 12000 Fused Core 2.7um 10000 Porous 3 um Fused Core 5um Porous 5 um Fused-Core 2.7µm Pressure (PSI) 8000 6000 4000 Dionex UHPLC Column: 150 x 4.6mm Mobile Phase: 60% Acetonitrile Temperature: 35ºC Sample: 10 LToluene Porous 3µm Fused-Core 5µm Porous 5µm 2000 0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 flow (ml/min) * Pressure includes instrument flow resistance 6
Particle Properties and Performance (150x4.6mm) Particle Type Shell thickness (µm) BET Surface Area (m 2 /g) Average Pore Diameter (Å) Plates* Pressure* (bar) 2.7 µm Ascentis Express Fused Core 2.7 µm Ascentis Express Peptide Fused Core 5 µm Ascentis Express Fused Core 0.5 135 90 38300 284 0.5 80 160 38300 284 0.6 90 90 28300 78 3 µm porous N/A 300 100 24200 309 5 µm porous A N/A 300 100 14600 100 Ascentis Express 5µm: 3µm efficiency with 5µm pressure 5 µm porous B N/A 170 120 14400 63 5 µm porous C N/A 450 100 15300 120 Fused-Core Outperforms Porous Particles * Plates measured at N max 7
Achieve 3µm Resolution with 5µm Fused-Core 2.000 2.7µm Fused-core 1.800 1.600 3µm Totally Porous Resolution 1.400 1.200 1.000 5µm Fused-core 5µm Totally Porous 0.800 0.600 N max flow lines C18 150 x 4.6mm 0.400 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Flow, ml/min 8
Ascentis Express 5µm: High Resolution and Speed 1.0 ml/min 80 bar 0 2 4 6 8 10 Time (min) Flow Plates Pressure ml/min psi 1.0 2.0 3.0 4.0 Time (min) 3.0 ml/min 250 bar 2.0 ml/min 160 bar 1.0 23,400 1200 1.5 23,500 1825 2.0 22,400 2450 2.5 20,800 3100 3.0 19,500 3750 1.0 2.0 3.0 4.0 Time (min) Ascentis Express C18 5 μm, 15 cm x 4.6 mm ID column.. Mobile phase: 50:50 water:acetonitrile. Test mixture: 5µL of uracil, acetophenone, benzene, toluene and naphthalene Agilent 1100, 35C, UV@250nm. 9
Achieve High Flow Rates with 400 bar Instruments* Porous 2 µm (estimated) Fused Core 2.7µm Porous 3.0 µm Fused- Core 5µm Porous 5.0 µm * Low instrument flow resistance 10
Ascentis Express 5 µm: Tools for 400 bar Instruments Column Pressure 40% Acetonitrile Column Pressure 60% Methanol ml/min: ml/min: ID L 0.5 1.0 1.5 2.0 2.5 3.0 ID L 0.5 1.0 1.5 2.0 2.5 3.0 4.6 250 56 112 168 220 272 321 4.6 250 94 187 276 362 >400 4.6 150 34 69 103 136 167 198 4.6 150 58 114 169 222 272 342 4.6 50 12 25 38 50 63 75 4.6 50 21 42 63 82 102 117 <80% of 400 bar pressure maximum 80-100% of 400 bar pressure maximum >100% of 400 bar pressure maximum Backpressure units are listed in bar (1 bar = 14.5 psi) Values reflect column backpressures only, instrument backpressures have been subtracted 11
Ascentis Express 5µm: Highest Plates/Pressure 360 360 135 80 5 µm Ascentis Express columns with Fused-Core particles show higher plates/pressure than 5 µm totally porous particles and much higher 40 plates/pressure than 3 µm totally porous particles Data collected on 150 x 4.6mm columns at the plate height minimum 12
Porous 5µm vs Ascentis Express 5µm Columns Agilent 1100, 35C, UV@250nm. Mobile phase: 50:50 water:acetonitrile. Test mixture: 5µL of uracil, acetophenone, benzene, toluene and naphthalene Popular C18 5 μm, 15 cm x 4.6 mm ID 1.0 ml/min, N=13,633 for last peak, 72 Bar 0 10 20 Time (min) Ascentis Express C18 5 μm, 15 cm x 4.6 mm ID 1.0 ml/min, N=23,422 for last peak, 80 Bar Fused-Core Particles have more plates/pressure than same-size porous particles 0 2 4 6 8 10 Time (min) 13
Ascentis Express: Highest Performing 5µm Column 1 2 3 4 5 Ascentis Express C18 5µm 38856 plates 155424 plates/meter 2380 psi (164 bar) k 4 = 2.3 1 2 3 4 Competitor 1 C18 5µm 28812 plates 115248 plates/meter 2020 psi (139 bar) k 4 = 3.2 All columns are 25cm x 4.6mm 5 0 10 20 Time (min) 1 2 3 4 5 Discovery C18 5µm 23120 plates 92480 plates/meter 1870 psi (129 bar) k 4 = 1.8 0 10 20 Time (min) 1 2 3 Competitor 2 C18 5µm 24479 plates 97916 plates/meter 1850 psi (128 bar) k 4 = 5.9 4 5 0 10 20 Time (min) 0 10 20 Time (min) 14
Comparable Loading Capacity: Fused-Core vs Porous Ascentis Express Columns 35000 30000 25000 2.7 µm Fused Core, 0.5 µm shell 1 Cl 4 Nitrobenzene Loading Columns: 4.6 x 150 mm Temperature: 30 C Mobile phase: 50/50 acetonitrile/water Plate Number 20000 15000 5 µm Fused Core, 0.6 µm shell 5 µm Totally porous 10000 5000 0.01 0.1 1 10 100 µg of 1 Cl 4 Nitrobenzene Fused-core particles: solute loading competitive with totally porous particles Particle size does not affect loading; note difference in column efficiency (Y axis) 15
Ascentis Express 5 µm Columns are Highly Stable 10000 Column: 4.6 x 50 mm of 5 µm HALO C18 fused-core Instrument: Shimadzu Prominence UFLC XR Flow rate: 1.8 ml/min, Injection Volume: 1 µl Mobile Phase: 50% ACN/50% water/0.1% TFA Temperature = 60 C 9000 8000 N 7000 6000 5000 naphthalene 1 chloro 4 nitrobenzene No performance change after 450 injections and about 30,000 column volumes of mobile phase. 4000 0 5000 10000 15000 20000 25000 30000 Column Volumes 16
Summary of Fused-Core Particles Features 2.7µm Fused-Core Replace sub-2µm and 3µm in fast assays & method development Compatible with optimized HPLC or UHPLC instruments Delivers about 250,000 N/m; uniform, rugged bed Efficiency plot flat like sub-2um columns for very fast separations Low pressure drop like 3µm particles 5µm Fused-Core Replace 3µm or 5µm particles in routine assays Compatible with optimized HPLC instruments Delivers about 150,000 N/m; uniform, rugged bed Efficiency plot flat like 3µm particles for fast separations Low pressure drop like 5µm particles Fused-Core Outperforms Porous Particles 17
Ascentis Express Fused-Core HPLC Phases 5µm Fused- Core Phases C18 C8 RPA Phenyl-Hexyl Cyano F5 5 HILIC 2.7µm Fused- Core Phases C18 Peptide ES-C18 C8 RP-Amide Phenyl-Hexyl ES-Cyano F5 5 HILIC (silica) Equivalency expected between particle phases 3 Nonpolar only (RP) 4 Medium to high polarity (enhanced selectivity RP) 2 Polar only (NP/ANP/HILIC) Phases in Green available now Phases in Red available in Nov 2012 Phases in Blue available soon 18
Column Selectivity
Resolution is Still Dominated by Selectivity (α) R s = N k k+1 4 Efficiency (new particles) α-1 α Retention (always critical) Selectivity (still dominates R S ) Resolution (R) 3.0 2.5 2.0 1.5 1.0 N k Efficiency equations: H = A + B/u + Cu + H IBW h = H/d P N = L/H 0.5 0.0 1.00 1.05 1.10 1.15 1.20 1.25 0 5K 10K 15K 20K 25K 0 5 10 15 20 25 * Yun Mao, PhD Dissertation (Peter Carr), University of Minnesota, 2001. N k 20
HPLC Selectivity Variables (Reversed-Phase)* Continuous variables (solvent): Solvent type: usually ACN or Me in water) ph Solvent strength (% organic) Additive type and concentration Temperature Discontinuous variable (column): Phase type and substrate Should analysts tweak the mobile phase with C18 or C8 or try different phases? Largest impact on selectivity is created by changing phase type, organic solvent type and ph (for acids and bases). * John Dolan, LC Resources, MCF 2009. 21
Alkyl Bonded Phase (C18 and C8) C18 reagents are large and can leave some silanols unreacted. C8 reagents are smaller and provide better silanol coverage. Primary phase reagent (very hydrophobic) R At ph >4, silanols can ionize and add cation-exchange character or anion repulsion. Nonpolar forces dominate retention. Endcap reagent Red = Hydrophobic Blue = Hydrophilic Free silanol Si O Hydrophobic selectivity O Si 22
Pentafluorophenyl (F5) Bonded Phase F5 is a Lewis acid or electron acceptor; π-π interaction can occur with solutes that are rich in electrons (Lewis bases). Dipolar interactions can occur along the ring edge. Due to aromatic rigidity, solute shape can determine selectivity. Solute retention can be dominated by either nonpolar or polar forces (multi-mode). Red = Hydrophobic Blue = Hydrophilic Free silanol π acid and strong dipole Endcap reagent Si O F F F F O Si Primary or main phase reagent 23
F5 is a Versatile and Highly Selective Phase PFP characteristics Both hydrophilic and hydrophobic character Dipole-dipole selectivity - interactions- attracts Pi donors (electron rich) Charge-transfer selectivity Ionic interactions Rigidity shape discrimination F F F Si O Si F F 24
Apparent Shape Selectivity with Ascentis Express F5 Peak id. 1,2 HO CH 3 H CH 3 O O CH 3 H O CH 3 1. Hydrocortisone 2. Prednisolone 3. Prednisone 3 H H O Hydrocortisone [*{BAN}; *{INN}; *{JAN}] Monoisotopic Mass = 362.209324 Da O CH 3 HO O H H Prednisone [*{BAN}; *{INN}] Monoisotopic Mass = 358.178024 Da Ascentis Express C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Time (min) 1 2 3 CH 3 H H H Mobile Phase: water:methanol (50:50, v/v) O Prednisolone [*{BAN}; *{INN}; *{JAN}] Monoisotopic Mass = 360.193674 Da Ascentis Express F5 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Time (min) 25
Selectivity Test Mix Results for C18 and F5 Columns* C18 and F5 cover different regions of selectivity space 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Ascentic Express C18 5µm Ascentic Express F5 5µm Ascentic Express C18 * Selectivity test conditions developed by Euerby (6) 26
RP Column Classification by Chemical Interaction Bonded Phase Hydrophobic H-Bonding Dipolar π-π Steric b Ion- Exchange c C18 Very Strong Weak No No No Weak C8 Strong Weak No No No Weak Amide Strong Strong Acceptor Moderate No Weak Very weak Phenyl Strong Weak Acceptor Weak Strong Donor Strong (Rigid) Weak Cyano Moderate Weak Acceptor Strong Weak No Weak PFP (F5) Moderate Moderate Acceptor Strong Strong Acceptor Strong (Rigid) Strong 27
New 5 Silica Bonded Phase for HILIC Solute retention dominated by polar forces (HILIC and Normal Phase modes). Selectivity similar to bare silica but ionization of surface silanols is suppressed. Retention is more related to partition and less related to ionexchange. Primary or main phase reagent Red = Hydrophobic Blue = Hydrophilic Si Si Free silanol O O 28
Fused-Core Column Selection Guidelines Ascentis Express
Method Development and Transfer RP mode HILIC mode
RP vs HILIC Modes: Visualizing Phase Polarity organic-rich layer at nonpolar surface Si-O-Si-R aqueous-rich layer at polar surface Si- Si- Si-O-Si-R Bulk mobile phase higher in aqueous content Si- Si- Bulk mobile phase higher in organic content Si- ACN Water Si- Water ACN Si-O-Si-R Si- RP conditions Si- Si- ANP/HILIC conditions Si-O-Si-R Si- Polar analytes can partition into aqueous-enriched phase in ANP/HILIC mode and be retained easily. Potential for polar and ionic interactions with silica surface exists with both modes but is very strong in ANP/HILIC mode 31
Using Log P Values as a Guide to Mode Selection* Aqueous Normal Phase (ANP/HILIC) employs a polar column in high organic solvent Reversed-Phase (RP) employs a nonpolar column in high aqueous solvent Region of mode overlap and probable dual-mode retention HILIC mode Both RP mode When logp is zero or negative, RP mode may not be a good choice for C18 columns due to weak stationary phase attraction. 32
HPLC Method Development Approaches New methods: select RP or HILIC mode based on RP gradient screening with C18 column (5-95% ACN). Solutes eluting at or near void volume indicate HILIC mode. Use logp values if sample structures are known. Methods can be easily transferred between 2.7µm and 5µm Fused-Core particles having same phase to maximize performance or minimize pressure. Existing methods: transfer to Fused-Core columns requires use of same phase to meet USP requirement (7); column equivalency must be shown. 33
System Suitability- USP Method Transfer Guidelines* Verify that system is adequate for the intended analysis Column Stationary phase- stay within L designation specified in USP monograph Particle size- decrease by 50% permitted; no increase permitted Column length- change by +/- 70% permitted Column inside diameter (ID)- maintain same linear velocity Mobile Phase: Composition- change in binary fraction by maximum of 10% permitted Flow rate- change of +/- 50% permitted for same column ID Gradient- adjustments to composition not recommended (?) Other variables (ph, temperature, etc.): see USP reference (7) * USPC Official Methods 2012, Section 621, Chromatography (7). 34
NSAIDs on C18 Columns with Different Silica Columns: 4.6 x 150 mm Instrument: Shimadzu Prominence UFLC XR Flow rate: 2.0 ml/min, Injection Volume: 2 µl, Detection: 254 nm; Temperature = 35 C Mobile Phase: A: 20 mm ph 2.5 Potassium Phosphate Same selectivity B: 50/50 ACN/Me; A:B = 48% A:52% B as retentive C18 Ascentis Express 5µm Pressure 240 bar N = 20,500 Porous 5µm Pressure 215 bar 0 2 4 6 More sensitivity Peak Identities (in order) 1. Acetaminophen 2. Aspirin 3. Salicylic acid 4. Tolmetin 5. Ketoprofen 6. Naproxen 7. Fenoprofen 8. Diclofenac 9. Ibuprofen N = 11,000 8 10 12 14 16 18 Time, min. 35
Taxols on F5 Columns with Different Silica mobile phase: (A) Water; (B) ACN; (60:40) flow rate: 1.5 ml/min Porous F5 5 µm 1 2 3 4 5 2690 psi 6 1 2 Porous F5 3 µm 4160 psi 3 4 5 6 1 2 Ascentis Express F5 5 µm 2720 psi 3 4 5 6 0 10 20 Time (min) 36
Ginsenosides with Isoeluotropic Conditions (same k) Porous C18 3µm 1 2 3 4 5 6 7 Initial 29% ACN, 2130 psi Ascentis Express C18 5 µm 1 2 3 4 5 6 7 Initial 25% ACN, 1470 psi Ascentis Express C18 2.7 µm Initial 27% ACN, 2700 psi 37
Catechins in Green Tea with C18 (USP Method) mau 0 20 HO O O HO O Epicatechin Gallate Ascentis Express C18 5µm 25cm x 4.6mm Peak Capacity = 308 0 10 20 30 40 50 60 70 80 90 Time (min) mau 0 20 Porous C18 5µm 25cm x 4.6mm Peak Capacity = 191 Ascentis Express 5µm shows more efficiency, peak capacity and sensitivity P C T G /P W 0 10 20 30 40 50 60 70 80 90 Time (min) 38
Faster Catechin Method on Ascentis Express C18 5um mau 0 200 400 15 cm x 4.6mm column at 1.2 ml/min (with gradient correction) Pressure=2262 psi mau 0 20 0 10 20 30 Time (min) 1 2 3,4 5 6 Reduction in run time from 90 min to 30 min while staying within USP guidelines and improving sensitivity 0 10 20 30 Time (min) 39
Nucleoside Experimental Conditions (HILIC) column: Ascentis Express 5, 10 cm x 2.1 mm, 2.7 µm (53757-U) mobile phase A: 5 mm ammonium acetate, ph 5.0 with acetic acid in 95:5, acetonitrile:water mobile phase B: 5 mm ammonium acetate, ph 5.0 with acetic acid in 80:20, acetonitrile:water Time %A %B 0 100 0 1 100 0 11 0 100 13 0 100 flow rate: 0.3 ml/min temp.: as listed det.: UV at 250 nm injection: 1 µl sample: 10 100 µg/ml in 95:5, acetonitrile:water 40
Nucleosides with Ascentis Express 5 (HILIC) No. Name tr 1 Ribothymidine 2.75 2 Uridine 3.07 3 2-Thiocytidine 4.62 4 2'-O-Methylcytidine 5.28 5 Pseudouridine 6.08 6 Inosine 6.50 7 5-Methylcytidine 7.53 8 Cytidine 7.79 9 Guanosine 8.60 10 3-Methylcytidine 9.02 11 1-Methyladenosine 10.29 12 7-Methylguanosine 11.11 Gradient: 5-20% water in acetonitrile Starting pressure: 1250 psi 1 2 3 4 5 Uridine logp -2 7 8 9 6 10 11 12 0 2 4 6 8 10 12 Time (min) 41
References and Acknowledgements 1. J. J. DeStefano, S. A. Schuster, J. M. Lawhorn, and J. J. Kirkland, Performance Characteristics of New Superficially Porous Particles, J. Chromatogr. A (2012), Accepted for Publication. 2. J. J. Kirkland, T. J. Langlois and J. J. DeStefano, Fused-Core Particles for HPLC Columns, American Laboratory, 39 (February 2007), 18-21. 3. G. Desmet, et. al., J. Chromatogr. A, 1161 (2007) 224-233. 4. G. Desmet, et. al., J. Chromatogr. A, 1217 (2010) 7074-7081. 5. F. Gritti and G. Guiochon, J. Chromatogr. A, 1228 (2012) 2-19. 6. M. R. Euerby and P. Petersson, J. Chromatogr. A, 994 (2003) 13 36. 7. United States Pharmacopeia 35, Section 621 Chromatography, United States Pharmacopeial Convention Inc., Rockville, Maryland, USA (2012).. Fused-Core performance data supplied by AMT, Inc., Wilmington, DE Other data supplied by Sigma-Supelco Applications Lab personnel CHROMASOLV is a registered trademark of Sigma-Aldrich Co. LLC Fused-Core is a registered trademark of Advanced Materials Technology, Inc. 42
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