Fused-Core TM Particle Columns provide UPLC performance on any PLC Major Contributors and Co-Authors. Bell, W. Campbell, W. Way, K. Buckendahl Supelco a ivision of Sigma Aldrich
Content Influence on Resolution & Van eemter Curve PLC Evolution of particle design Fused-Core TM particle Technology Column performance - System compatibility (pressure requirements) - Comparison to Sub 2µm and 3µm materials 2 Fused-Core is a trademark of Advanced Materials Technology Inc. Ascentis is a trademark of the Sigma-Aldrich Corp.
Resolution in PLC Key Influence Factors 3 Efficiency N α k R= N 4 Retention k k +1.. Selectivity α-1 α - Increase column length - ecrease particle size (N ~ 1/d p ) - Change stationary phase - Change mobile phase solvent - Change buffer p - Increase (weaker solvent) - ecrease (stronger solvent) Resolution (R) Zhao, J.. and P.W. Carr. Analytical Chemistry, 1999. 71(14): p. 2623-2632 3.0 2.5 2.0 1.5 1.0 0.5 α 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
van eemter-kurve: = A + B/u + Cu B = 2γ m C = (f 1 d P2 +f 2 d f2 ) A = 2λd P 4 u
Evolution of PLC Column Particle esign Irregular ifficult to pack, easily clogged and not very rugged Pellicular Thin porous layer showed low capacity Totally Porous Current PLC workhorse Fused-Core tm The future of PLC columns? 5 Fused-Core is a trademark of Advanced Materials Technology, Inc.
Pressure & Efficiency are reciprocal to the particle size Influence of d P on P >> N Effiiciency bar 6 30,000 25,000 20,000 15,000 10,000 5,000 0 400 350 300 250 200 150 100 50 0 0 5 10 15 20 0 5 10 15 20 d p (μm) N P 1 2 d p 1 d p 2x efficiency 4x higher pressure Smaller particles are not the simplest solution and have draw backs. d P (µm) psi Bar N 1.8 5889 406 27,500 2.5 3089 213 20,000 3 2118 146 16,500 5 769 53 10,000 10 189 13 5,000 15 87 6 3,750 20 44 3 2,500 10 cm column, 3 mm/s linear flow ow much separation power do you have commonly on your PLC- System?
Fused Core-Technology igher Efficiency 1 2 4 3 Agilent 1200 Ascentis Express C18, 15cm x 4.6mm, 2.7 µm 1.0mL/min, 254nm, RT, 10µL inj 4. Toluol N = 30,738 3. Benzol N = 31,696 2. Acetophenon N = 33,786 1. Uracil (dead time marker) Pressure = 183 bar (2690 psi) 0 1 2 3 4 min 7
Ascentis Express Fused-Core Particle Technology Fused-Core particle technology invented by Jack Kirkland Porous silica layer fused to solid core 0.5 µm Ascentis Express PLC columns use this newly engineered particle 2.7 µm 1.7 µm 2.7 micron Silica particle 1.7 micron solid core 0.5 micron porous shell C18 and C8 phases available at launch (RP-Amide later in 2007) 8
Fused Core Technology Reducing dispersion (increasing efficiency) A solid core at the center of a particle, the potential diffusion path length of an analyte molecule is effectively shortened A larger particle then can theoretically generate similar efficiency of a smaller totally porous particle without generating high backpressures 9
Fused-Core Vs. Totally Porous: Improved Peak Shape 0.5 µm Migration Path In Particle 1.5 µm 2.7 µm 1.7 µm 10 Shorter diffusion path reduces axial dispersion and minimizes peak broadening
System Compatibility Comparison of Pressure for ifferent Particles = A + B/u + Cu ΔP = 1000Fη ΔP = L t o πr 2 d 2 p 16,000 35.00 16,000 14,000 1.7 µm 30.00 12,000 ETP (μm) 25.00 20.00 15.00 Pressure (psi) 10,000 8,000 6,000 2.7 µm FC 10.00 4,000 3 µm 5.00 2.7 µm Ascentis Express 2,000 1 2 3 4 5 Mobile Phase Velocity (mm/sec) 2 4 6 8 10 12 Mobile Phase Velocity (mm/sec) 11
van eemter Ascentis Express 2.7 µm Vs. 3 µm Small Molecule Study: Naphthalene: M.W. = 130 8.00 Naphthalene 7.00 h = / d p h 6.00 5.00 4.00 3.00 Acentis C8 3 µm Ascentis Express C18 2.7 µm Ascentis Express C8 2.7 µm 2.00 1.00 12 Reduced plate hight to compare columns with diff. Particle sizes 0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 v (mm/s) Columns: 50 x 4.6 mm Mobile phase: 55/45 ACN/Water Sample: Naphthalene (20 µg/ml), dissolved in 55/45 ACN/water Injection: 5 µl etection: 254 nm Temperature: 30 C Instrument: Agilent 1100.
Smaller Particles Exponentially Increase Pressure Pressure rop Vs. Particle Size bar 450 400 350 300 250 200 150 10 0 50 0 1.8 µm ~ 400 bar (6,000 psi) 3 µm ~ 150 bar (2,250 psi) 5 μm ~ 50 bar (750 psi) 0 5 10 15 2 0 2 5 d p (μm) 13 10 cm column, 3 mm/s linear velocity
Frit Requirements for PLC columns Column Work horse igher Efficiency Particle size (μm) Frit size (μm) 5 2 3 0.5 UPLC 1.6-2.0 0.2 Ascentis Express 2.7 2 Ascentis Express 3µm Porus material Ascentis Express narrow particle distribution allows for 2 μm-frits Like 5 μm-columns Ascentis Express-Columns can efficiently be protected by 0.5 μm-inline-filter. 14
igh efinition () Resolution Compared to 5µm porus particle column Sub 2 µm columns can provide up to 1.55 x increase in resolution for an 8 fold increase in pressure Ascentis Express Fused-Core (FC) columns can provide 1.55 x increase in resolution for only a 4 fold increase in pressure dp N Rs factor* Pressure (psi) Pressure (bar) 5μm 10.000 1 750 50 3μm 14.000 1,18 2.250 150 2.7μm FC 24.000 1,55 2.800 190 1.8μm 24.000 1,55 6.000 400 15 10cm columns * RS normalised to 5µm column
Twice the Efficiency at the System Limit 2.7 µm Fused-Core Vs. 3 µm Totally Porous Particles Ascentis Express C18 2.7 µm 65 % ACN N = 237,740 p/m or N = 35,661 p/col Psi (approx 4,000) 1 2 3 4 Efficiency Comparison / Columns: 150 x 4.6 mm Mobile Phase: Acetonitrile / Water Flow: 1.5 ml/min Injection: 2.0 μl etection: 220 nm Agilent 1100 PLC System 1. p-hydroxy ethylbenzene 2. Napthalene 3. p-xylene 4. Biphenyl 0 2 4 Ascentis C18, 3 µm 72.5 % ACN N = 140,631p/m or N = 21,095 p/col Psi (approx 4,000) 1 2 3 4 0 2 4 16 NB: Ascentis C18 and Ascentis Express C18 have different Surface Chemistries
mau 0 20 40 60 80 100 120 140 mau 0 20 40 60 80 100 120 140 17 TO11/IP6A Carbonyl-NP Mix Almost Twice the Sensitivity Ascentis Express C18, 2.7 µm Peak 8 N = 260,720 p/m N = 39,108 p/col 0 2 4 6 8 10 12 Ascentis C18, 3 µm Peak 8 N = 146,587p/m N = 21,988p/col 8 0 2 4 6 8 10 12 8 Sensitivity Gap Column: 150 x 4.6 mm I.. Mobile phase: Ascentis Express C18 2.7 µm: 25:75, water: acetonitrile Ascentis C18 3 µm: 30:70, water: acetonitrile Flow rate: 1.0 ml/min. Temp.: 30 C et.: UV at 365 nm Injection: 1 µl Sample: 47285-U TO11/IP6A Carbonyl-NP Mix as indicated below in 40:60, water: acetonitrile Peak Is 1. Formaldehyde-2,4-NP (105 µg/ml) 2. Acetaldehyde-2,4- NP (76.4 µg/ml) 3. Acrolein-2,4- NP (63.2 µg/ml) 4. Acetone-2,4- NP (61.5 µg/ml) 5. Propionaldehyde-2,4- NP (61.5 µg/ml) 6. Crotonaldehyde-2,4- NP (53.6 µg/ml) 7. Butyraldehyde-2,4- NP (52.5 µg/ml) 8. Benzaldehyde-2,4- NP (40.5 µg/ml) 9. Isovaleraldehyde-2,4- NP (46.4 µg/ml) 10. Valeraldehyde-2,4- NP (46.4 µg/ml) 11. o-tolualdehyde-2,4- NP (37.5 µg/ml) 12. m-tolualdehyde-2,4- NP (37.5 µg/ml) 13. p-tolualdehyde-2,4- NP (37.5 µg/ml) 14. exaldehyde-2,4- NP (42 µg/ml) 15. 2,5-imethylbenzaldehyde-2,4- NP (35 µg/ml) NB: Ascentis C18 and Ascentis Express C18 have different Surface Chemistries
igher Sample Throughput on Traditional PLC Systems* 0 2 4 6 0 2 4 6 0 2 4 6 Conventional C18 25 cm x 4.6 mm I.., 5 μm 1.0 ml/min., N= 22147 Pressure: 128 bar (1880 psi) 0 2 4 6 0 2 4 6 0 2 4 6 0 10 20 30 40 Ascentis Express C18 10 cm x 4.6 mm I.., 2.7 μm 1.0 ml/min., N = 22694 Pressure: 167 bar (2450 psi) Method requirement was N >20.000 0 2 4 6 8 10 Ascentis Express C18 10 cm x 4.6 mm I.., 2.7 μm 1.5 ml/min., N = 21297 Pressure: 248 bar (3645 psi) *Agilent 1100 PLC System 18 0 2 4 6 8 10
igh Speed: Increased Flow Rate Ascentis Express Vs. sub-2 µm at System Pressure Limit Ascentis Express C18 0.3 ml/min 45 % acetone. 2130 psi N = 12,500 Sub-2 µm Column 2 0.3 ml/min 51 % acetone. 9500 psi N = 12,170 1 2 3 1.0 2.0 3.0 4.0 1 2 4 4 5 Ascentis Express C18 0.6 ml/min 45 % acetone. 4450 psi N = 8,500 1 2 4 Mobile Phase: water : acetonitrile; isoelutropic for β-estradiol Columns: 100 x 2.1 mm Flow: variable et: 200 nm Inj: 1µL Elution order: 1. Estriol 2. β-estradiol 3. Contaminant 4. Estrone 5. Estrone degradant 3 5 3 5 19 1.0 2.0 3.0 4.0 1.0 2.0 All column efficiencies have been compromised by system dispersion, however the pressure associated with the Ascentis Express column when run at twice the flow rate. It is still at only half that of the sub 2 µm column.
Twice the Efficiency at the System Limit: 4,000 6,000 psi System Ascentis Express C18 100 mm length 45 % acetonitrile 4500 psi N = 8,500 p/col C18 Sub-2 µm Column 50 mm length 46 % acetonitrile 5200 psi N = 3,790 p/col 1 1 2 2 3 4 1.0 2.0 4 5 Mobile Phase: water : acetonitrile; isoelutropic for β-estradiol Columns: 2.1 mm I Flow: 0.6 ml/min et: 200 nm Inj: 1µL Elution order: 1. Estriol 2. β-estradiol 3. Contaminant 4. Estrone 5. Estrone degradant Both column efficiencies have been compromised by system dispersion however N is double for the longer column as would have been predicted at the same pressure 3 5 20 0.2 0.4 0.6 0.8 1.0 1.2
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Ascentis Express Achieve 100,000 Plates under 7,000 PSI 1 Column 15cm x 4.6mm Agilent 1200, 1.0mL/min, 254nm, ambient temp, 10uL 4. N Toluene = 30,738 3. N Benzene = 31,696 2. N Acetophenone = 33,786 Pressure = 183 bar (2690 psi) 2 columns 30cm N Toluene = 63,212 N Benzene = 64,785 N Acetophenone = 67,390 Pressure = 350 bar (5145 psi) N Toluene = 91,311 3 columns 45cm 2 3 4 N Benzene = 94,979 N Acetophenone = 101,586 Pressure = 473 bar (6953 psi) 0 2 4 6 8 10 12 14 22
Ascentis Express separates deuterated Isomeres Agilent 1200 55cm x 4.6mm (>100,000 N in 14 min) 60% Acetonitrile 1.1mL/min, 254nm, 50 C, 10uL 4 columns B B B Toluene N = 104,235 Benzene N = 106,096 Acetophenone N = 117,475 Pressure = 473 bar (7,000 psi) B 0 2 4 6 8 10 12 14 Toluol N = 105,657 10 Benzol N = 106,260 6 Benzol N = 105,235 Acetophenon N = 118,567 Pressure = 473 bar (7,000 psi) 10 0 2 4 6 8 10 12 14 23
24 euterated Analogues of Toluene and Benzene C 3 C 3 C 3 C 3
igh-efficiency Separations: euterated Analogues of Toluene and Benzene Length: 45 cm Pressure: 10500 psi N (acetophenone) = 73400 1.8 µm, C18 0 10 20 Length: 60 cm Pressure: 10550 psi N (acetophenone) = 113810 2.7 µm, Fused Core 25 0 10 20
26 Further examples of high resolution using Ascentis Express
Increased resolution over 3 µm particles Ascentis Express C18 (2.7μm) 6 0 2 4 6 Efficiency (N) Peak 9: 13160 2990 psi columns: 10 cm x 2.1 mm I.. mobile phase A: 100 mm ammonium acetate (p 7.0; titrated with ammonium hydroxide) mobile phase B: water mobile phase C: methanol online mixing: Ascentis Express C18: A:B:C = 10:28:62 3 µm, C18: A:B:C = 10:25:65 (isoelutropic for peaks 6 & 9) flow rate: 0.3 ml/min. temp.: 55 C det.: UV at 250 nm injection: 1 µl C18 3 µm 6 Efficiency (N) Peak 9: 8290 1750 psi Peak I 1. Uracil 2. Nordoxepin (50 mg/l) 3. esipramine (50 mg/l) 4. Nortiptyline (50 mg/l) 5. oxepin (50 mg/l) 6. Norclomipramine (50 mg/l) 7. Imipramine (50 mg/l) 8. Amitriptyline (50 mg/l) 9. Clomipramine (50 mg/l) 0 2 4 6 27
Carbonic Anhydrase Tryptic igest on Ascentis Express C18 40μL Injection 1.0mL/min flow @ 35ºC 215nm etection Response time >0.010min Gradient: MP A 5% ACN w/0.1% TFA MP B 80% ACN w/0.095% TFA 2 min hold @ 0% B 58 min gradient from 0% B to 50% B 20 min gradient from 50% B to 85% B Ascentis Express 15x4.6mm 2.7μm C18 15x4.6mm 3μm 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 28
Greater peak resolution with Ascentis Express Magnified regions: each pair is at the same scale 2 14 16 18 20 22 24 26 28 12 14 16 18 20 22 24 26 28 C18 3μm Ascentis Express C18 8 30 32 34 36 38 40 2829 30 32 34 36 38 40
ILIC on Ascentis Express Erythromycin and Related Compounds by O erythromycins.esp 1 3 3 C O 3 C O C 3 O 3 C C 3 O N C 3 2 4 3 C O O C 3 O O O C 3 C 3 O 0 5 10 15 Retention 3 C O O C 3 C 3 30 Column Name Ascentis Express Si Length 15 cm iameter 0.05 cm Particle Size 2.7 um Mobile Phase A 2 O; 100mM ammonium formate p Mobile Phase A 3 Mobile Phase B acetonitrile Gradient 90B Temperature 35 C Injection Volume 5 ul
Comparison of Ascentis Express ILIC and porous particle based silica phases Ascentis Express ILIC 1 2 3 4 1. Norephedrine 2. Ephedrine 3. Pseudoephedrine 4. Synephrine 1 2 3 4 Ascentis Silica 31 0 2 4 6 8 10 Column Ascentis Express ILIC Plates/Column - Synephrine 18500 Ascentis Silica 12878 Other ILIC 10013 0 2 4 6 8 10 12 14 4 1 Other porous ILIC 2 3 0 2 4 6 8 10 12 14
32 Ascentis Express Stability A Customers Experience
Summary Ascentis Express columns with 2.7 µm Fused-Core Particles offer the chromatographer a real Speed Advantage - Greater resolving power per unit pressure on any system Almost double the efficiency of 3 µm columns with totally porous particles Close to half the back pressure of sub 2 µm particles possible use of 2x longer columns - Increase the speed of analysis while maintaining resolution Use a column of half the length or smaller while maintaining the same or better resolving power than 5 µm & 3 µm columns Get the same speed as sub 2 µm columns but at half the pressure Or increase flow and double the speed (or better) at the same pressure - Benefit from the ruggedness and durability of traditional PLC columns 33
Acknowledgements r. illel Brandes r avid Bell ugh Cramer r. Richard A. enry r. Wayne Way r. Russel Gant r. Paul Ross r. William Campbell r. A. aniel Jones, MSU 34
Thank you! C18 C8 ILIC RP-Amide (coming soon) 35 Seminar Offer 25% off for a Test Column