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Outperforming Sub-2 Um Totally Porous Particles Using Fused-Core Technology

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Outperforming Sub-2 Um Totally Porous Particles Using Fused-Core Technology Outperforming Sub-2-µm Totally Porous Particles using Fused-Core Technology Robert E. Moran, Stephanie A. Schuster, Barry E. Boyes, William L. Miles Advanced Materials Technology Inc., Wilmington, DE 19810 Presented at EAS 2014 Abstract Comparing van Deemter Plots (h) Ballistic Separation of Anticoagulants Steroids Separation : HALO vs. TPP Explosive Separation : HALO vs. TPP Reduced Plate Height vs. Mobile Phase Velocity Plots Column : 2.1 x 100 mm Flow rate : 0.4 mL/min Sample : Column : 2.1 x 50 mm PFP Temperature : 30 °C Sample : Hydrocortisone Columns: 50 x 2.1 mm; Instrument: Shimadzu Nexera; Solute: naphthalene Peak Identities : Mobile Phase : 72/28 H2O/MeOH Temperature : 42 °C 1. HMX Column : 2.1 x 30 mm HALO 2 C18, 2µm, 90 Å Flow rate : 1.1 mL/min Detection : 240 nm Predisolone 2. RDX Mobile phase: Halo - 50/50 ACN/water, k = 6.3; o 1. Uracil Mobile Phase : 57% H2O/ 43% MeOH Cortisone Fused-core particles have shown distinct advantages over comparable Mobile phase A : 20 mM formic acid Temperature : 45 C Instrument : Shimadzu Nexera Detection : 254 nm 3. 1,3,5-Trinitrobenzene 1.6 m SPP - 48.5/51.5 ACN/water, k = 6.3; 1.7 m SPP - 47/53 ACN/water, k = 6.2 2. 6,7-dihydroxycoumarin Flow rate : 0.3 mL/min Injection volume : 1.0 µL Prednisone o Mobile phase B : 50/50 ACN/MeOH Detection : 254 nm Injection volume : 1.0 µL 4. 1,3-Dinitrobenzene 1.7 m TPP - 48.5/51.5 ACN/water, k=6.3; Temperature: 35 C; Injection volume: 0.2 L 3. 4-hydroxycoumarin Dexamethasone 4 totally porous particles (TPP) for separating molecules of all sizes and Gradient : Time %B Injection : 0.2 µL 250 5. Nitrobenzene 4. Coumarin 3 HALO 2 C18, 2µm, 90 Å 6. Tetryl 0 – 0.06 20 Max Pressure : 430 bar 78 5. 6-chloro-4-hydroxycoumarin 200 Pressure: 550 bar 7. 2,4,6-Trinitrotoluene characteristics. The introduction of a new 2.0 µm particle size to the 7.0 HALO 2 PFP, 2µm, 90 Å 0.06 – 1.06 20 – 75% 6. Warfarin 68 1 8. 2-Amino-4,6-Dinitrotoluene 7. Coumatetralyl Pressure: 281 bar Pw = 0.071 9. 4-amino-2,6-dinitrotoluene 58 150 2 Pw = 0.103 6.5 SPP Halo 2.0 m 5 10. 2,4-Dinitrotoluene Fused-core product line continues to advance high speed, high efficiency 35 8. Coumachlor SPP Halo 2.7 m 48 7 10 11. 2,6-Dinitrotoluene 100 6 8 6.0 SPP 1.6 m 3 Plates = 7800 12. 2-Nitrotoluene 7 38 9 separations into the future. These 2.0 µm Fused-core particles permit SPP, 1.7 m 30 11 Pw = 0.168 13. 4-Nitrotoluene 5.5 TPP, 1.7 m 4 28 50 12 14 14. 3-Nitrotoluene Absorbance (mAU) Absorbance 13 separation speeds and resolution improvements greater than sub-2-µm 18 5.0 25 2 5 0 8 totally porous particles, but with about 20% lower column back pressure. 4.5 6 -50 20 ‐2 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 012345678910 4.0 8 Based on chromatographic theory, the trend of using smaller particle sizes 58 250 15 1 TPP C18, 1.7µm, 130 Å 3.5 TPP PFP, 1.8µm, 120 Å has been accepted as a way to increase efficiency. This report describes 48 200 Pressure: 640 bar 10 Pressure: 430 bar Absorbance (mAU) Absorbance Reduced Plate Height 3.0 4 Narrower and 38 Plates = 4300 150 3 the effect of these new particles on multiple factors of separation 1 P = 0.140 2.5 w taller peaks at 5 28 2 100 Pw = 0.094 9,10 15% lower performance, including reduced plate height, column efficiency, back 5 2.0 18 7 8 Industry 6 Pw = 0.213 0 50 11 pressure 12 1.5 Data fitted using van Deemter equation leading 13 14 Absorbance (mAU) Absorbance pressure, and resolution. (mAU)Absorbance 8 (mAU) Absorbance 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Engineered ‐5 0 1.0 efficiency for high Time (min) ‐2 0123456789101112 over broad 012345678910 -50 Faster separation 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 performance Linear Mobile Phase Velocity, mm/sec Time (min) range of flow with high Time (min) rates efficiency Pressure vs. Flow Rate Carbonyl-DNPH High Resolution Separation Peptide Separation : HALO comparison Protein Separation : HALO vs. TPP Mobile Phase/Column Pressure Plots Column : 2.1 x 150 mm, HALO 2 C18, 2µm, 90 Å Flow rate : 0.5 mL/min Sample : DNPH Derivatized Columns : 3.0 x 50 mm Mobile Phase A : 90/10 H2O/ACN/0.1% TFA Sample: Gly-Tyr Columns : 4.6 x 100 mm Flow rate : 3 mL/min Peak Identities: Columns: 50 x 2.1 mm, C18: Instrument: Shimadzu Nexera; Solute: naphthalene Carbonyl Compounds – Val-Tyr-Val 1. Ribonuclease A 13.7 kDa Mobile Phase A : H2O Temperature : 30 °C Instrument : Agilent 1100 Mobile Phase B : 30/70 H2O/ACN/0.1% TFA Instrument : Agilent 1200 SL* Mobile Phase A : water/0.1% TFA 2. Cytochrome c 12.4 kDa Mobile phase: Halo, 50/50 ACN/water, k=6.3; SPP 1.6 m, 48.5/51.5 ACN/water, k=6.3 Mix of Options #1 and #2 Met-Enkephalin Injection Volume : 5 µL Mobile Phase B : acetonitrile/0.1% TFA o Mobile Phase B : 80/20 ACN/THF Detection : 360 nm Injection Volume : 2 µL Gradient : 0-55% B in 10 min. Angiotensin II 3. Lysozyme 14.3 kDa SPP 1.7 m, 47/53 ACN/water, k=6.2; Temperature: 35 C; Injection volume: 0.2 L (19 out of 20 major Gradient : Time %B Injection volume : 1.0 µL components resolved) Detection : 220 nm Flow rate : 0.6 mL/min Leu-Enkephalin Detection : 215 nm Gradient : 23-85% B in 1 min 4. α-Lactalbumin 14.2 kDa o 5. Catalase 250 kDa total; 0 – 5 min 45 Temperature : 30 °C Ribonuclease A Temperature : 60 C *3 + 6 µL heat exchangers 900 Insulin tetramer of ~60 kDa each 85 345 5 – 20 min 45 – 90 VWD1 A, Wavelength=220 nm (1034-1\QA_0-55_000112.D) 0.0065 mAU 75 0.0071 800 Halo 2.0 m HALO Protein ES-C18, 3.4 µm, 400 Å 300 HALO 2 Peptide ES-C18 9.079 1.6 µm 65 Halo 2.7 m 250 Pressure: 234 bar 295 2 µm, 160 Å Pw = 0.0419 SPP 1.6 m 200 Pw = 0.0328 55 700 Pressure: 208 bar 7.904 0.0065 SPP 1.7 m Peak Capacity : 180 Columns for 150 0.693 45 100 0.0069 6.330 1.7 µm 6.548 5.450 0.0082 245 Max Pressure : 281 bar 3.016 50 35 600 every 9.207 0 0.452 25 2.0 µm instrument 0 1 2 3 4 5 6 7 8 9 min 15 195 VWD1 A, Wavelength=220 nm (1034-1\HALO27_55_00104.D) 500 (mAU) Absorbance mAU 5 300 HALO Peptide ES-C18 9.076 ‐5 145 250 400 2.7 µm, 160 Å P = 0.0413 P = 0.0570 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 200 w w 0.661 Pressure: 111 bar 150 7.918 85 95 100 6.435 300 6.120 5.205 50 2.668 75 9.201 Column Pressure, bar 2.7µm Totally Porous C18, 3 µm, 300 Å 0.437 0.0102 0 65 Pressure: 244 bar 0.0114 200 45 0 1 2 3 4 5 6 7 8 9 min VWD1 A, Wavelength=220 nm (1034-1\HALO5_55_000105.D) 55 mAU Absorbance (mAU) Absorbance 45 0.0104 100 300 ‐5 HALO-5 Peptide ES-C18 8.777 35 0.0107 250 P = 0.0457 Pw = 0.0595 0 2 4 6 8 10 12 14 16 18 5 µm, 160 Å w 0.0150 Time (min) 200 25 0 150 0.645 Pressure: 47 bar 7.672 15 Peak Identities : Formaldehyde-2,4-DNPH, Acetaldehyde-2,4-DNPH, Acetone-2,4-DNPH, Acrolein-2,4-DNPH, Propionaldehyde-2,4-DNPH, 100 Absorbance (mAU) Absorbance 6.227 5.983 5.119 Crotonaldehyde-2,4-DNPH, Butyraldehyde-2,4-DNPH, Benzaldehyde-2,4-DNPH, Cyclohexanone-2,4-DNPH, Isovaleraldehyde-2,4-DNPH, 50 2.761 5 8.905 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.425 Valeraldehyde-2,4-DNPH, o-Tolualdehyde-2,4-DNPH, m-Tolualdehyde-2,4-DNPH, p-Tolualdehyde-2,4-DNPH, Hexaldehyde-2,4-DNPH, 2,5- 0 ‐5 Dimethylbenzaldehyde-2,4-DNPH, Heptaldehyde-2,4-DNPH, Octyl aldehyde-2,4-DNPH, Nonanal-2,4-DNPH, Decyl aldehyde-2,4-DNPH Mobile Phase Flow Rate, mL/min 0 1 2 3 4 5 6 7 8 9 min 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) Incredible high High resolution pressure for protein separations Halo 2µm High Pressure Stability stability Plates per Bar Peptide Separation : Peak Capacity Peptide Peak Capacity : HALO vs. TTP Conclusions Column : 3.0 x 150 mm Penta-HILIC Mobile Phase : Plates per Pressure Required Columns : 4.6 x 150 mm Flow rate : 4.0 mL/min Peak Identities (in order) : Columns : 2.1 x 150 mm Peak Identities (in order) : Instrument : Shimadzu Nexera 88/12 ACN/ 0.1M Ammonium Formate pH:3 Flow rate : 0.5 mL/min 1.
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