Superficially Porous Silica Particles with Wide Pores for Biomolecular Separations B. M. Wagner, S. A. Schuster, B. E. Boyes, J. J. Kirkland Advanced Materials Technology, Inc. Wilmington, DE, USA
Study of Wide-pore Fused-Core particles developed to show the effect of: 1. Particle size and shell thickness on column efficiency for proteins 2. Stationary phase on protein separation performance 3. Pore size in separating large proteins 4. Shell thickness, particle type, particle size on sample loading 5. Particle type (Fused-core, totally porous) on column efficiency for proteins 6. Stability of columns with wide-pore Fused-core particles
Physical characteristics of Fused-Core particles Fused-Core Particle Particle Size, µm Pore Size, Å BET Surface Area, m 2 /g Shell Thickness, μm % Porosity Pore volume, cm 3 /g Halo 2.7 90 135 0.5 75 0.26 Halo Peptide 2.7 160 80 0.5 75 0.29 Wide-pore 2.7 400 30 0.35 59 0.23 Wide-pore 2.7 400 14 0.2 46 0.11 Wide-pore 3.4 400 10 0.2 31 0.068
Halo Wide-pore Fused-core Particles Solid Core 2.0 µm 0.35 µm Shell with pores
Effect of Particle on Performance Columns: 4.6 x 100 mm; Temperature: 60 o C Mobile phase: 23.9% acetonitrile/76.1% aqueous trifluoroacetic acid, 0.1% Agilent 1100 with autosampler 30 25 2.7 mm, 0.35 mm shell; k = 3.6 3.4 mm, 0.2 mm shell; k = 1.8 Data fitted to Knox equation Plate Height, H, mm 20 15 Solute: ribonuclease A (13.7 kda) 10 5 0 1 2 3 4 5 6 7 Linear Mobile Phase Velocity, mm/sec
Effect of Particle on Performance Columns: 4.6 x 100 mm; Temperature: 60 o C Mobile Phase: 23.9% acetonitrile/76.1% aqueous trifluoroacetic acid, 0.1% Agilent 1100 with autosampler 11 10 9 2.7 mm, 0.35 mm shell; k = 3.6 3.4 mm, 0.2 mm shell; k = 1.8 Data fitted to Knox equation Reduced Plate Height, h 8 7 6 5 4 Solute: ribonuclease A (13.7 kda) 3 2 1 0 1 2 3 4 5 6 7 Linear Mobile Phase Velocity, mm/sec
Effect of Bonded Phase Columns: 2.1 x 100 mm Instrument: Agilent 1200 SL Injection Volume: 1 µl Detection: 215 nm Mobile Phase: 20 70% ACN/water/0.1% TFA in 20 min. Flow rate: 0.3 ml/min Temperature: 60 C Sample: In order 1.Ribonuclease A MW = 13.7 kda 2.Cytochrome C MW = 12.4 kda 3.Bovine Serum Albumin MW = 66.4 kda 4.Apomyoglobin MW = 17.0 kda 5.Enolase MW = 46.7 kda 6.Phosphorylase B MW = 97.2 kda C4 ES-C8 ES-C18 0 2 4 6 8 10 12 14 16 18 20 Time (min.)
Pore Size Distribution of Fused-Core Particles 0.45 0.40 0.35 dv/dlog(w) Pore Volume, [cm^3/g*a] 0.30 0.25 0.20 0.15 0.10 0.05 0.00 10 100 1000 10000 Pore Width, [A]
Columns: 4.6 x 100 mm Instrument: Agilent 1100 Flow rate: 1.5 ml/min Injection Volume: 5 µl Detection: 220 nm Mobile Phase: A: 10% ACN / 0.1% TFA B: 70% ACN / 0.1% TFA Gradient: 0% B to 50% B (15min) Temperature = 30 C Peak widths at 50% height given for ribonuclease A and insulin Effect of Pore Size Peak Identities: In order 90 Å C18 0.1953 0.0649 0.0582 0.0368 1.Gly-Tyr 2.Val-Tyr-Val 3.Met-Enk 4.Angiotensin II 5.Leu-Enk 6.Ribonuclease A 7.Insulin MW = 238.2 g/mol MW = 379.5 g/mol MW = 573.7 g/mol MW = 1046.2 g/mol MW = 555.6 g/mol MW = 13700 g/mol MW = 5800 g/mol 160 Å ES-C18 ES-C8 0.0534 0.0384 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min.)
Large Protein Separations Columns: 2.1 x 100 mm Instrument: Agilent 1200 SL Flow rate: 0.3 ml/min Injection Volume: 1 µl Detection = 215 nm Mobile Phase: A: Water/0.1% TFA B: ACN/ 0.1% TFA Gradient: 30% B to 70% B in 10 min. Temperature = 60 C Sample: In order 1. Cytochrome C MW = 12.4 kda 2. Ferritin MW = 443 kda 3. β-amylase MW = 200 kda 4. Myosin MW = 500 kda [220 kda monomer] 3 4 C4 ES-C8 ES-C18 2 1 0.0433 0.0348 0 1 2 3 4 5 6 7 8 9 10 Time (min.)
Effect of Particle Type on Sample Loading Columns: 4.6 x 100 mm; Temperature: 60 o C; Agilent 1100: Injection: 5 µl Mobile phase- A: water/0.1% trifluoroacetic acid, B: acetonitrile/0.1% trifluoroacetic acid Gradient: 37-47 % B in 10 min; Flow rate: 0.5 ml/min 0.20 0.18 3.4 µm, 0.2 µm shell, 0.35 µm shell 0.16 Peak Width, min. 0.14 0.12 0.10 0.08 Solute: myoglobin(17 kda) 0.06 0.04 0.02 0.1 1 µg of myoglobin
Protein Separations Fused-Core vs. Totally Porous Columns: 4.6 x 100 mm; Temperature: 60 C Mobile phase: A = water/0.1% TFA; B = Acetonitrile/0.1% TFA Gradient: 20-70% B in 10 min.; Flow rate = 1.5 ml/min; Detection = 215 nm; Injection = 5 µl Absorbance (mau) Absorbance (mau) 140 120 100 80 60 40 20 140 120 100 80 60 40 20 3 µm Totally Porous 300 Å C18 0.0752 0.0388 Sample: In order 1.Bovine Serum Albumin MW = 66.4 kda 2.Apomyoglobin MW = 17.0 kda 3.Enolase MW = 46.7 kda 0.0437 0 4.5 5.0 5.5 6.0 6.5 7.0 Fused-Core ES-C18 0.0510 0.0249 0.0248 0 4.5 5.0 5.5 6.0 6.5 7.0 Time (min.)
Fused-Core Particle Stability Column: 2.1 x 100 mm ES-C8; Temperature: 60 C Mobile phase: A = water/0.1% TFA; B = 70% ACN/30% water/0.1% TFA; Gradient: 9-55% B in 10 min.; Flow rate = 0.5 ml/min; Detection = 220 nm; Injection = 1 µl, Retention times given for each peak, Peak widths at half height for selected peaks (min.) Peak Identities: In order 1.Gly-Tyr 2.Val-Tyr-Val 3.Met-Enk 4.Angiotensin II 5.Leu-Enk 6.Ribonuclease A 7.Insulin MW = 238.2 g/mol MW = 379.5 g/mol MW = 573.7 g/mol MW = 1046.2 g/mol MW = 555.6 g/mol MW = 13700 g/mol MW = 5800 g/mol 6.92 w ½ = 0.0307 8.64 0.59 w ½ = 0.0288 w ½ = 0.0310 1.48 2.78 3.65 4.54 Injection 362 0.5 w ½ = 0.0285 w ½ = 0.0318 1.50 2.81 3.69 4.55 Injection 1 6.92 w ½ = 0.0321 8.6 0 1 2 3 4 5 6 7 8 9 10 Time, min.
Rabbit Skeletal Myosin Columns: 2.1 x 100 mm; Temperature: 80 C Mobile phase: A = water/0.1% TFA; B = Acetonitrile/0.1% TFA Gradient: 35-65% B in 15 min.; Flow rate = 0.45 ml/min.; Detection = 215 nm; Injection = 1 µl 9 8 Absorbance (mau) Absorbance (mau) 7 6 5 4 3 2 1 0-1 9 8 7 6 5 4 3 2 1 0-1 Light chain subunits @ 17-25 kda 0 2 4 Time (min.) 6 8 10 Structure adapted from Alberts, et al., Molecular Biology of the Cell ( Garland Science 2008) Heavy chain subunits @ ~220 kda Fused-Core 160 Å ES-C18 Fused-Core ES-C18
Conclusions from Study Chromatographic characteristics of wide-pore particles: 1. Particles with pores effective for efficiently separating proteins without restricted diffusion 2. C4 and C8 may be preferred for separating proteins 3. Thicker-shell particles have greater mass loading properties, but somewhat poorer efficiency than thinner-shell particles 4. Fused-core particles have performance advantages over totally porous particles for separating proteins 5. Columns of particles are both efficient and stable