Fused-Core Particles:

Fused-Core Particles: Varying Shell Thickness and Pore Size Stephanie A. Schuster; Joseph J. Kirkland; Brian M. Wagner; Barry E. Boyes; William L. Johnson; Timothy J. Langlois; Joseph J. DeStefano Advanced Materials Technology, Wilmington, DE 19810 USA

Overview Fused-core particles Changes to pore size Changes to shell thickness Conclusions Future Directions

Fused-Core Particles: Varying Pore Size Porous Shell Solid Core x d p y x (µm) y (µm) d p (µm) Pore Size (Å) Surface Area (m 2 /g) 1.7 0.5 2.7 90 150 1.7 0.5 2.7 160 80

Particle Size Distributions 3500 3000 2500 2000 Standard Halo 90 A mode = 2.80 um, SD = 0.14 Number 1500 Halo Peptide 160 A mode = 2.82 um, SD = 0.14 1000 500 0-500 0 2 4 6 Particle Diameter, [µm]

Effect of Pore Size on Peptide and Small Protein Separations w=0.0516 Standard HALO C18 90 Å w=0.0861 w=0.0819 w=0.5411 w=0.4671 w=0.0491 w=0.0516 HALO Peptide ES-C18 160 Å w=0.0457 w=0.0837 w=0.1043 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min.) 1. Leu-enk (555 g/mol) 2. Bovine Insulin (5733 g/mol) 3. Human Insulin (5808 g/mol) 4. Cytochrome C (12,400 g/mol) 5. Lysozyme (14,300 g/mol) Columns: 4.6 x 100 mm Flow rate: 1.5 ml/min Temperature: 30 C A: 0.1% TFA/10% ACN, B: 0.1% TFA/70% ACN Gradient: 15% to 50% B in 15 min. Injection volume: 5 µl Detection: 220 nm

Effect of Pore Size on Efficiency 16 14 Reduc ced Plate Height, h 12 10 8 6 4 2 β-amyloid (1-38) MW: 4100 Da 90 Å 179% lower β-amyloid (1-38) MW: 4100 Da 160 Å Leu-Enk MW: 555 Da 160 Å 0 0 1 2 3 4 5 6 7 Mobile Phase Velocity, mm/sec Columns: 4.6 x 100 mm HALO C18, 2.7 µm, 90 Å 4.6 x 100 mm HALO Peptide ES-C18, 2.7 µm, 160 Å Mobile Phase: Leu-Enk: 21% ACN/79% Water/0.1% TFA β-amyloid (1-38) 160 Å : 29% ACN/71% Water/0.1% TFA β-amyloid (1-38) 90 Å : 27% ACN/73% Water/0.1% TFA Temperature: 60 C Detection: 215 nm

High Mobile Phase Velocity LC/MS Analysis of a Tryptic Digest 9.56 Relative Abundance 100 90 80 70 60 50 40 30 20 10 0 NL: 5.18E5 Base Peak F: ITMS + c ESI Full ms [300.00-2000.00] 2.47 1127.67 5.33 1549.90 7.97 684.20 8.42 704.49 752.35 10.63 804.51 11.28 748.41 13.35 690.43 14.17 909.03 HALO Peptide ES-C18 160 Å 0 2 4 6 8 10 12 14 16 18 20 Time (min.) 16.54 943.13 19.49 1617.64 DJ_Halo_stem_ApoMyoglobin_3pmol_AF_1_051810 #1945-1971 RT: 12.79-12.95 AV: 11 NL: 6.88E4 F: ITMS + c ESI Full ms [300.00-2000.00] 754.52 100 Relative Abundance 90 80 70 60 50 40 30 20 10 0 941.18 395.19 494.21 690.02 1037.00 1232.29 1506.56 1604.58 1833.39 1963.06 400 600 800 1000 1200 1400 1600 1800 2000 m/z Halo Peptide ES-C18, 0.2 mm ID x 50 mm, Flow Rate 9 µl/min., 2-45% B in 15 minutes, 3 pmol apomyoglobin digest in 2 µl; A: 0.1 % Formic Acid/10 mm Ammonium Formate B: 0.1% Formic acid in Acetonitrile

Fused-Core Particles: Varying Shell Thickness Porous Shell Solid Core x d p y X (µm) y (µm) d p (µm) Surface Area (m 2 /g) Pore Size (Å) 1.7 0.5 2.7 80 160 1.7 0.3 2.3 49 160

Particle Size Distributions 4000 Num mber 3500 3000 2500 2000 Mode: 2.31 µm Shell: 0.3 µm S.D: 0.13 µm Pore Size: 160 Å for both Mode: 2.74 µm Shell: 0.5 µm S.D: 0.14 µm 1500 1000 500 In spite of the change in particle size, the standard deviation remains 5.5% of the mean. 0 0 1 2 3 4 5 6 Particle Diameter (µm)

Effect of Shell Thickness on Sample Loading Effic ciency (at Half Height) 25000 20000 15000 10000 5000 LH-RH MW: 1182 Da Shell: 0.5 µm LH-RH MW: 1182 Da Shell: 0.3 µm LHRH on 2.7 um 160 A LHRH on 2.3 um 160 A 0 Pore Size: 160 Å for both 0.01 0.1 1 10 100 Mass (µg) Column: 4.6 x 100 mm SP-C8 Mobile Phase: Isocratic: 2.7 µm: 17% ACN/83% Water/0.1% TFA 2.3 µm: 16.5% ACN/83.5% Water/0.1% TFA Flow rate: 1.0 ml/min Temperature: 60 C Detection: 220 nm LC System: Agilent 1100 Sample: Luteinizing Hormone-Releasing Hormone (LH-RH) MW = 1182

Effect of Shell Thickness on Efficiency 6.0 5.5 2.3 u m P a rtic le s 2.7 u m P a rtic le s Reduced Reduc Plat ced te Height, Plate Height, h h 5.0 4.5 4.0 3.5 3.0 β-amyloid (1-38) MW: 4100 Da Shell: 0.5 µm 27% lower β-amyloid (1-38) MW: 4100 Da Shell: 0.3 µm 2.5 2.0 Pore Size: 160 Å for both 0 1 2 3 4 5 6 7 Mobile M o b ile Phase P h a svelocity, e V e c ity, mm/sec m m /s e c Columns: 4.6 x 50 mm SP-C8, 2.7 µm, 0.5 µm shell, 160 Å and 2.3 µm, 0.3 µm shell, 160 Å Mobile Phase: 27.4% ACN/72.6% Water/0.1% TFA Temperature: 60 C Detection: 215 nm

Effect of Shell Thickness on Efficiency 1 6 1 4 1 2 2.3 µ m P a rtic le s 2.7 µ m P a rtic le s RNase A MW: 13,700 Da Shell: 0.5 µm Reduced Reduced Pla d ate Plate Height, Height, h h 1 0 8 6 48% lower RNase A MW: 13,700 Da Shell: 0.3 µm 4 2 Pore Size: 160 Å for both 0 1 2 3 4 5 6 7 MMobile o b Phase P h a svelocity, e V e cmm/sec ity, m m /s e c Columns: 4.6 x 50 mm SP-C8, 2.7 µm, 0.5 µm shell, 160 Å and 2.3 µm, 0.3 µm shell, 160 Å Mobile Phase: 24.5% ACN/75.5% Water/0.1% TFA Temperature: 60 C Detection: 215 nm

Effect of Shell Thickness on Efficiency 16 14 RNase A (13.7 kda), 0.5 µm 12 Redu uced Plate Height, h 10 8 6 RNase A (13.7 kda), 0.3 µm β-amyloid (1-38) (4.1 kda), 0.5 µm 4 2 0 β-amyloid (1-38), (4.1 kda), 0.3 µm Pore Size: 160 Å for all 0 1 2 3 4 5 6 7 Mobile Phase Velocity, mm/sec Columns: 4.6 x 50 mm SP-C8, 2.7 µm, 0.5 µm shell, 160 Å and 2.3 µm, 0.3 µm shell, 160 Å Mobile Phase: RNase A: 24.5% ACN/75.5% Water/0.1% TFA β-amyloid (1-38): 27.4% ACN/72.6% Water/0.1% TFA Temperature: 60 C Detection: 215 nm

Effect of Shell Thickness on Peak Capacity Peak capacity was calculated by averaging the peak widths of the labeled tryptic digest peaks below and using the following equation: n pc = t t f i W 4σ Shell: 0.3 µm Pore Size: 160 Å n pc : 224 P max : 204 bar t i T97-102 T146-153 T18-32 T64-78 T104-119 Shell: 0.5 µm Pore Size: 160 Å n pc : 229 P max : 148 bar T97-133 T97-118 t f Columns: 4.6 x 50 mm SP-C8 2.7 µm 160 Å 4.6 x 50 mm SP-C8 2.3 µm 160 Å Mobile Phase: A: Water/0.1% TFA B: 80% ACN/20% Water/0.1% TFA Gradient: 5-60% B in 30 min. Flow rate: 2.4 ml/min Temperature: 60 C Detection: 220 nm LC System: Agilent 1100 Sample: Apo-myoglobin tryptic digest

Conclusions Increasing the pore size of the Fused-core particles improves the mass transfer for larger molecular weight solutes Different property Fused-core particles can be produced with extremely narrow size distributions Decreased shell thickness improves the mass transfer for larger molecular weight solutes Degree of improvement is a function of molecular size; analytic details under investigation (diffusion dependence) Sample load/retention decreased by lower surface area

Future Studies 1) Investigate the effect of even larger pores on particle characteristics for separating large molecules 2) Explore the advantages and disadvantages of smaller fused-core particles 3) Determine the practical role of shell thickness for (1) and (2)

Acknowledgements Advice and insight from Jack Kirkland Barry Boyes Joe DeStefano Tim Langlois Brian Wagner Financial support from SB-IR grant Z-R44- GM077688-02