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1 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 USA

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

3 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)

4 Particle Size Distributions Standard Halo 90 A mode = 2.80 um, SD = 0.14 Number 1500 Halo Peptide 160 A mode = 2.82 um, SD = Particle Diameter, [µm]

5 Effect of Pore Size on Peptide and Small Protein Separations w= Standard HALO C18 90 Å w= w= w= w= w= w= HALO Peptide ES-C Å w= w= w= 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

6 Effect of Pore Size on Efficiency Reduc ced Plate Height, h 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 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

7 High Mobile Phase Velocity LC/MS Analysis of a Tryptic Digest 9.56 Relative Abundance NL: 5.18E5 Base Peak F: ITMS + c ESI Full ms [ ] HALO Peptide ES-C Å Time (min.) DJ_Halo_stem_ApoMyoglobin_3pmol_AF_1_ # RT: AV: 11 NL: 6.88E4 F: ITMS + c ESI Full ms [ ] Relative Abundance 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

8 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)

9 Particle Size Distributions 4000 Num mber 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 In spite of the change in particle size, the standard deviation remains 5.5% of the mean Particle Diameter (µm)

10 Effect of Shell Thickness on Sample Loading Effic ciency (at Half Height) 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 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

11 Effect of Shell Thickness on Efficiency 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 β-amyloid (1-38) MW: 4100 Da Shell: 0.5 µm 27% lower β-amyloid (1-38) MW: 4100 Da Shell: 0.3 µm Pore Size: 160 Å for both 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

12 Effect of Shell Thickness on Efficiency 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 % lower RNase A MW: 13,700 Da Shell: 0.3 µm 4 2 Pore Size: 160 Å for both 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

13 Effect of Shell Thickness on Efficiency RNase A (13.7 kda), 0.5 µm 12 Redu uced Plate Height, h RNase A (13.7 kda), 0.3 µm β-amyloid (1-38) (4.1 kda), 0.5 µm β-amyloid (1-38), (4.1 kda), 0.3 µm Pore Size: 160 Å for all 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

14 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 T T T18-32 T64-78 T Shell: 0.5 µm Pore Size: 160 Å n pc : 229 P max : 148 bar T T 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

15 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

16 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)

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

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