Standard Halo vs. Halo Peptide
Fused-Core® Particles for the Fast, High Resolution Separation of Peptides
Fused-Core Particle Analysis Electron Micrographs of Halo Peptide
Standard Halo vs. Halo Peptide 3500
3000
4.9% RSD
2500
Halo Peptide mean = 2.85 um, SD = 0.14 Standard Halo mean = 2.82 um, SD = 0.14
2000
1500
1000
500
0 0 ‐500
1
2
3
4
5
Particle Diameter, [µm]
6
Fused-Core® Peptide Particles Silica --------------------High Purity Type B Overall diameter---------------------2.8 µm Solid core diameter------------------1.7 µm Porous shell thickness---------------0.5 µm Avg. pore diameter-------------------16 nm Surface area, nitrogen---------------70 m2/g Pore volume ------------------------0.23 mL/g Particle density ---------------------1.3 cm3/g
Fused-Core Pore Analysis Standard Halo vs. Halo Peptide N2 Absorption
Fused-Core Pore Size Analysis Standard Halo vs. Halo Peptide Inverse SEC Test Conditions: Columns: 4.6 x 150 mm Mobile Phase: THF Flowrate: 1.0 ml/min Samples: polystyrene standards Detection: UV at 254 nm
1000000
Log Molecular Weight
100000
Fused-core Peptide Silica 150 A 10000
Standard fused-core silica 90 A
1000
Relative Retention Volume 100
Column Efficiency Solute: beta‐amyloid (1‐38) MW = 4131
Halo, 9 nm pores
Halo Peptide, 16 nm pores
Columns: 4.6 x 100 mm; Particle size: 2.7 μm Mobile Phase: 50% ACN/50% water/0.1% TFA Temperature: 25°C Agilent 1100 with autosampler
Columns: 4.6 x 100 mm; Particle size: 2.7 μm Standard Halo: 27% ACN/73 % water /0.1% TFA; k = 3.0 at 1.0 mL/min Halo Peptide: 29% ACN/71% water /0.1% TFA; k = 3.0 at 1.0 mL/min Temperature: 60°C Instrument: Agilent 1100 with autosampler using both 3 and 6 µL heat exchangers
Sample Loading Column: 4.6 x 100 mm Halo Peptide ES-C18; T= 60 °C Sample: Bovine insulin, MW= 5733; Inj vol = 10 µL Mobile Phase: 0.1% TFA/31.5% ACN, Flow Rate: 1 mL/min
Halo Peptide, 16 nm
Halo, 9 nm
Column Stability Column: 2.1 x 100 mm, Halo Peptide ES-C18; Flow Rate: 0.5 mL/min; T= 60 °C A: 0.1% TFA; B: 0.1% TFA/70%; ACN; Gradient: 9-55% B in 10 min.; Inj vol: 5 µL (Gly-Tyr, Val-Tyr-Val, Met-enk, Angio II, Leu-enk, RNAase, P. Insulin) Shimadzu UFLC XR
Injection 775 Injection 600 Injection 400 Injection 200 Injection 1
t (min)
Halo Peptide ES-C18 Protein and Peptide Separations Column: 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: 0% to 50% B in 15 min.; Injection volume: 5 µL w =0.0303
w =0.0686
Standard Halo w =0.0372
w =0.0413
w =0.0294
w =0.0437
Standard Halo w=0.0819
w =0.0400 w =0.0449
w =0.0433
w=0.0861
w =0.2257
Halo Peptide w =0.0376
w=0.0516
w =0.0392
w =0.0777
w=0.0516
Halo Peptide
w=0.5411
w=0.4671
w=0.0491 w=0.1043
w=0.0457 w=0.0837
w =0.0384
Sample 1 Gly-Tyr, Val-Tyr-Val, Met-enk, Angiotensin II, Leu-enk Ribonuclease, Porcine Insulin
Sample 2 Leu-enk Bovine Insulin, Human Insulin, Cytochrome C, Lysozyme
Tryptic Digest Column: 2.1 x 100 mm, Halo Peptide ES-C18; Flow Rate: 0.25 mL/min; T= 45 °C A: 0.1% TFA; B: 0.1% TFA/80% ACN; Gradient: 5-65% B in 120 min.; Sample: 15 µL (1 µg/µL) apo-myoglobin digest Agilent 1200 SL npc = 212 Pmax = 124 bar
ti
Minutes
Peak capacity [npc] calculated as tf – ti ; Peaks with * used for W4σ
W4σ
tf
Rapid Separation at High Temperature Column: 2.1 x 50 mm Halo Peptide ES-C18; Flow: 0.5 mL/min; A: 0.1% TFA; B: 0.1% TFA/80% ACN; Gradient: 15-50% B in 12.5 min.; Sample: 5 µL (250-500 ng) A Peptides
100 °C
90 °C
A(12-28)
80 °C
70 °C
better recovery at higher temps
Longer Columns for High Resolution Column: 2 @ 2.1 x 100 mm Halo Peptide ES-C18; Flow: 0.5 mL/min; T= 45 °C A: 0.1% TFA; B: 0.1% TFA/80% ACN; Gradient: 5-65% B in 120 min.; Sample: 15 µL (1 µg/µL) tryptic digests
Apo-myoglobin
Transferrin
2 columns in tandem
npc = 297 Pmax = 476 bar
Solute: beta-amyloid (1-38) MW = 4131
Halo, 9 nm pores
Halo Peptide, 16 nm pores
Columns: 4.6 x 100 mm; Particle size: 2.7 μm Standard Halo: 27% ACN/73 % water /0.1% TFA; k = 3.0 at 1.0 mL/min Halo Peptide: 29% ACN/71% water /0.1% TFA; k = 3.0 at 1.0 mL/min Temperature: 60°C Instrument: Agilent 1100 with autosampler using both 3 and 6 µL heat exchangers
7.0
6.5
Solute: beta‐amyloid (1‐38) MW = 4131
6.0
3 µm, 300 Å
Reduced Plate Height, h
5.5
5.0
4.5
4.0
3.5
3.0
Halo Peptide, 2.7 µm, 160 Å
2.5
2.0
1.5 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Mobile Phase Velocity, mm/sec
Columns: 4.6 x 100 mm; Particle size: 3.0 μm and 2.7 μm : 28% ACN/72 % water /0.1% TFA; k = 3.6 at 1.0 mL/min Halo Peptide: 29% ACN/71% water /0.1% TFA; k = 3.0 at 1.0 mL/min Temperature: 60°C Instrument: Agilent 1100 with autosampler using both 3 and 6 µL heat exchangers
Halo Peptide ES-C18 Comparison with 90 Ångstrom Halo C18 • For peptides > 5 aa residues, retention is increased on the Halo Peptide ES-C18 column. • For larger molecules, peak widths are greatly reduced due to improved mass transfer. Column: 4.6 x 100 mm; Flow rate: 1.5 mL/min; Temperature: 30° C A: 10% ACN/90%Water/0.1% TFA;B: 70% ACN/30% Water/0.1 % TFA Gradient: 0% to 50% B in 15 min.; Injection volume: 5 µL; Sample 1 w =0.0303
Standard Halo
w =0.0686 w =0.0392
w =0.0372 w =0.0413
w =0.0294
Halo Peptide w =0.0376
Column: 4.6 x 100 mm; Flow rate: 1.5 mL/min; Temperature: 30° C A: 10% ACN/90%Water/0.1% TFA;B: 70% ACN/30% Water/0.1 % TFA Gradient: 15% to 50% B in 15 min.; Injection volume: 5 µL; Sample 2
w=0.0516 w =0.0437
w =0.0433
w=0.0861
w=0.0819
w =0.2257
w =0.0400 w =0.0449
Standard Halo
w=0.0516
Halo Peptide
w=0.5411
w=0.4671
w=0.0491 w=0.1043
w=0.0457 w=0.0837
w =0.0777 w =0.0384
Sample 1
Sample 2
Gly-Tyr (238 Da), Val-Tyr-Val (380 Da), Met Enkephalin (574 Da), Angiotensin II (1046 Da) Leu Enkephalin (556 Da), Ribonuclease (13.7 kDa), Porcine Insulin (5807 Da)
Leu Enkephalin. Bovine Insulin (5734 Da), Human Insulin (5808 Da). Cytochrome C (12.4 kDa), Lysozyme (14.4 kDa)
Conclusions Fused-core silica packing materials with enlarged pore size (160 Å) have been successfully created. These packing materials exhibit a surprisingly narrow particle size distribution and high physical stability. Columns prepared from the moderate pore size Halo particles exhibit excellent separation efficiencies, for both small and larger molecules. A highly robust material, Halo Peptide ES-C18, has use for peptide and small protein separations.
Fused-Core Particle Analysis Electron Micrographs of Halo Peptide
Standard Halo vs. Halo Peptide 3500
3000
4.9% RSD
2500
Halo Peptide mean = 2.85 um, SD = 0.14 Standard Halo mean = 2.82 um, SD = 0.14
2000
1500
1000
500
0 0 ‐500
1
2
3
4
5
Particle Diameter, [µm]
6
Fused-Core® Peptide Particles Silica --------------------High Purity Type B Overall diameter---------------------2.8 µm Solid core diameter------------------1.7 µm Porous shell thickness---------------0.5 µm Avg. pore diameter-------------------16 nm Surface area, nitrogen---------------70 m2/g Pore volume ------------------------0.23 mL/g Particle density ---------------------1.3 cm3/g
Fused-Core Pore Analysis Standard Halo vs. Halo Peptide N2 Absorption
Fused-Core Pore Size Analysis Standard Halo vs. Halo Peptide Inverse SEC Test Conditions: Columns: 4.6 x 150 mm Mobile Phase: THF Flowrate: 1.0 ml/min Samples: polystyrene standards Detection: UV at 254 nm
1000000
Log Molecular Weight
100000
Fused-core Peptide Silica 150 A 10000
Standard fused-core silica 90 A
1000
Relative Retention Volume 100
Column Efficiency Solute: beta‐amyloid (1‐38) MW = 4131
Halo, 9 nm pores
Halo Peptide, 16 nm pores
Columns: 4.6 x 100 mm; Particle size: 2.7 μm Mobile Phase: 50% ACN/50% water/0.1% TFA Temperature: 25°C Agilent 1100 with autosampler
Columns: 4.6 x 100 mm; Particle size: 2.7 μm Standard Halo: 27% ACN/73 % water /0.1% TFA; k = 3.0 at 1.0 mL/min Halo Peptide: 29% ACN/71% water /0.1% TFA; k = 3.0 at 1.0 mL/min Temperature: 60°C Instrument: Agilent 1100 with autosampler using both 3 and 6 µL heat exchangers
Sample Loading Column: 4.6 x 100 mm Halo Peptide ES-C18; T= 60 °C Sample: Bovine insulin, MW= 5733; Inj vol = 10 µL Mobile Phase: 0.1% TFA/31.5% ACN, Flow Rate: 1 mL/min
Halo Peptide, 16 nm
Halo, 9 nm
Column Stability Column: 2.1 x 100 mm, Halo Peptide ES-C18; Flow Rate: 0.5 mL/min; T= 60 °C A: 0.1% TFA; B: 0.1% TFA/70%; ACN; Gradient: 9-55% B in 10 min.; Inj vol: 5 µL (Gly-Tyr, Val-Tyr-Val, Met-enk, Angio II, Leu-enk, RNAase, P. Insulin) Shimadzu UFLC XR
Injection 775 Injection 600 Injection 400 Injection 200 Injection 1
t (min)
Halo Peptide ES-C18 Protein and Peptide Separations Column: 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: 0% to 50% B in 15 min.; Injection volume: 5 µL w =0.0303
w =0.0686
Standard Halo w =0.0372
w =0.0413
w =0.0294
w =0.0437
Standard Halo w=0.0819
w =0.0400 w =0.0449
w =0.0433
w=0.0861
w =0.2257
Halo Peptide w =0.0376
w=0.0516
w =0.0392
w =0.0777
w=0.0516
Halo Peptide
w=0.5411
w=0.4671
w=0.0491 w=0.1043
w=0.0457 w=0.0837
w =0.0384
Sample 1 Gly-Tyr, Val-Tyr-Val, Met-enk, Angiotensin II, Leu-enk Ribonuclease, Porcine Insulin
Sample 2 Leu-enk Bovine Insulin, Human Insulin, Cytochrome C, Lysozyme
Tryptic Digest Column: 2.1 x 100 mm, Halo Peptide ES-C18; Flow Rate: 0.25 mL/min; T= 45 °C A: 0.1% TFA; B: 0.1% TFA/80% ACN; Gradient: 5-65% B in 120 min.; Sample: 15 µL (1 µg/µL) apo-myoglobin digest Agilent 1200 SL npc = 212 Pmax = 124 bar
ti
Minutes
Peak capacity [npc] calculated as tf – ti ; Peaks with * used for W4σ
W4σ
tf
Rapid Separation at High Temperature Column: 2.1 x 50 mm Halo Peptide ES-C18; Flow: 0.5 mL/min; A: 0.1% TFA; B: 0.1% TFA/80% ACN; Gradient: 15-50% B in 12.5 min.; Sample: 5 µL (250-500 ng) A Peptides
100 °C
90 °C
A(12-28)
80 °C
70 °C
better recovery at higher temps
Longer Columns for High Resolution Column: 2 @ 2.1 x 100 mm Halo Peptide ES-C18; Flow: 0.5 mL/min; T= 45 °C A: 0.1% TFA; B: 0.1% TFA/80% ACN; Gradient: 5-65% B in 120 min.; Sample: 15 µL (1 µg/µL) tryptic digests
Apo-myoglobin
Transferrin
2 columns in tandem
npc = 297 Pmax = 476 bar
Solute: beta-amyloid (1-38) MW = 4131
Halo, 9 nm pores
Halo Peptide, 16 nm pores
Columns: 4.6 x 100 mm; Particle size: 2.7 μm Standard Halo: 27% ACN/73 % water /0.1% TFA; k = 3.0 at 1.0 mL/min Halo Peptide: 29% ACN/71% water /0.1% TFA; k = 3.0 at 1.0 mL/min Temperature: 60°C Instrument: Agilent 1100 with autosampler using both 3 and 6 µL heat exchangers
7.0
6.5
Solute: beta‐amyloid (1‐38) MW = 4131
6.0
3 µm, 300 Å
Reduced Plate Height, h
5.5
5.0
4.5
4.0
3.5
3.0
Halo Peptide, 2.7 µm, 160 Å
2.5
2.0
1.5 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Mobile Phase Velocity, mm/sec
Columns: 4.6 x 100 mm; Particle size: 3.0 μm and 2.7 μm : 28% ACN/72 % water /0.1% TFA; k = 3.6 at 1.0 mL/min Halo Peptide: 29% ACN/71% water /0.1% TFA; k = 3.0 at 1.0 mL/min Temperature: 60°C Instrument: Agilent 1100 with autosampler using both 3 and 6 µL heat exchangers
Halo Peptide ES-C18 Comparison with 90 Ångstrom Halo C18 • For peptides > 5 aa residues, retention is increased on the Halo Peptide ES-C18 column. • For larger molecules, peak widths are greatly reduced due to improved mass transfer. Column: 4.6 x 100 mm; Flow rate: 1.5 mL/min; Temperature: 30° C A: 10% ACN/90%Water/0.1% TFA;B: 70% ACN/30% Water/0.1 % TFA Gradient: 0% to 50% B in 15 min.; Injection volume: 5 µL; Sample 1 w =0.0303
Standard Halo
w =0.0686 w =0.0392
w =0.0372 w =0.0413
w =0.0294
Halo Peptide w =0.0376
Column: 4.6 x 100 mm; Flow rate: 1.5 mL/min; Temperature: 30° C A: 10% ACN/90%Water/0.1% TFA;B: 70% ACN/30% Water/0.1 % TFA Gradient: 15% to 50% B in 15 min.; Injection volume: 5 µL; Sample 2
w=0.0516 w =0.0437
w =0.0433
w=0.0861
w=0.0819
w =0.2257
w =0.0400 w =0.0449
Standard Halo
w=0.0516
Halo Peptide
w=0.5411
w=0.4671
w=0.0491 w=0.1043
w=0.0457 w=0.0837
w =0.0777 w =0.0384
Sample 1
Sample 2
Gly-Tyr (238 Da), Val-Tyr-Val (380 Da), Met Enkephalin (574 Da), Angiotensin II (1046 Da) Leu Enkephalin (556 Da), Ribonuclease (13.7 kDa), Porcine Insulin (5807 Da)
Leu Enkephalin. Bovine Insulin (5734 Da), Human Insulin (5808 Da). Cytochrome C (12.4 kDa), Lysozyme (14.4 kDa)
Conclusions Fused-core silica packing materials with enlarged pore size (160 Å) have been successfully created. These packing materials exhibit a surprisingly narrow particle size distribution and high physical stability. Columns prepared from the moderate pore size Halo particles exhibit excellent separation efficiencies, for both small and larger molecules. A highly robust material, Halo Peptide ES-C18, has use for peptide and small protein separations.
