Waters BioSuite Peptide Analysis Columns - Manual (Page 2)

[ Care and Use ManUal ]
BioSuite Peptide Analysis Columns 2
d. BioSuite C
18
3 μm, PA-B
BioSuite C
18
3 μm, PA-B is a 300Å, high ligand density, monofunctional,
silica-based column.
• Outstanding performance when separating complex digests
containing hydrophillic, hydrophobic, as well as basic peptides
• Superior peak shapes and acpacity for peptide separations using
TFA containing eluents (e.g., 0.1% TFA)
• Good choice for the separation of larger peptide fragments
generated by non-trypsin endoproteases (e.g., Lys-C)
This section contains recommended operating conditions and specifications
for Waters’ BioSuite C
18
3 μm PA-A and C
18
3.5 μm PA-B Columns.
e. Operating Conditions
1. Shipping Solvent: BioSuite C
18
3 μm PA-A Column: 60%
Acetonitrile (CH
3
CN)/40% H
2
0 BioSuite C
18
3.5 μm PA-B Column:
50% CH
3
CN/50% H
2
0
2. Pressure: BioSuite Peptide Analysis Columns can tolerate
pressures of up to 6,000 psi (400 bar or 40 Mpa) although
pressures greater than 4,000-5,000 psi should be avoided in
order to maximize column and HPLC system seal lifetimes.
Effect of Flow Rate and Separation Temperature on Observed System
Backpressure: BioSuite Peptide Analysis Columns contain 3 μm or 3.5 μm
particles. With HPLC, column i.d. and length being equal, observed HPLC
system backpressures when using columns packed with either 3 μm or 3.5 μm
particles are higher than those observed when using columns packed with
either 5 μm or 10 μm particles. As indicated in Table 1, chromatography
performed at an elevated temperature (e.g., 40 °C) via the use of a column
heater will significantly reduce the observed system backpressure when
using a BioSuite Peptide Analysis Columns. When performing peptide separa-
tions at ambient temperature (especially on BioSuite 2.1 mm x 250 mm or
4.6 mm x 250 mm Peptide Analysis Columns), reduced operating flow rates
may be required to avoid exceeding the maximum recommended operating
pressure for the HPLC system.
Table 1: Effect of Flow Rate and Separation Temperature on HPLC System
Backpressure using BioSuite Peptide Analysis Columns (250 mm length)
Note:
1. Observed HPLC System backpressures were recorded using a Waters
Alliance ® HPLC System. System backpressures may vary from reported
values depending on your HPLC system and configuration.
BioSuite C
18
, 3 μm PA-A (4.6 mm x 250 mm) with H
2
0/CH
3
CN (60/40) Eluent
Effect of Different Flow Rates at 40 °C on HPLC System Backpressure
Flow (mL/min) System Pressure (psi) Column Temp (°C)
0.50 1200 40
0.75 1750 40
1.00 2400 40
1.25 3050 40
1.50 3700 40
Effect of Different Separation Temperatures at 1.5 mL/min on HPLC System
Backpressure
Flow (mL/min) System Pressure (psi) Column Temp (°C)
1.50 4400 30
1.50 4050 35
1.50 3700 40
1.50 3400 45
1.50 3100 50
BioSuite C
18
, 3.5 μm PA-A (2.1 mm x 250 mm) with H
2
0/CH
3
CN (60/40)
Eluent
Effect of Different Flow Rates at 40 °C on HPLC System Backpressure
Flow (mL/min) System Pressure (psi) Column Temp (°C)
0.10 1000 40
0.16 1650 40
0.20 2030 40
0.26 2650 40
0.31 3330 40
Effect of Different Separation Temperatures at 0.31 mL/min on HPLC System
Backpressure
Flow (mL/min) System Pressure (psi) Column Temp (°C)
0.31 4000 30
0.31 3600 35
0.31 3330 40
0.31 3030 45
0.31 2800 50