Owner's manual

[ 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