BioHPLC Column Selection Guide Cover - Agilent Technologies

Capillary and Nano Columns
Proteins in a complex sample by 2-D HPLC
with Nano HPLC columns
Column: ZORBAX 300SB-C18
5065-9913
0.3 x 5 mm, 5 µm
Column: ZORBAX 300SB-C18
5065-9911
0.075 x 150 mm, 3.5 µm
Mobile Phase: Quaternary Pump: 3% Acetonitrile/0.1% Formic acid
Nanopump: A = Water, 0.1% Formic acid,
B = ACN, 0.1% Formic acid
Flow Rate: Quaternary Pump: 30 µL/min
Nanopump: 300 nL/min
Gradient: Quaternary Pump: Isocratic
Nanopump:
6 min = 3% B, 120 min = 60% B, 125 min = 80% B,
130 min = 80% B, 131 min = 3% B, 140 min = 3% B
Tryptic digest of bovine serum albumin (BSA).
The base peak chromatograms show a
selection of fractions from a 2-D HPLC
separation. Single chromatograms represent
peptides from BSA eluting at a given salt
concentration followed by enrichment and
reversed-phase chromatography.
112 www.agilent.com/chem/biohplc
Intens.
x10
1.0
6
0.5
x10 5
6
4
2
x10
6
5
4
2
x10
1.0
6
0.5
MS Conditions: Source: Nano ESI, drying gas flow: 5 L/min, drying gas temp: 225 °C
Ion Trap: Skim: 1:35 V, cap exit offset: 115 V, octopole 1:12 V,
octopole 2:3.5 V, trap drive: 80 V. ICC: on, averages: 4, max accu time:
150 ms; target 60.000, ion mode positive, MS/MS mode.
Sample: Tryptic Digest of bovine serum albumin
Volume: 1 to 8 µL
Salt Step Elution: 8 mL of 10 mM-100 mM KCI (10 mM increments),
125 mM, 150 mM, 200 mM, 300 mM, 500 mM, 1 M.
5 10 15 20
Time (min)
25 30 35 40
30 mM KCI
60 mM KCI
75 mM KCI
120 mM KCI
LCCN004