BioHPLC Column Selection Guide Cover - Agilent Technologies

Baseline expansion of a separation
of protein standards
Column: Agilent Bio-Monolith CM15,
5.5 x 15 mm
Mobile Phase: A: 10 mM Na2HPO4, pH 6.0
B: A + 0.5 M NaCl or just 0.5 M Na2HPO4, pH 6.0
Flow Rate: 2 mL/min
Gradient: 0.5 min hold with mobile phase A followed by a
linear gradient to 45% B in 15 min (elapsed time
15.5 min); then 60% B at 15.6 min continued to
20 min. Column flushed with 100% B for 15 min
before re-equilibration for the next run.
pH Gradient: A: 5 mM Na2HPO4, buffer pH 5.5
and B: 40 mM NA2HPO4 (not buffered, pH 8.9).
2% B/min at 1 mL/min for 15 min, followed by
a column wash with 90% B for 5 min.
Detector: UV at 220 nm
Sample: One mg each/mL in mobile phase A.
1. RNAse from bovine pancreas (pI 9.6)
2. Cytochrome c from bovine heart (pI 10.37-10.8)
3. Lysozyme from chicken egg (pI 11.35) (0.5 mg)
mAU
14
12
10
8
6
4
mAU
14
12
10
8
6
4
ion-exchange Chromatography
4 6 8 10 12 14 16 18 min
4
4.483
4.968
4.284
4.520
4.688
4.945
5.653
6.357
6.834
1 2 3
A with NaCl gradient
7.832
6.482
7.059
7.604
8.162
9.072
9.271
10.335
12.170
B with Na 2HPO 4 gradient
6 8 10 12 14 16 18 min
Instrument: Agilent 1200 SL with diode array detector B shows a better resolution of protein contaminants.
16.410
16.901
UHPLC/HPLC
3