BioHPLC Column Selection Guide Cover - Agilent Technologies

Reversed-Phase HPLC
Higher resolution of intact monoclonal antibody
Column: ZORBAX RRHD 300SB-C8
857750-906
2.1 x 50 mm, 1.8 µm
Mobile Phase: A: H20:IPA (98:2) + 0.1% TFA (v/v)
B: IPA:ACN:H20 (70:20:10) + 0.1% TFA (v/v)
Flow Rate: Between 0.5 mL/min and 1.0 mL/min
Gradient: Multi-segmented and linear elution
Temperature: 80 ºC
Detector: Agilent 1290 LC Infinity system with auto injector
(ALS), binary pump and thermostatted oven
and diode array detector (DAD)
Detection: UV, 225 nm
Higher resolution of oxidation study
Column: ZORBAX RRHD 300SB-C18
857750-902
2.1 x 50 mm, 1.8 µm
Mobile Phase: A: 0.1% TFA
B: 0.01% TFA + 80% ACN
Flow Rate: 1.0 mL/min
Gradient: 33 to 50% B, 0 to 4 min
Detector: 1290 Infinity LC with diode array detector at 280 nm
Sample: Insulin, insulin chain A and chain B, oxidized
(bovinesigma, 1 mg/mL)
Tips & Tools
Typical mobile phases for protein and peptide separations combine a very low pH with TFa (or other acids)
to solubilize proteins. stableBond columns have extremely long lifetimes under these conditions. They are
available in 300Å pore size for proteins up to 00-500 kDa.
20 www.agilent.com/chem/biohplc
mAU
250
Gradient A
0.5 mL/min
200
150
Optimized resolution
100
50
0
Shoulder Resolved
0 2 4 6 8 10 min
mAU
350
mAU
100
Gradient B
1.0 mL/min
250
Increased
High speed anaysis time
150
sensitivity
50
0
mAU
17.5
15
12.5
10
7.5
5
2.5
0
0 2 4 6 8 10 min
0.148
oxidized insulin
chain A
1.384
80
60
40
20
0
oxidized insulin
chain B insulin
1.648
1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 min
Expanded View
subspecies of oxidized insulin chain B
It is evident that the oxidized insulin chains are resolved from insulin in under 2 minutes using the Agilent ZORBAX RRHD 300SB-C18 2.1 x 50 mm,
1.8 µm column.
min