BioHPLC Column Selection Guide Cover - Agilent Technologies

Improved reproducibility of monoclonal antibodies
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
B: IPA:ACN:H20 (70:20:10), 0.1% TFA
Flow Rate: 1.0 mL/min
Temperature: 80 ºC
Detector: 1290 Infinity LC with diode array detector at 225 nm
Sample: MAb
Reversed-Phase HPLC
0 0.5 1 1.5 2 2.5 3 min
Gradient timescale
Time (min) % Solvent B
500
pressure
gradient wash and
re-equilibration region
0.00 25
300
3.00 35
4.00 90
100
UV
5.00 25 0 5 min
Excellent column reproducibility and protein recovery using Agilent ZORBAX 300SB-C8.
Increased resolution for peptide mapping
Column: ZORBAX 300SB-C18
858750-902
2.1 x 100 mm, 1.8 µm
Mobile Phase: A: 0.1% TFA
B: 0.01% TFA + 80% ACN
Flow Rate: 0.5 mL/min
Gradient:
Temperature: 50 ºC
2% B for 1 min, 2 to 45% B
for 8.8 min, 45 to 95% B
for 0.2 min, 95% B for 2 min,
95 to 2% B for 0.2 min
Detector: 1290 Infinity LC with diode array
detector at 280 nm
Sample: Enzymatic protein digest (MAb)
Norm
350
300
250
200
150
100
50
0
-50
mA
400
300
200
100
mA
700
1st injection
150th injection
pre- and post-150
injection column
blank run overlays
1 2 3 4 5 6 7 8 9 min
The longer 100 mm Agilent ZORBAX RRHD 300SB-C18 column provides maximum resolution for protein digests – in this sample the total run
time, including washing and equilibration, is under five minutes.
bar
750
500
250
UHPLC/HPLC
2