BioHPLC Column Selection Guide Cover - Agilent Technologies
BioHPLC Column Selection Guide Cover - Agilent Technologies
BioHPLC Column Selection Guide Cover - Agilent Technologies
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Rapid human polyclonal IgG quantification<br />
using the <strong>Agilent</strong> Bio-Monolith Protein A HPLC column<br />
<strong>Column</strong>: Protein A<br />
5069-3639<br />
5.2 x 4.95 mm<br />
Mobile Phase: PBS buffer, pH 7.4<br />
0.5 M acetic acid, pH 2.6<br />
Flow Rate: 1 mL/min<br />
Relative absorbance [mAU]<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
Gradient: Stepwise gradient: 100% buffer<br />
A-100% buffer B-100% buffer A (0.5 min each step)<br />
0<br />
0<br />
0 0.5 1 1.5<br />
Time [min]<br />
2 2.5 3<br />
affinity Chromatography<br />
Detector: A high pressure gradient HPLC system, <strong>Agilent</strong> 1200 - UV at 280 nm<br />
Sample: Human Plasma diluted with binding buffer (PBS buffer, pH 7.4)<br />
The selectivity of the Bio-Monolith Protein A column for the IgG from human plasma. IgG binds to protein A, a 100% buffer B step gradient is<br />
applied, and IgG elutes at 1.5 min.<br />
SDS PAGE analysis of fractions from the separation.<br />
Key:<br />
Lane 1: Whole serum prior to separation<br />
Lane 2: IgG standard<br />
Lane 3: Peak 1 (flow-through fraction)<br />
Lane 4: Peak 2 (Protein A-bound fraction; i.e. IgG1 and IgG2)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
% Gradient<br />
UHPLC/HPLC<br />
9
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