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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size exclusion Chromatography (seC)<br />
Pore Size Choice<br />
The choice of media pore size will influence the resolution in SEC. As the separation is based on differences in molecular size in solution the sample<br />
must be able to permeate the porous structure of the particles – if the pore size is too small the samples will be excluded from the pores and elute<br />
in the void volume of the column and if too large then all will be able to fully permeate the particles and so there will be very little separation.<br />
Pore size choice: Proteins<br />
<strong>Column</strong> A: Bio SEC-3, 100Å<br />
5190-2503<br />
4.6 x 300 mm, 3 µm<br />
<strong>Column</strong> B: Bio SEC-3, 150Å<br />
5190-2508<br />
4.6 x 300 mm, 3 µm<br />
<strong>Column</strong> C: Bio SEC-3, 300Å<br />
5190-2513<br />
4.6 x 300 mm, 3 µm<br />
Mobile Phase: 50 mM Na 2HPO 4, 50 mM NaH 2PO 4<br />
+ 0.15 M NaCl, pH 6.8<br />
Flow Rate: 0.35 mL/min<br />
Detector: UV @ 220 nm<br />
Sample: BioRad Gel Filtration Standards Mix<br />
Pore size choice: Mouse IgG<br />
<strong>Column</strong> A: Bio SEC-3, 100Å<br />
5190-2503<br />
4.6 x 300 mm, 3 µm<br />
<strong>Column</strong> B: Bio SEC-3, 150Å<br />
5190-2508<br />
4.6 x 300 mm, 3 µm<br />
<strong>Column</strong> C: Bio SEC-3, 300Å<br />
5190-2513<br />
4.6 x 300 mm, 3 µm<br />
Mobile Phase: 50 mM Na 2HPO 4, 50 mM NaH 2PO 4<br />
+ 0.15 M NaCl, pH 6.8<br />
Flow Rate: 0.35 mL/min<br />
Detector: UV @ 220 nm<br />
Sample: Mouse IgG<br />
0 www.agilent.com/chem/biohplc<br />
mAU<br />
2500<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
mAU<br />
1400<br />
1200<br />
1000<br />
800<br />
600<br />
400<br />
200<br />
0<br />
100Å<br />
150Å<br />
0 2.5<br />
100Å<br />
150Å<br />
300Å 1<br />
2<br />
3<br />
4<br />
5.0 7.5 10.0 12.5 15.0 17.5 20.0<br />
PoreSz_Proteins<br />
300Å<br />
5<br />
6<br />
1. Thyroglobulin Aggregates<br />
2. Thyroglobulin<br />
3. IgA<br />
4. γ-globulin<br />
5. Ovalbumin<br />
6. Myoglobin<br />
7. Vitamin B12<br />
7<br />
0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0<br />
PoreSz_Mouse<br />
1<br />
2<br />
3<br />
1. Dimer<br />
2. Monomer<br />
3. Monomer Fragment<br />
4. Azide<br />
4
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