BioHPLC Column Selection Guide Cover - Agilent Technologies

ion-exchange Chromatography
Exceptional separating power
Column: Agilent Bio SCX, stainless steel
5190-2423
4.6 x 50 mm, 3 µm
Buffer: 10 mM Phosphate, pH 6.0
Flow Rate: 0.5 mL/min
Gradient: 0-1.0 M NaCl, 15 min
Detector: 280 nm
The hydrophilic, polymeric layer and densely packed ion-exchange functional
groups provide extremely sharp peak shapes and high resolution of a mixture
of proteins with a broad range of isoelectric points (pI).
Separation of protein standards on Agilent 3 µm ion-exchange columns
by cation-exchange chromatography
Column A: Agilent Bio SCX, NP 3, 4.6 x 50 mm, SS
Column B: Agilent Bio WCX, NP 3, 4.6 x 50 mm, SS
Column C: Agilent Bio MAb, NP 3, 4.6 x 50 mm, SS
Mobile Phase: A: 10 mM NaH2PO4.2H2O, pH 5.70
B: A + 1 M NaCl
Flow Rate: 0.5 mL/min
Gradient: 0 min - 100% A : 0% B
25 min - 0% A : 100% B
Temperature: Ambient
Detector: Agilent Infinity 1260 Bio-inert HPLC system -
with diode array detector at 220 nm
Sample: Cytochrome c, ribonuclease A, lysozyme and protein mix
52 www.agilent.com/chem/biohplc
mAU
40
30
20
10
1
0 2 4 6 8 10 12 min
Illustration that Bio WCX, SCX and MAb columns are capable of producing protein separations
2
3
4
5
1. Ovalbumin, 4.6 pl
2. Ribonuclease A, 8.7 pl
3. Cytochrome c, 9.6 pl
4. Aprotinin, 10.0 pl
5. Lysozyme, 11.0 pl
N > 100,000/50 mm for Lysozyme
Agilent column Peak number Peak name RT [min] Height [mAU] Area [mAU*s] Plates Width [min] Resolution
Bio WCX NP, 3 µm 1 Cytochrome c 7.86 124 1833 7844 0.2089 -
2 RNase A 9.03 241 3358 10800 0.2044 3.32
3 Lysozyme 13.13 636 7274 44488 0.1466 13.73
Bio SCX NP, 3 µm 1 RNase A 7.06 396 2616 39847 0.0832 -
2 Cytochrome c 7.66 297 2778 28920 0.1060 1.08
3 Lysozyme 10.49 763 7186 44828 0.1167 1.37
Bio MAb NP, 3 µm 1 Cytochrome c 10.04 203 2369 21814 0.1600 -
2 RNase A 11.37 256 2690 33314 0.1467 3.11
3 Lysozyme 12.59 652 6616 56734 0.1244 5.28
SCX
WCX
MAb
1
2
1
2
1
3
2
3
3