14
IEC
Applications with TSKgel 5PW Cation Exchange
Columns
Comparison of strong and weak Cation Exchangers
Differences in selectivity between strong (TSKgel SP-5PW)
and weak (TSKgel CM-5PW) cation exchangers are demon-
strated in Figure 16 which is a separation of globular pro-
teins.
Semi-preparative Purification of Lipoxidase
The purification of 200 mg of crude lipoxidase on a
21.5 mm ID TSKgel SP-5PW column is illustrated in
Figure 17. Scale-up is simplified as only the particle size
changes from 10 μm (7.5 mm ID) to 13 μm (21.5 mm ID) or
20 μm (55 mm ID) colum.
IEC
TSKgel 5PW SERIES
Selectivity of TSK-GEL strong and weak cation exchangers
Columns:
Sample:
Elution:
Flow Rate:
TSKgel SP-5PW and TSKgel CM-5PW, 7.5 mm ID x 7.5 cm L
1. trypsinogen, 2. ribonuclease A, 3. a-chymotrypsinogen,
4. cytochrome C, 5. lysozyme
60 min linear gradient from 0 mol/L to 0.5 mol/L NaCl in
0.02 mol/L phosphate, pH 7.0
1.0 mL/min
Minutes
CM-5PW
SP-5PW
2
1
3
4
4
5
2 1
3
4
4
5
0
40
20
selectivity on TSKgel strong and weak cation
exchangers
Columns:
TSKgel SP-5PW
and
TSKgel CM-5PW
, 7.5 mm ID x 7.5 cm L;
Sample: 1. trypsinogen, 2. ribonuclease A, 3.
a
-chymotrypsinogen, 4. cyto-
chrome C, 5. lysozyme;
Elution: 60 min linear gradient from 0 mol/L to 0.5 mol/L NaCl in 0.02 mol/L
phosphate, pH 7.0; Flow rate: 1.0 mL/min; Detection: UV @ 280 nm
figure 16
Semi-preparative purification of lipoxidase
Minutes
Column:
Sample:
Elution:
Flow Rate:
Detection:
Recovery:
TSKgel SP-5PW, 21.5mm ID x 15cm
crude lipoxidase, 200mg
120min linear gradient from 0mol/L to 0.5mol/L Na
2
SO
4
in
0.02mol/L acetate, pH 4.5
4.0mL/min
UV @ 280nm
Lipoxidase activity collected between the two
vertical lines was 84%
0
30
60
semi-preparative purification of lipoxidase
Column: TSKgel SP-5PW, 21.5 mm ID x 15 cm L; Sample: crude lipoxidase,
200 g; Elution: 120 min linear gradient from 0 mol/L to 0.5 mol/L Na
2
SO
4
in
0.02 mol/L acetate, pH 4.5; Flow rate: 4.0 mL/min; Detection: UV @ 280 nm;
Rec very: Lipoxidase activity collected between the tw vertical lines was
84%.
figure 17
