Iec
IEC
55
figure 16
Separation of nucleosides by ion-exchange chromatography on
TSKgel SP-2SW
A.
pH 3.5
1
1
2
2
3
3
5
10
Separation of nucleosides by ion-exchange chromatography
on TSKgel SP-2SW
Column:
TSKgel SP-2SW 4.6mm ID x 25cm
Sample:
Nucleoside Standards: 1) Guanosine, 2) Cytidine, 3) Adenosine
Mobile Phase: A) 0.1 mol/L sodium citrate - phosphoric acid buffer, pH 3.5
B) 0.1 mol/L sodium citrate - acetic acid buffer, pH 4.25
Flow Rate:
0.75 mL/min
Temperature: 23°C
Detection:
UV detection @ 260nm
inj.
inj.
0
5
10
0
B.
pH 4.25
Minutes
Minutes
figure 14
Selectivity on TSKgel strong and weak cation exchangers
Selectivity of TSK-GEL strong and weak cation exchangers
Columns:
Sample:
Elution:
Flow Rate:
Detection:
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
UV @ 280 nm
Minutes
CM-5PW
SP-5PW
2
1
3
4
4
5
2 1
3
4
4
5
0
40
20
Columns:
TSKgel SP-5PW
and
TSKgel CM-5PW
, 7.5 mm ID x 7.5 cm L;
Sample: 1. trypsinogen, 2. ribo uclease A, 3. a-chymotrypsin gen, 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
TSKgel SP-5PW and TSKgel CM-5PW
Differences in selectivity between strong (TSKgel SP-5PW) and weak
(TSKgel CM-5PW) cation exchangers are demonstrated in
Figure 14
which is a separation of globular proteins.
The purification of 200 mg of crude lipoxidase on a 21.5 mm ID TSKgel
SP-5PW column is illustrated in
Figure 15
. 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) columns.
TSKgel SP-2SW, CM-2SW and CM-3SW
Silica-based cation exchangers are typically used in the separation
of low molecular weight compounds such as pharmaceuticals,
nucleotides, catecholamines, and small peptides. For example,
Figure 16
shows the separation of nucleosides on TSKgel SP-2SW.
Column: TSKgel SP-5PW, 21.5 mm ID x 15 cm L; Sample: crude lipoxidase,
200 mg; 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;
Recovery: Lipoxidase activity collected between the two vertical lines was
84%.
Column: TSKg l SP-2SW 4.6 mm ID x 25 cm L
Sample: Nucleoside Standards: 1) Guanosine, 2) Cytidine, 3) Adenosine
Mobile phase: A) 0.1 mol/L sodium citrate - phosphoric acid buffer, pH 3.5
B) 0.1 mol/L sodium citrate - acetic acid buffer, pH 4.25
Flow rate: 0.75 mL/min
Semi-preparative purification of lipoxidase
Minutes
Column:
Sample:
Elution:
Flow Rate:
Detection:
Recovery:
TSKgel SP-5PW, 21.5m 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
figure 15
Semi-preparative purification of lipoxidase
