Basic HTML Version
HIC
80
Modulation of selectivity
The addition of organic solvents or chaotropic agents in the final buffer
can improve separations. However, relative elution positions may
change. Therefore, add chaotropic agent and organic solvent in small
quantities. See
FIGURE 4
for the effect of chaotropic agents and organic
solvents on the HIC separation of two different samples.
Antibiotics
The TSKgel Ether-5PW column was used to determine the relative
purity of the antibiotic components C-1027 and C-1027-AG as shown in
Figure 5
. Antibiotic C-1027 is composed of a protein consisting of many
hydrophobic and hydroxyamino acids with a non-protein chromophore.
Antibiotic C-1027-AG is composed of the hydrophobic and hydroxyamino
acids without the chromophore.
figure 4
Effect of urea and isopropanol on the separation of commercial lipoxi-
dase and a standard protein mixture
Effect of ure and isopropanol on the separation of
commercial lipoxidase and a standard protein mixture
Column:
TSKgel Phenyl-5PW, 7.5mm ID x 7.5cm
Sample:
A & B: commercial lipoxidase
C & D: protein mixture:
1. cytochrome C; 2. myoglobin
3. ribonuclease A; 4. lysozyme
5.
α
-chymotrypsinogen; 6.
α
-chymotrypsin
Elution:
A: 60min linear gradient from 0.1mol/L phosphate buffer
containing 1.5mol/L (NH
4
)
2
SO
4
(pH 7.0) to 0.1mol/L
phosphate buffer (pH 7.0)
B: 60min linear gradient from 0.1mol/L phosphate buffer
containing 1.5mol/L (NH
4
)
2
SO
4
(pH 7.0) to 0.1mol/L
phosphate buffer containing 2mol/L urea (pH 7.0)
C: 60min linear gradient from 0.1mol/L phosphate buffer
containing 1.8mol/L (NH
4
)
2
SO
4
(pH 7.0) to 0.1 mol/L
phosphate buffer (pH 7.0)
0
30
60
90 0
30
60
90
0
30
60
0
30
60
A
B
D
C
1
2
3
4
5
6
1
2
3
4
5
6
Minutes
Minutes
Minutes
Minutes
Applications - TSKgel hic columns
Column: TSKgel Phenyl-5PW, 7.5 mm ID x 7.5 cm L;
Sample: A & B: commercial lipoxidase, C & D: protein mixture: 1. cytochrome
C, 2. myoglobin, 3. ribonuclease A, 4. lysozyme, 5.
a
-chymotrypsinogen,
6.
a
-chymotrypsin; Elution: A: 60 min linear gradient from 0.1 mol/L phosphate
buffer containing 1.5 mol/L (NH
4
)
2
SO
4
(pH 7.0) to 0.1 mol/L phosphate buffer
(pH 7.0), B: 60 min linear gradient from 0.1 mol/L phosphate buffer containing
1.5mol/L (NH
4
)
2
SO
4
(pH 7.0) to 0.1 mol/L phosphate buffer containing 2 mol/L
urea (pH 7.0), C: 60 min li ear gradient from 0.1 mol/L phosphate buffer con-
taining 1.8 mol/L (NH
4
)
2
SO
4
(pH 7.0) to 0.1 mol/L phosphate buffer (pH 7.0),
D: 60min linear gradient from 0.1 mol/L phosphate buffer containing 1.8 mol/L
(NH
4
)
2
SO
4
(pH 7.0) to 0.1 mol/L ph sphate buffer (pH 7.0) containing 7% iso-
propanol; Flow Rate : A & B: 0.5 mL/min; C & D: 1.0 mL min;
Temp.: 25°C; Detection: UV@280nm
figure 5
Purification of anti-tumor antibiotic
Purification of anti-tumor antibiotic
Column:
TSKgel Ether-5PW, 7.5mmID x 7.5cm
Sample:
C-1027
C-1027-AG
concentration: 1mg/mL
Injection:
20µL
Elution:
linear gradient from 1.5mol/L to 0mol/L (NH
4
)
2
SO
4
in 0.1mol/L phosphate buffer, pH 7.0
Flow Rate:
0.8mL/min
Detection:
UV @ 220nm
Minutes
0
10
20
5
15
25
0.06
0.04
0.02
0
Absorbance @ 220nm
Column: TSKgel Ether-5PW, 7.5 mm ID x 7.5 cm L; Sample: C-1027, C-1027-AG
concentration: 1 mg/mL; Injection: 20 μL; Elution: linear gradient from
1.5 mol/L to 0 mol/L (NH
4
)
2
SO
4
in 0.1 mol/L phosphate buffer, pH 7.0;
Flow Rate: 0.8 mL/min; Detection: UV@220nm