HIC
73
hic
figure 9
Effect of urea and isopropanol on the separation of commercial
lipoxidase and a standard protein mixture
Effect of ur a nd 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
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 L/min;
Temp.: 25°C; Detection: UV @ 280 nm
Monoclonal Antibodies
Monoclonal antibodies (mAbs) play a part in many research, diagnostic,
and therapeutic applications. Monoclonal antibodies are generally the
most hydrophobic proteins in ascites fluid and cell culture supernatant.
Figure 7
shows typical results from the screening of two mAbs using a
TSKgel Ether-5PW column.
TSKgel Phenyl-5PW
RNAs
Figure 8
illustrates the separation of 16S and 23S ribosomal RNA on
a TSKgel Phenyl-5PW column. The approximate molecular weights of
these RNAs are 560,000 and 1,100,000 Da, respectively.
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 9
for the effect of chaotropic agents and organic
solvents on the HIC separation of two different samples.
Applications - TSKgel hic columns
figure 8
Retain large RNAs on TSKgel Phenyl-5PW
Retain large RNAs on TSKgel Phenyl-5PW
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel Phenyl-5PW, 7.5mm ID x 7.5cm
16S and 23S rRNA from E. coli, 0.05mg in 0.1mL
60min linear gradient from 2mol/L to 0mol/L (NH
4
)
2
SO
4
in 0.1mol/L phosphate buffer, pH 7.0
0.5mL/min
UV @ 280nm
Minutes
0
30
60
16S rRNA
23S rRNA
Column: TSKgel Phenyl-5PW, 7.5 mm ID x 7.5 cmL;
Sample:16S and 23S rRNA from E. coli, 0.05 mg in 0.1 mL; Elution: 0 min
linear gradient from 2 ol/L to 0 mol/L (NH
4
)
2
SO
4
in 0.1mol/L phosphate
buffer, pH 7.0; Flow rate: 60.5 mL/min; Detection:UV @ 280 nm
figure 7
Monoclonal antibody purification
Retention time (minutes)
0
50
110
Mouse
lgG, Kappa
Detector response (AU)
Column: TSKgel Ether-5PW, 10 µm, 8.0 mm ID × 7.5 cm, glass
Mobile phase: 67.5 min isocratic load and wash with 1 mol/L (NH
4
)
2
SO
4
in
1 mol/L glycine, 0.5 mol/L phosphate buffer, pH 7.0, followed by a 37.5 min
linear gradient from 1.0 mol/L to 0 mol/L (NH
4
)
2
SO
4
in 1.0 mol/L glycine, 0.05
mol/L phosphate, pH 7.0; Flow rate: 1.0 mL/min; Detection: UV @ 280 nm,
3.0 AUFS; Injection vol.: 50 mL; Sample: 25 mL raw cell culture supernatant,
200 mg total protein, 15 mg total antibody diluted to 50 mL with initial
elution buffe
