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HIC
81
hic
RNAs
Figure 6
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.
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 mol/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@280nm
Column: TSKgel Phenyl-5PW, A.) 7.5 mm ID x 7.5 cm L and B.) 21.5 mm
ID x 15 cmL; Sample: 1. myoglobin, 2. ribonuclease A, 3. lysozyme, 4.
a
-chymotrypsinogen, 5.
a
-chymotrypsin; Elution: 60 min linear gradient
from 1.8 mol/L to 0 mol/L (NH
4
)
2
SO
4
in 0.1 mol/L phosphate buffer, pH 7.0;
Flow Rate: 0.5 mL/min (7.5 mm ID) or 4 mL/min (21.5 mm ID); Detection:
UV@280nm
Proteins
Figure 7
compares the resolution of standard proteins on analytical
and preparative TSKgel Phenyl-5PW columns. Different flow rates
compensated for the change in particle size and column dimensions.
High resolution was obtained on both columns.
Antibody Fragments
Figure 8
shows the separation of Fab and Fc fragments of an antibody
on TSKgel Butyl-NPR. The appearance of additional Fc fragments is due
to the oxidation of methionine residues by 0.10% t-butylhydroperoxide
(tBHP). The numbers above the Fc peaks correspond to the number of
oxidized residues in each fragment.
Column: TSKgel Butyl-NPR, 4.6 mm ID x 3.5 cm L; Elution: Buffer A: 2
mol/L (NH
4
)
2
SO
4
, 20 mmol/L Tris, pH 7, Buffer B: 20 mmol/L Tris, pH 7;
Gradient: linear from 10 % B to 100 % B in 34 minutes; Flow rate:1 mL/min;
Temperature: 30°C
figure 7
Scale up to preparative separations
figure 6
Retain large RNAs on TSKgel Phenyl-5PW
Retain large RNAs on TSKgel Phenyl-5PW
Column:
Sample:
Elution:
Flow Rate:
Detecti n:
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
Scale up to preparative separations
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKg l Phenyl-5PW, A.) 7.5mm ID x 7.5cm and
B.) 21.5mm ID x 15cm
1. myoglobin, 2. ribonuclease A, 3. lysozyme,
4.
α
-chymotrypsinogen, 5.
α
-chymotrypsin
60min linear gradient from 1.8mol/L to 0mol/L (NH
4
)
2
SO
4
in 0.1mol/L phosphate buffer, pH 7.0
0.5mL/min (7.5mm ID) or 4mL/min (21.5mm ID)
UV @ 280nm
Minutes
30
60
0
30
60
1 2
3
4
5
1
2
3
4
5
A. Analytical Column
B. Preparative Column
0
Minutes
figure 8
Separation of Fab and Fc fragments on TSKgel Butyl-NPR
Separation of F
ab
and F
c
fragments on
TSKgel Butyl-NPR
Column:
Elution:
Gradient:
Flow rate:
TSKgel Butyl-NPR, 4.6mm ID x 3.5cm
Buffer A: 2mol/L (NH
4
)
2
SO
4
, 20mmol/L Tris, pH7
Buffer B: 20mmol/L Tris, pH7
linear from 10%B to 100%B in 34 minutes
1mL/min
mAU
100
50
0
10 12 14 16
Time (min)
mAU
100
50
0
10 12 14 16
Time (min)
mAU
100
50
0
10 12 14 16
Time (min)
F
ab
peaks
F
c
peaks
1
2
0
C - Incubated with 0.1% tBHP
B - Incubated with 0.01% tBHP
A - Incubated with 0% tBHP (control)
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Applications - TSKgel hic columns