49
HIC
Figure 11 compares the selectivities of the TOYOPEARL
Phenyl-600M,
TOYOPEARL Phenyl-650M and an Agarose
Phenyl resin.
For smaller molecules such as lysozyme (14,300 Da) the
even narrower pore diameter TOYOPEARL SuperButyl-550C
resin (derived from the 50 nm pore diameter TOYOPEARL
HW-55) is recommended. A comparison of the DBC of
TOYOPEARL SuperButyl-550C with other TOYOPEARL HIC
resins is shown in Figure 12.
The TOYOPEARL Phenyl-600M resin also has a high DBC
for lysozyme (Figure 13). The engineered higher dynamic
binding capacity of the 600 and 550 series HIC products for
their specific targets and the selectivity differences induced
by the smaller mean pore size of the respective beads can
have a dramatic impact on process economics.
ToyoScreen prepacked columns for process
development
ToyoScreen columns packed with the full range of our
TOYOPEARL HIC
products are available in 1 mL and 5 mL
resin volumes. They provide a
convenient way to screen
different resins for both target retention and
recovery.
Multiple columns can be connected in series for addi-
tional
separation. Please see the ordering information at
the end of this section or contact us for more information
on these products.
ToyoScreen RoboColumns are miniaturized chromato-
graphic columns for operation with a robotic liquid
handling system, such as the Freedom EVO
®
from TECAN.
This approach allows automated high-throughput, small-
scale biochromatographic separations of protein samples
by running up to eight individual columns simultaneously.
ToyoScreen RoboColumns packed with the most popular
TOYOPEARL HIC media are available with 200 µL and 600 µL
resin volumes.
hydrophobic interaction
chromatography
figure 11
Selectivity comparison of phenyl-type resins
Column:
7.5cm ID X 7.5cm
Sample:
1 g/L RNase A (1), lysozyme (2)
and
α
-chymotrypsinogen A (3)
Sample load:
100µL
Gradient:
60 min. linear gradient from buffer A toB
Buffer A:
1.8mol/L (NH
4
)
2
SO
4
+ 0.1mol/L
sodium phosphate (pH 7.0)
Buffer B:
0.1mol/L sodium phosphate (pH 7.0)
Linear velocity: 136cm/hr
Temperature:
25°C
Toyopearl Phenyl-650M
0
0
50
100
150
10
20
30
40
50
60
70
UV @ 280 nm (mAU)
Elution volume (mL)
1
2
3
Toyopearl Phenyl-600M
Phenyl Agarose
Column: 7.5 mm ID x 7.5 c L; Sample: 1 g/L RNase A (1), lysozyme (2)
and
a
-chymotrypsinogen A (3); Sample load: 100 µL;
Gradient: 60 min. linear gradient fro buffer A to B;
Buffer A: 1.8 mol/L (NH
4
)
2
SO
4
+ 0.1 mol/L sodium phosphate (pH 7.0);
Buffer B: 0.1 mol/L sodium phosphate (pH 7.0);
Linear velocity: 136 cm/h (1.0 mL/min); Temperature: 25 °C;
Detector: UV @ 280 nm
Selectivity comparison of phenyl-type resins
figure 12
Typical Dynamic Binding Capacities for Lysozyme
Binding capacity (mg/mL)
(10% Breakthrough)
Ether-650M
12.5
Phenyl-650M
27.5
Butyl-650M
32.2
Hexyl-650C
33.2
PPG-600M
44.2
SuperButyl-550C
54.3
Conditions
Column size:
7.8mm ID x 20cm
Sample:
1mg/mL Lysozyme in 0.1mol/L Phosphate Buffer +
1.8mol/L Sodium Sulfate (pH7.0)
Linear Velocity: 100cm/h
Detection:
UV @ (280nm)
0
0.01
0.02
0.03
Absorbance (UV:280nm)
0
20
40
60
80
Lysozyme loaded (mg/mL)
Phenyl-650M
Butyl-650M
Ether-650M
PPG-600M
Hexyl-650C
SuperButyl-550C
Binding capacity (mg/mL)
(10% Breakthrough)
Ether-650M
12.5
Phenyl-650M
27.5
Butyl-650M
32.2
Hexyl-650C
33.2
PPG-600M
44.2
SuperButyl-550C
54.3
Column size: 7.8 mm ID x 20 cm L; Sample: 1 mg/mL Lysozyme in 0.1 mol/L
phosphat buffer + 1.8 mol/L sodium sulfate (pH 7.0); Linear Velocity: 100
cm/h (0.8 mL/min); Detection: UV @ 280 nm
Typical Dynamic Binding Capacities for Lysozyme
