12
SEC
The TOYOPEARL HW-type resin range spans peptide
and protein molecular weights between 100 - 50,000,000
Daltons. Each TOYOPEARL HW-type resin displays a typical
calibration curve and exclusion limit for globular proteins
(Figure 2).
TOYOPEARL HW-65 and HW-55 resins are the base beads
for many TOYOPEARL products listed in the later sections
of this catalog. A
product having “-650” in its name uses
the TOYOPEARL HW-65 resin for the base bead. Products
having “-550” are derived from the TOYOPEARL HW-55
bead. The impact of pore size and its relationship to
dynamic binding capacity and resin selectivity for a partic-
ular protein application is discussed in subsequent catalog
sections.
See the hydrophobic interaction chromatography section
of this catalog for TOYOPEARL PPG-600, TOYOPEARL
Phenyl-600 and
TOYOPEARL Butyl-600 resins which
are functionalized on the
TOYOPEARL HW-60 bead (not
available for use in SEC).
Particle size
Resolution increases with decreasing particle size (Figure 3).
Resin particle size is proportional to HETP and inversely
proportional to the column efficiency and resolution of two
peaks.
Most TOYOPEARL HW-type resins are available in three
particle size ranges:
S-grade = 20 - 40 µm (Superfine)
F-grade = 30 - 60 µm (Fine)
C-grade = 50 - 100 µm (Coarse)
When the highest resolution is needed, the smaller S and F
grade beads are preferred for process SEC. For desalting,
where the resin is used in a filtration mode to remove the
target from a buffer, the C grade is primarily employed
because of its better flow dynamics at lower operating
pressures.
Some TOYOPEARL HW-type products are also manufac-
tured in “M-grade” (40-90 µm) and “EC-grade” (100-300 µm)
to be used as the base beads for functionalized materials.
These special grades are not commercially offered as SEC
products.
Please note that for the functionalized base beads in later
catalog
sections a “C-grade” is specified as a 50-150 µm
bead and is not the SEC “C-grade” range of 50-100 µm.
size exclusion
chromatography
Calibration curves for globular proteins on Toyopearl resins
Ve / Vc
HW-75
HW-65
HW-55
HW-50
HW-40
Log MW (Da)
7
6
5
4
3
2
1
0.30 0.40 0.50 0.60 0.70 0.80
Column: 22 mm ID x 30 cm L; Sample: Protein standards; Mobile phase:
0.06 mol/L phosphate buffer (pH 7.0) in 0.06 mol/L KCI; Legend: Ve = elution
volume, Vc = column volume; Detection: UV @ 280 nm
figure 2
Calibrationcurves forglobularproteinsonTOYOPEARL
Comparison of resolution on different particle sizes of
Toyopearl HW-55
1
2
3
4
C-grade
F-grade
S-grade
1
2 3
4
1
2
3
4
1
1.5
2
2.5
Elution Time (hr)
Column: TOYOPEARL HW-55, 26 mm ID x 70 cm L
Sample: 1. Thyroglobulin (0.3 %), 2.
g
-Globulin (0.3 %), 3.
b
-Lactoglobulin
(0.3 %), 4. Cytochrome C (0.1 %); Mobile phase: 33.3 mmol/L phosphate
buffer (pH 7.0), 0.2 mol/L NaCI; Flow rate: 106 mL/h (20 cm/h); Inj. vol.: 1 mL;
Temperature: 25 °C; Detection: UV @ 280 nm
figure 3
Comparison of resolution on different particle sizes
of toyopearl hw-55
