10
SEC
size exclusion
chromatography
The role of Size Exclusion Chromatography in
process purification
Size exclusion chromatography (SEC), also known as gel
filtration,
separates molecules in an aqueous mobile phase
according to their physical size in solution as they pass
through a porous structure.
Molecules with a diameter
greater than the largest pores within the resin material are
unable to enter the particle. Because they are excluded
from the pores they travel quickly through the column and
elute first. Smaller molecules, which are able to access
pores within the resin particles, permeate a larger acces-
sible volume within the column and are eluted later, in
order of decreasing molecular weight.
SEC is applicable in final polishing steps where a target
protein is being separated from its aggregates or other
significantly different molecular weight impurities. Another
application area is the desalting of the purified target
protein.
Tosoh Bioscience offers a number of TOYOPEARL HW-type
products for size exclusion chromatography (Table I). These
same SEC TOYOPEARL HW-type products are chemically
modified with functional groups to make the TOYOPEARL
products shown in the later sections of this catalog.
Much of the information in this catalog section pertains to
the available pore sizes, fractionation ranges, and particle
sizes of the TOYOPEARL HW-type products, along with
their physical and chemical properties. This data not only
aids in the evaluation of the best resin for a SEC process
step, but also lends insight into understanding the physical
nature and the selection process of all TOYOPEARL resins.
Resin chemistry
TOYOPEARL size exclusion resins are highly hydroxylated
polymethacrylic polymer beads (Figure 1). Their surface
hydroxyl groups render them very hydrophilic and useful
for protein separations. TOYOPEARL products including
the functionalized materials seen in
later catalog sections,
have the least non-specific binding of any chromatographic
resin. This is of particular note for separations such as blood
factors where backbone interactions with the feedstock
may result in decreased recovery of the targets. Their semi-
rigid polymeric nature also gives them better pressure-flow
characteristics than softer materials such as agarose.
Pore size
Commercial TOYOPEARL HW-type size exclusion materials
are
available in 5 pore sizes covering 5 different fraction-
ation ranges. The choice of TOYOPEARL HW products
depends on the molecular weight of the feedstock compo-
nents. Tables I and II show this information for proteins,
dextrans and PEG polymers.
Resin Chemistry of Toyopearl SEC resins
(Hydroxylated Acrylic)
CH
3
CH
3
C CH
2
CH
2
C
C=O
C=O
O
O
R-(OH)
R-(OH)
O
O
C=O
C=O
C
C
CH
2
CH
2
CH
3
CH
3
R = Hydroxylated
Aliphatic Group
Resin chemistry of toyopearl sec resins
(hydroxylated acrylic)
figure 1
features
Benefits
small particles available
high resolution
hydrophilic porous polymer structure
minimal non-specific adsorption effects
narrow particle size distribution
high performance SEC – more efficient separations
better pressure-flow characteristics
good mechanical stability
excellent flow characteristics in large
industrial size columns
chemically stable (pH 2 – 14)
constant packing volume over a wide range of salt
concentrations
compatible with organic solvents, can be cleaned in place (CIP)
with acid or base
stable polymer may be run at elevated temperature (4 - 60 °C)
autoclavable at 121 °C
identical resin structure to TSKgel HPLC resins
direct scale-up from TSKgel HPLC columns
