Size exclusion chromatography (SEC) and dynamic light scattering (DLS) are common technologies in
protein laboratories. SEC is used routinely for the
purification, identification, and quantification of
protein mixtures, while batch DLS is used for pre-column
size and polydispersity measurements, along
with aggregate quantification. Until now, coupling
of the two technologies has been difficult—largely
because the eluted protein sample concentrations
from SEC are often well below the detection limit
of most DLS instruments. The recent introduction
of a chromatography flow mode for the high-sensitivity Zetasizer® Nano particle characterization system (Malvern® Instruments, Westborough, MA)
changes this situation. By overcoming the problem
of detection limits, it makes the coupling of SEC and
DLS technologies a practical and attractive option.
Combining the high resolution of chromatography
with the ease of data acquisition from DLS has the
advantage of allowing rapid peak identification.
It also confers the ability to perform absolute size
exclusion chromatography (ASEC), whereby protein
size can be measured in real time, under flow,
as the samples are eluted from the column, rather
than relying on column calibration or additional
data from a second detector. Good estimates of
molecular weight can then be computed rapidly
on the basis of protein size. This has the practical
advantage of negating the need for molecular
weight standards with conventional SEC in order
to identify oligomeric species or determine protein
purity. In short, ASEC allows:
- Confirmation of oligomeric state—monomer,
- Purity determination
- Quantification of mixtures
- Confirmation of protein identity
- Detection of low-molecular-weight impurities.
As shown in the examples that follow, ASEC provides
a valuable new tool for protein laboratories.
Figure 1 - Setup for absolute size exclusion chromatography.
In standard size exclusion chromatography, after the
column, the eluent flows through one or more detectors,
typically monitoring ultraviolet light absorbance
and refractive index in order to detect the presence of
molecular species. For absolute size exclusion chromatography,
a DLS instrument, namely a Zetasizer Nano,
simply needs to be connected after the last detector
(Figure 1). The absolute nature of the measurement
means that no column calibration is required and the software makes the entire measurement process
simple and straightforward. For the majority of
protein separations in aqueous media, no extra
parameters are required. Size is plotted in real time
as material is eluted from the column, and the
molecular weight of each peak is calculated automatically
at the end of the measurement.
The other detector outputs can be displayed
simultaneously with the light scattering data,
and by determining the volume delay between
detectors during the setup procedure, the data
output from all detectors is aligned.
The conversion of molecular size to molecular
weight using DLS is particularly simple and accurate
for globular materials such as proteins, as
demonstrated by the examples that follow.
Protein samples were prepared in an appropriate
buffer and loaded onto a Superdex 200 column
(GE Healthcare, Uppsala, Sweden). Each measurement
used a flow rate of 0.5 mL/min. Dynamic
light scattering measurements were accumulated
continuously and analyzed every 3 sec. The Zetasizer
Nano software recorded and stored all correlation
functions and intensity values.
Bovine serum albumin
Bovine serum albumin (BSA) of expected molecular
weight 67 kDa was prepared in 0.1 M phosphate
buffered saline at a concentration of 8 mg/mL; 500 μL (4 mg of protein) was loaded onto
the Superdex 200 column. Such a quantity of
BSA slightly overloaded the column, leading to
nonideal separation of the component oligomers.
Figure 2 - Hydrodynamic diameter (z-average, nm, in blue) and light
scattering intensity (kilo counts per second, in green) versus elution volume
for BSA, injected onto a Superdex 200 column.
Figure 2 shows the z-average size (DLS) and the
light scattering intensity trace. The monomer
peak eluted between 15.2 and 16.0 mL, corresponding
to a hydrodynamic diameter of 7.4 nm.
This is very close to the expected size for this molecule.
On the basis of this size, the computed molecular
weight prediction was 72 kDa, slightly larger than
the 67 kDa expected. Nevertheless, this estimate
clearly confirms that this material is the BSA monomer,
rather than any other oligomer.
Figure 3 - Eluted size distribution from BSA in PBS. In the analysis
of the elution profile, the majority by intensity is found to be a monomer.
Results can also be interpreted in terms of size distribution
to give an indication of the relative composition
of the original sample. Figure 3 shows the size distribution
of the BSA sample. The majority of the protein
(60.2% by intensity) is contained in the
monomer elution peak. The next peak
is the dimer with 20% of the signal. This
distribution is a much higher resolution
than can be obtained by a “batch” measurement,
i.e., measurement of the sample
without separation by SEC.
Figure 4 - Hydrodynamic diameter (z-average, nm, in
blue) and light scattering intensity (kilo counts per second, in
green) versus elution volume for IgG4, injected onto a Superdex
An antibody (IgG4) of expected
molecular weight around 150 kDa was
prepared in 0.1 M ammonium bicarbonate
buffer (pH not adjusted) at a
concentration of 7 mg/mL; 100 μL (0.7
mg of protein) was loaded onto the
Superdex 200 column.
Figure 5 - Hydrodynamic diameter (z-average, nm, in
blue) and light scattering intensity (kilo counts per second, in
green) versus elution volume for carbonic anhydrase.
The main monomer peak eluted between 13.3
and 14.2 mL, corresponding to a hydrodynamic
diameter of 11.6 nm (Figure 4). This is close to
the expected size for this molecule. A prediction
of the molecular weight from the measured size
leads to 200 kDa, rather more than the nominal
150 kDa expected. The higher size is due to the
Y-shape of the IgG4 molecule—antibodies do not
give an ideal fit with the globular protein family
model. A typical compact globular protein of 150
kDa would have a size of 10 nm. Nevertheless, the
estimate of 200 kDa clearly confirms the presence
of the monomer.
Carbonic anhydrase of expected molecular weight
29 kDa was prepared in phosphate buffered saline at
a concentration of 5 mg/mL; 100 μL (0.5 mg of protein)
was loaded onto the Superdex 200 column.
The single peak, eluted between 18.0 and 18.6
mL, had a z-average size of 4.81 nm diameter
(Figure 5), corresponding to an estimated molecular
weight of 26.2 kDa for a globular protein.
This simple size measurement confirmed the
main elution peak as that of the carbonic anhydrase
The latest generation of DLS systems, specifically
the Zetasizer Nano, allow the coupling of dynamic
light scattering and size exclusion chromatography
in the form of absolute SEC. A powerful, easy-to-use
protein characterization tool, ASEC has the advantage
of providing a rapid and direct measurement of
protein size without the need for costly and time-consuming
column calibration. In addition, since
size versus mass relationships for globular proteins
are well known, DLS measurements of the hydrodynamic
radius can also be used to calculate molecular
weight. The same technique is applicable to other
types of chromatographic measurement, such as
affinity or ion exchange chromatography, thereby
enhancing the experimental return.
Figure 6 - Zetasizer Nano.
About dynamic light scattering
Dynamic light scattering is a powerful technique for determining structural characteristics such as molecular weight, size, melting point, and charge (zeta potential) of particles in solution. One of its great advantages is its ability to measure a large population of particles in a very short time period, with no manipulation of the dispersing medium.The Zetasizer Nano (Figure 6) is a benchtop instrument that includes both the hardware and software for combined dynamic, static, and electrophoretic light scattering measurements. The system was designed specifically to meet the low concentration and sample volume requirements typically associated with pharmaceutical and biomolecular applications, along with the high concentration requirements for colloidal applications. With a size range of 0.6 nm to 6 μm, and a concentration range of 0.1 ppm to 30% w/v, its specifications for sample size and concentration far exceed those of other commercially available DLS instruments.
Dr. Nobbmann is Nanometrics Specialist, Malvern
Inc., 117 Flanders Rd., Westborough, MA
01581, U.S.A.; tel.: 508-768-6400; fax: 508-768-6403;