GPC/SEC is a vital analytical
technique for characterizing
Gel permeation/size exclusion chroma- tography (GPC/SEC) is a vital ana- lytical technique used to characterize
synthetic and natural polymers, including
biologically important macromolecules
such as proteins and DNA. Evolving challenges—such as the development of smart,
novel polymers with exacting performance
specifications; the replacement of conventional polymers with those that are more
environmentally benign; and the formulation of biopharmaceuticals—tax the capabilities of traditional GPC/SEC and invite
advances in the technology.
A critical tool for molecular
A basic understanding of how GPC/
SEC works provides an important basis
from which to assess the boundaries of
the technique and the value of recent
GPC/SEC is a two-step analytical
technique in which samples are separated into fractions on the basis of
hydrodynamic size, followed by characterization of the fractions. Separation is
achieved by passing the sample through
a packed chromatography column.
Detecting the amount of sample in each
sized fraction enables determination of
the size distribution and, more importantly, molecular weight and molecular
weight distribution data.
Traditional GPC/SEC systems use a single concentration detector; most commonly a refractive index (RI) detector. Column
calibration, using appropriate standards,
provides the correlation needed to estimate
a molecular weight distribution from mea-
surements of the amount of sample (con-
centration) in each sized fraction. Howev-
er, the resulting molecular weight data are
only accurate if the relationship between
molecular size and weight is the same for
the sample as for the standard. This is a
crucial limitation, most especially for novel
materials where there are often no appro-
priate standards, and where absolute rather
than relative data may be critical.
In recent years, the limitations of
a single detector set-up have been
addressed by increasing the use of
multiple detectors. For example, a light
scattering detector, in combination with
a concentration detector, enables direct
measurement of the absolute molecular
weight of the eluting molecules, eliminating the need for column calibration
standards. Further complementary
additions include a viscometer, to enable
the measurement of structural features,
such as branching or conformation, and
a photodiode array (PDA) detector for
detailed investigation of the distribution
of chromophores as a function of molecular size/weight.
The two-step nature of GPC/SEC
means the associated instrumentation is
well-suited to progressive enhancement.
In many laboratories, the evolution of the
technique can be seen from the design of
successively attached detectors. However,
today’s busy laboratories typically demand
levels of accuracy and productivity that
can’t be delivered by such “user-developed”
systems, often with multiple software
packages and complex data integration.
It’s increasingly important that all users,
often a variety of researchers and analysts
measuring very different sample types, can
quickly and easily access high quality data.
Innovations in the latest GPC/SEC systems answer to these requirements with
easier-to-use integrated systems that deliver greater sensitivity and accuracy, while
requiring only a small amount of sample,
often a critical requirement at early stages
Faster, more precise separation
Precisely resolved separation of the
sample is a critical first step in any GPC/
SEC experiment, and has a defining
influence on the overall accuracy of the
data. The performance of the separation
module directly influences the stability
of the analysis baseline and, consequently, the signal-to-noise ratio achieved
during detection. Better separation performance translates directly into more
accurate measurements (Table 1).
In novel separation modules—such
as Malvern Instruments’ OMNISEC
RESOLVE—each element of the system—
Tailoring GPC/SEC to Today’s Applications
Performance improvement The practical value Features to look for
Better baseline stability and
improved signal-to-noise ratio.
Improved sensitivity. High-performance pumps for low pulsation.
Efficient degasser performance.
Quicker set-up and reduced
time to changeover from one
sample type to another, as well
as lower sample usage.
Ease-of-use, especially when
analyzing a range of different
sample types and/or when
samples are precious.
Low-volume degassers and injector designs with
minimal or zero sample loss.
Precise temperature control. Ease-of-use, especially for
samples that are temperature
sensitive, or when using high
viscosity solvents. Better reso-
lution, higher accuracy.
Precise temperature control of the three key
instrument regions: all detectors and the
inter-detector tubing connecting them; the
autosampler storage tray ( 4 C required for
proteins); and the column oven that can be
control at C7, and up to C5.
Table 1: Improvements in separation performance: Summarizing
the latest features and the benefits they bring.
Source: Malvern Instruments