The Challenge of Separating and Analyzing Polysaccharide Mixtures
Utilizing Asymmetric Flow Field-Flow Fractionation (AF4) Coupled to
Multiangle Light Scattering (MALS)
Claudia Zielke and Lars Nilsson
Dept. of Food Technology Engineering and Nutrition, Faculty of Engineering LTH, Lund
University, Sweden, [email protected]
Polysaccharides are biopolymers which often have complex characteristics in regard to – besides
others – size, molar mass, structure and properties in aqueous solutions. These characteristics may
influence the functionality of these substances and, thus, understanding them is essential. Separation
and analysis of polysaccharides and especially of complex polysaccharide mixtures can be very
challenging and only few analytical techniques are suitable for the task. Challenges typically relate to
broad size and molar mass distributions but often also to the tendency of the substances to form supramolecular aggregates. AF4 in combination with MALS and dRI (differential refractive index)
detection is a well suited method but molar mass and size determination can still be challenging when,
for instance, analyzing complex samples and heterogeneous mixtures of polysaccharides.
Nevertheless, careful and systematic method development is a requirement in order to obtain high
quality data.
One often observed – but often as artifact of data treatment neglected – phenomenon in AF4 is a
downturn in the molar mass vs. elution time at high retention. The results in this work show that this
phenomenon is a correct measurement, where same molar mass species are separated due to their
different hydrodynamic radii. Furthermore, these effects are discussed in regard to disturbances such
as shifts in retention time and peak distortion due to phase separation in mixtures and changing
channel positions.
Fig. 1: Often observed downturn in the molar mass (grey dotted) vs. elution time plot of
polysaccharides at high retention (Displayed: AF4-MALS-dRI results, black – Rayleigh ratio from
MALS, red – dRI signal)