Multi-angle light scattering (MALS) measures the molar mass and size distributions of heterogeneous biopolymers, as well as branching via conformational analyses.


Multi-angle light scattering coupled to size exclusion chromatography (SEC-MALS) or field-flow fractionation (FFF-MALS) is the most robust and reliable method for characterizing heterogeneous distributions typical of biopolymers such as polysaccharides, hyaluronic acid, cellulose or polylactic acid.

Since MALS determines molar mass and size without regard for elution time, it does not depend on identifying and qualifying well-characterized molecular standards for each type of biopolymer and solvent. The analysis of mass and size at each elution volume permits ASTRA® software to calculate various characteristic values of the distribution such as weight-, number- or z-averaged mass and size.

Is your biopolymer branched or otherwise structured? Quantitative determination of conformation can also be obtained from MALS analysis through the measured relationship between size and molar mass.


An important component of the blood coagulation chain, heparin requires careful, complete characterization. Light scattering offers several methods in which to do so:

Hydroxyethyl starch (HES)

HES is a polysaccharide-based blood replacement manufactured from corn and potato raw materials. The two sources result in HES with different conformations arising from differing degrees of branching. SEC-MALS not only determines accurate molecular weight independent of conformation, it also quantifies the conformation and branching ratio for a deeper understanding of the two HES processes. As described by Karyakin et al., SEC-MALS is an essential component of HES quality control.

Wood, pulp and paper products

Some interesting analytical challenges are posed by biopolymers from wood pulp and similar natural sources.


Lignins tend to absorb laser light and fluoresce. Both phenomena lead to erroneous measurements of molar mass and size, unless the instrumentation and software can overcome these sources of error. The DAWN offers an optional infrared laser plus narrow bandpass optical filters on the detectors to minimize both total fluorescence and the amount reaching the detectors. In addition, the 'Forward Monitor' detector in the DAWN measures laser absorption by the sample to allow the ASTRA software to correct the measured molar mass.


Cellulose forms large, rod-shaped nanocrystals. While they may not be accommodated by standard GPC, these are readily separated by the Eclipse™ DualTec™ field-flow fractionation system, then measured downstream with a DAWN and possibly the WyattQELS integrated dynamic light scattering module. The shape of the nanocrystals is assessed from the dependence of molar mass on size, then fed back into the analysis to refine the accuracy of mass and size distributions.


Light scattering is an excellent means for assessing the degradation of biopolymers as a result of exposure to heat, light, high or low pH, and other stimuli.

Aggregation and fragmentation

Some common forms of biodegradation are aggregation and fragmentation, both readily characterized with high information content via SEC-MALS. Simply take aliquots of a biopolymer sample before and after exposure to environmental or chemical stress and inject onto an appropriate GPC column followed by MALS and dRI detection. The analysis can provide several means of quantifying degradation, e.g. shifts in Mn, Mw and Mz, representing the number-, weight- and z-averaged molecular weights of the sample, respectively. Biodegradation may also lead to changes in molecular conformation, indicated in SEC-MALS by the ratio of rms radius Rg to molecular weight.

Particle formation

Another manifestation of biodegradation is the formation or dissociation of particulates. For a quick assessment of nanoparticle populations, DLS is ideal, requiring little sample and very little time for preparation an measurement. While the size of monodisperse particles can be measured accurately by DLS, size distributions tend to be more qualitative than quantitative. A more thorough analysis is provided by FFF-MALS, which separates both soluble and insoluble components with excellent resolution so they may be analyzed by downstream light scattering detectors.


While very slow processes are amenable to analysis by SEC-MALS via periodic sampling from a reaction vessel, batch MALS, batch DLS and CG-MALS offer alternative approaches to analyzing kinetics of more rapid reactions.

If there is enough time between initiating the reaction, and mixing and pipetting to a cuvette, batch MALS or batch DLS can work. The Calypso can perform this automatically with a dead time of just a few seconds.

Absolute molar mass & size

Agilent StackOnly multi-angle light scattering (MALS) can determine the absolute molar mass and size distributions of heterogeneous polymers independently of retention time and molecular standards, and regardless of non-ideal column interactions. That's because MALS measures molecular weight and rms radius (a.k.a. radius of gyration) directly from first principles. All you need is a convenient means of size-based separation preceding the on-line MALS detector.

Couple a DAWN MALS detector and an Optilab refractive index concentration detector to your favorite size exclusion chromatography (SEC) or gel permeation chromatography (GPC) system to create a SEC-MALS absolute characterization tool for polymers. Wyatt detectors interface with HPLC systems for all major vendors.

Molar Mass v. Time

Hydrodynamic Radius v. Time

For more advanced separation capabilities consider the advantages of field-flow fractionation coupled to MALS detectors – FFF-MALS. Wyatt's Eclipse DualTec and Eclipse AF4 FFF systems separate nanograms to milligrams over sizes from 1 to 1000 nm, without shear or non-ideal column interactions.


Molecules smaller than 10 nm in radius require a ViscoStar differential viscometer to determine size or else a WyattQELS dynamic light scattering module embedded in the MALS detector for size measurements.

radius v. elution plot

Figure 1. RMS radius vs. elution volume plot of acrylic copolymer.


Even if the polymer is not branched, information regarding conformation is available in the relationship between molar mass and size, or between the ratio of rms radius Rg to hydrodynamic radius Rh.

Figure 2. Polystyrene, PMMA, cellulosic rods and hyaluronic acid exhibit different slopes corresponding to different conformations.

Figure 3. Theoretical relationship between mass and radius for different conformations.


Figure 4. Structure of an HB polyester polyol with an Mw of 10,976 g/mol and 10 OH groups. Three major components of fatty acid methyl esters are also displayed (J. Milic et al., ILSC 2012).

Zimm plots

Zimm Plot

Some polymers are too fragile to run through a gel permeation chromatography (GPC) column without degradation, e.g., a macroligand which can dissociate from labile metals. Others, such as large PMMA molecules, may be too large for GPC. These materials can still be characterized by means of batch MALS, which determines weight-average molecular weight Mw and Z-averaged rms radius Rg,z. Accurate determination of these values requires a 'Zimm plot' analysis, i.e., a measurement of light scattering intensity as a function of angle and concentration, without separation.

The traditional method for making Zimm plots involves manual preparation of a series of aliquots with increasing concentrations. Batch MALS measurements are made in a scintillation vial using the Batch Conversion Kit. Alternatively, in the microbatch method the aliquots are injected into the MALS flow cell. In either case ASTRA software analyzes the data via a global fit of all angles and concentrations to a single light scattering equation. The results include Mw, rg,z and the second virial coefficient A2 which indicates solute-solute and solute-solvent interactions.

Several convenient methods for making Zimm plots utilize automation to create a series of dilutions from a single stock solution, injecting the samples into the MALS and RI flow cells, acquiring the concentration and light scattering data automatically and calculating the three parameters. This automation may be carried out with the Calypso composition-gradient system or by programming an autosampler with a large injection loop.

Selected references

Al-Assaf, S.; Phillips, G. O.; Williams, P. A.; du Plessis, T. A. Application of ionizing radiations to produce new polysaccharides and proteins with enhanced functionality. Nucl. Instrum. Meth. B  2007, 265, 37-43.

Alftrén, J.; Peñarrieta, J. M.; Bergenståhl, B.; Nilsson, L. Comparison of molecular and emulsifying properties of gum arabic and mesquite gum using asymmetrical flow field-flow fractionation. Food Hydocolloid.  2012, 26, 54-62.

Andres-Brull, M.; Al-Assaf, S.; Phillips, G. O.; Jackson, K. Optimisation of asymmetrical flow-field fractionation for the characterization of gum arabic (Acacia sengal var senegal) and comparison with gel permeation chromatography. Anal. Methods  2013, 5, 4047-4052.

Peng, Y.; Zhang, L. Characterization of a polysaccharide-protein complex from Ganoderma tsugae mycelium by size-exclusion chromatography combined with laser light scattering. J. Biochem. Bioph. Meth.  2003, 56, 243-252.

Shah, P. N.; Min, J.; Kim, H.-J.; Park, S.-Y.; Lee, J.-S. Chiroptical properties of graft copolymers containing chiral poly(n-hexyl isocyanate) as a side chain. Macromolecules  2011, 44, 7917-7925.

Karyakin, A. V.; Beksaev, S. G.; Flegontov, P. A. Quality Control of Polysaccharide Molecular-Weight Distributions Using Exclusion Chromatography with a Multi-Angle Laser Light Scattering Detector. Pharmaceutical Chemistry Journal  2014, 48(7), 470-477.