Dynamic light scattering (DLS) is a popular method used for measuring the size and size distribution of particles and molecules. Over the years, several myths have evolved regarding the value of light scattering and its usage in various applications. This article exposes some of these common misconceptions and provides answers for each.
DLS and SLS are Same Methods – False
DLS and Static light scattering (SLS) are two different techniques and measure different things. DLS investigates the fluctuations of scattered light over time and processes this signal by means of an auto-correlation technique, which in turn gives a particle's translational diffusion coefficient (DT) and then determines its size distribution, mean hydrodynamic radius (Rh), or polydispersity.
In SLS, the total scattered intensity of light is measured as a virtue of concentration or angle. This is usually summarized in a Debye or Zimm. It is then possible to measure the radius of gyration (Rg), the weight average molecular weight (Mw), and the second virial coefficient (A2) for the samples under study.
Molecular Weight Measurements through SLS Need Multiple Angles – False
SLS can examine either the concentration or angular-dependent scattering relationship, or both concentration and angle. It is a common misconception that multi angle instrumentation is important for absolute molecular weight measurements.
SLS measurements can be performed by examining the intensity of light as either a virtue of concentration or angle. When below the size limit of 1/20 the wavelength of the laser, there will be no angular dependence in the scattered light and no extra information can be obtained by adding multiple detectors.
SLS Offers Improved Resolution than DLS when Analyzing Different Oligomeric States – False
SLS can measure molar mass or weight average molecular weight and needs fractionation or separation technique to resolve different oligomeric states.
Likewise, when DLS is combined with a separation or fractionation technique, higher resolution can also be obtained over batch-mode measurements when the sample contains oligomers.
However, both SLS and DLS techniques produce the weight average molecular size or weight of everything in the batch sample in the absence of a separation technique.
DLS Analysis Needs Dilute Samples for Successful Measurement - False
Although some light scattering devices need dilute samples to disperse within the optimal response range for the detector(s), contemporary light scattering instruments can employ a method of auto-attenuation of laser or alter the measurement position inside the sample to off-set multiple scattering and sample concentration without the necessity for altering the sample.
DLS is not Suitable for Samples Containing Aggregated Materials - False
As the size of a particle increases, the amount of light dispersed by the particle also increases considerably. If some amount of light (X) is scattered by a particle of a given size, the particle size doubles the scattered light intensity to 84x more light.
This makes DLS very sensitive in the detection of even trace amounts of agglomerates or aggregates, making it extremely useful in a variety of applications. The detection and quantitation of these larger aggregates is as important as the characterization of the main particle or molecule itself.
Commercial Electrophoretic Light Scattering Measurements Denature Biological Samples – False
When loading or studying biological samples, special care may be required, and appropriate instruments will automatically enable flexibility and user discretion in selecting the exact measurement settings.
Based on the conductivity of the measured sample, instruments with good zeta potential can automatically adjust the applied voltage to produce a field strength that causes electrophoresis and yet is gentle enough to not denature biological samples.
Always Filter Samples before Light Scattering Analysis - False
Although it is better to filter samples before light scattering analysis to ensure they are dust free, it is not needed in the majority of instruments. Older DLS systems using 90-degree scattering angles indeed made it difficult to perform DLS measurements in the presence of dust or aggregates.
However the current generation of instruments, particularly those integrated with data quality factor indicators, backscatter optical designs, and dust rejection logic, are capable of performing analyzes on samples containing dust and aggregates.
DLS Data Interpretation is Difficult - False
Modern DLS instruments produce data and data reports that are user-friendly and usually contain a quality factor that enables users to assign a degree of confidence to the quality of the data.
When DLS data is difficult to interpret, it is mostly due to a lack of understanding of the sample instead of what the data is essentially reporting.
When Using MALS system, More Angles Means Greater Accuracy - False
The number of angles in a multi-angle light scattering (MALS) detector is not the main basis of data accuracy. It has been established that when a system contains more angles, the molecular weight will be more precise.
However, it is the precision of the extrapolation to 0° that eventually leads to more precisely calculated Rg values and molecular weight, and while having more angles is important, it is the lowest angles that are most vital.
DLS Analysis Should Report the Same Particle Size as that Reported by TEM - False
DLS is capable of determining the molecule or particle’s hydrodynamic radius i.e. the hydrated molecule or particle as it travels via a suspending medium, and it is an intensity-weighted size.
Transmission electron microscopy (TEM) creates an image of particles as electrons, which are transmitted via the sample and result in a Number-weighted size distribution. Intensity-weighted particle size is more influenced by the larger particles in the sample, while number-weighted particle size is more influenced by the present of smaller particles.
As such, Intensity-weighted sizes will be larger when compared to Number-weighted sizes in the absence of other sampling problems. In fact, both sizes are correct as they are simply measuring and reporting different things.
Conclusion
DLS is a non-calibrated technique and hence does not require calibration. This technique is well established for measuring the size and size distribution of particles/molecules.
The above answers should help to dispel some doubts related about the value and usage of light scattering in various applications.
This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.
For more information on this source, please visit Malvern Panalytical.