Dynamic Light Scattering (DLS), sometimes referred to as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering, is a technique classically used for measuring the size of particles typically in the sub-micron region, dispersed in a liquid. The sensitivity of some modern systems is such that it can also now be used to measure the size of macromolecules in solution.
In this new webinar, I summarise how the technique works, what its size and concentration limits are and discuss its advantages and disadvantages.
This webinar was well-attended and I received several questions, I thought that I will list some of them in the Q&A below.
Q: What are the main reasons for a low intercept in the correlation function?
The intercept value is the signal/ background ratio and the value will vary according to the sample and instrument optical configuration. Appropriate values are between 10% and 100% (i.e. 0.1 to 1.0). A lower than expected value may indicate too high or too low a sample concentration, absorption or fluorescence of the sample. Too high a sample concentration may lead to multiple scattering effects that will reduce the intercept value. The NIBS configuration of the Zetasizer Nano S/ZS/ZSP allows for the path length of the laser beam to be minimized, which, in turn, will minimize any multiple scattering present. If there is low excess scattering i.e. the difference in scattering between the dispersant and the particles dispersed in the dispersant, then the intercept will be low. The presence of sample fluorescence or absorption can also reduce the intercept. In the case of fluorescence, there is the option of installing a narrow band filter into the instrument which will remove any fluorescent light present.
Q: Can I use the size data (size value) from number-weighted analysis? Is that reliable?
DLS measures the time-dependent fluctuations in the intensity of scattered light and therefore the fundamental information obtained from the technique is based upon the intensity of scattered light. The z-average diameter is the intensity-weighted mean diameter obtained from the cumulants analysis as defined in ISO22412 (2017) and the primary size distribution is based upon intensity.
Whilst the transformation of the measured intensity distribution to volume or number seems straightforward, DLS users are strongly cautioned to be careful not to over analyze the results. The DLS technique tends to overestimate the width of the peaks in the distribution and this effect is magnified in the transformations to volume and number. The volume and number size distributions should only be used for estimating the relative amounts of material in separate peaks as the means and particularly the widths are less reliable. Therefore, it is recommended to use the intensity size distribution for reporting the size of each mode in the distribution but to use the volume or number data for reporting the relative amounts of each particle family in the sample.
Q: What is the difference between dynamic light scattering and static light scattering?
Dynamic light scattering measures the time-dependent fluctuations in the scattered light intensity which allows the determination of the translational diffusion coefficients (i.e. Brownian motion) and hence particle/molecular size.
Static light scattering measured the time-averaged intensity in the scattered light. In the Zetasizer product range, the time-averaged intensity of the scattered light is measured as a function of sample concentration (i.e. molecular solutions) and from this, the absolute molecular weight of a macromolecular solution is determined. In the Mastersizer product range, the time-averaged intensity of scattered light is measured as a function of angle and this allows the determination of the particle size.
Q: Does the z average depend on the concentration of the particle suspension?
The answer to this question is yes, it can. DLS assumes that the particles/molecules being measured are undergoing random, Brownian motion. This is true at a dilute sample concentration. However, as the sample concentration increases, other effects can happen such as multiple scattering, restricted diffusion, particle-particle interactions. Whereas the influence of multiple scattering can be minimized by using backscatter detection and measuring close to the wall of the cuvette, restricted diffusion and particle-particle interactions are more problematic to understand. Normally, restricted diffusion can be compensated for by using the sample, rather than dispersant, viscosity. Particle-particle interactions are much more complex and cannot be compensated for.
The size obtained from a DLS measurement should be independent of sample concentration (ISO22412 (2017)). However, when the size obtained for any sample is dependent upon the concentration of the sample, a dilution series should be performed and the measured diffusion coefficient of each sample concentration should be plotted as a function of concentration and the data extrapolated to zero concentration. The value obtained at zero concentration is the true diffusion coefficient (i.e. mean size) of the sample. This is known as a dynamic Debye plot and is discussed in ISO22412 (2017). If you have the advanced protein feature key installed with your Zetasizer software, the Calculators contain a dynamic Debye plot.
I have attached some documents which discuss concentration effects in more detail.
- Application of Dynamic Light Scattering (DLS) to Protein Therapeutic Formulations: Principles, Measurements, and Analysis – 2. Concentration Effects and Particle Interactions
- FAQ – What is multiple scattering?
- FAQ – What is restricted diffusion?
Q: What is the minimum size measurable be DLS?
The lower size limit of dynamic light scattering depends on many factors such as the optical configuration of the instrument, laser wavelength/power, detector sensitivity, sample concentration and excess level of scattering. This latter point is the difference in scattering between the dispersant used and the molecule/particles in the dispersant. The greater the level of excess scattering, the easier it is to measure. The smallest size we have measured on a Zetasizer is 0.6nm (peak mode) and I have attached an application note which gives details of such measurements.
- Application note “Measuring Sub Nanometre Sizes Using Dynamic Light Scattering“
Q: What does an intercept > 1 mean?
This usually means that your sample contains very large particles/aggregates/dust which are causing an interference with the measurements. We refer to this as number fluctuations and I have attached an FAQ which discusses this in more detail. What is certain is that the sample is not suitable for DLS and that the large material must be removed before the measurements are taken.
On the same subject: