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Dynamic Light Scattering
Particle analysis for quality control

Dynamic light scattering (DLS) is an established method in the measuring range of nanometers and submicrometers. Using a laser it characterizes particle sizes and particle size distributions in suspensions and emulsions to determine the hydrodynamic radius of the molecules. In the following article we will explain what dynamic light scattering (DLS) and its variants are, clarify its theoretical principle, talk about fields of application and also show where DLS reaches its limits as an particle measurement technology and which alternatives there are.

Table of contents

What is dynamic light scattering? Basic facts about DLS

In 1827, when Robert Brown observed tiny pollen particles from evening primroses in liquids and gases through a microscope, he couldn’t believe his eyes. As the first human being he became witness to what later became known as Brownian motion, named after him. What he managed to see was the sudden thermal movement of the organelles. This is  dynamic light scattering: DLS uses a laser to measure the random movement of particles which occurs when macromolecules interact with the molecules of a solvent. By optical detection this measurement provides insights into particle sizes and particle size distributions of suspensions and emulsions in the nanometer and submicrometer range, as smaller particles move faster inside the liquid than bigger ones. Furthermore, with dynamic light scattering it is possible to observe changes to particle sizes over time.

How does dynamic light scattering work?

In the DLS method a monochromatic laser beam is directed through a polarizer. If the phase position of the laser is precise enough, no polarizer is needed. Afterwards, the laser hits the sample that is located in a cuvette. As soon as the laser beam hits small particles inside the sample it is diffracted and scattered in all directions. The interaction between the particles and the laser beam forms scattered waves and when these waves overlap interferences occur, which can be directed through another polarizer (if one is present). Subsequently, a photo-electron multiplier (specific measurement diodes – mostly Avalanche photo-diodes for their light enhancing qualities) captures the interferences at a certain angle. For dynamic light scattering measurements this process is repeated multiple times over a period of time. As the intensity of the scattered light varies, the fluctuation of smaller particles is faster, while bigger particles show higher amplitudes between the minimum and the maximum. The measurement results are hidden in these fluctuations and interferences and can be converted into particle sizes and particle sizes distributions through autocorrelation. For dynamic light scattering the autocorrelation function proceeds linearly at the beginning and then decreases exponentially. This indicates the movement of a particle. Hidden in this exponential decrease of the autocorrelation function is the information for particle size. The curve decreases quickly for small particles and slowly for bigger particles.

Dynamic Light Scattering Illustration of the structure
Dynamic Light Scattering Illustration of the structure

The theory of dynamic light scattering

The theoretical principle of dynamic light scattering is based on measuring Brownian motion. Thanks to this physical model from the 19th century we know that particles in a viscous medium randomly move in all directions and thereby collide with the particles of the solvent. Energy is passed on through these collisions of molecules, which causes particle movement. As this transfer of energy is relatively constant, it has a bigger influence on smaller particles so they move much faster than bigger ones. The already discussed autocorrelation function serves as a mathematical description of the fluctuations of the scattered light and is used to determine the diffusion coefficient. For this you compare the intensity of the scattered light at two different moments on the same intensity track to receive the correlation function. Subsequently, if you measure the speed of the particles by the dynamic light scattering method and also use further factors that influence the movement of the particles you can determine the hydrodynamic radius. Some of these necessary factors are the viscosity of the dispersion as well as its temperature. The Stokes-Einstein equation relates the particle speed and the particle size to the hydrodynamic radius. The results may be impacted by sedimentation though.

D = kBT / 6πηRH

Methods of dynamic light scattering

There are different methods to interpret DLS signals and also various ways to detect light signals. While for homodyne detection the scattered light itself acts as a reference, the heterodyne detection principle uses the interference of light that has been redirected from the sample compared with a controlled reference. For interpretation of a DLS signal you may rely on a time-dependent autocorrelation function (mostly for homodyne measurements), as can be seen for photon correlation spectroscopy (PCS) or photon cross-correlation spectroscopy (PCCS) or you may rely on a method called frequency power spectrum (FPS), which gives more accurate results.

Photon correlation spectroscopy (PCS):
The technical base of the DLS measurement technique

For the process of dynamic light scattering measurement particles are irradiated by a laser. In doing so large and small particles scatter the light in interactions. As a result scattered waves are forming and can be measured by photon correlation spectroscopy (PCS). Because the particles move randomly the distances between them changes and interferences occur (the individual scattered waves overlap). Due to optical interferences all sub-waves combine to an overall scattered wave. The intensity of this combined scattered wave fluctuates between the constructive interference (maximum) and the destructive interference (minimum). The signal of the scattered light is recorded optically by a photon-detector and from a certain angle over a period of time and autocorrelated to determine the particle size distribution. Dynamic light scattering correlates the recorded scattering signal with a measurement from a previous moment within the autocorrelation. The fluctuations of the recorded signal of the scattered light (frequency shifts) delivers information about the particle movement and the particle size distribution.

Dynamic Light Scattering Illustration of the structure of a PCS device - photon correlation spectroscopy
Dynamic Light Scattering Illustration of the structure of a PCS device - photon cross correlation spectroscopy

The measurement of multiple scattering in highly concentrated samples with photon cross-correlation spectroscopy (PCCS)

If you decide to use a conventional photon correlation spectroscopy for particle analysis, you will see its limits as soon as you try to measure highly concentrated samples. Because if the laser beam is scattered more than once when using dynamic light scattering it has effects on the scattering wave and negatively impacts the measurement results of the detector. It is possible to reduce this impact by greatly diluting the DLS samples, but then again this influences the characteristics of the particles. To avoid such distortions when analyzing particles via dynamic light scattering a photon cross-correlation spectroscopy is used for highly concentrated samples. To separate the singly scattered light from the multiply scattered light during measurement, the laser beam is split into two partial beams with exactly the same intensity, which are then overlapped inside the sample. Furthermore, both scattered waves are measured by photo-detectors and allow the measurement of highly concentrated samples by the dynamic light scattering-method without dilution. In this way the negative effects of multiple scattering are eradicated and it is possible to determine the stability of dispersions and opaque emulsions and suspensions.

Frequency power spectrum (FPS)

If more accurate measurements for particle characterization are needed and PCS and PCCS are not able to deliver this accuracy, DLS signals can also be transformed into a frequency power spectrum by the Fast Fourier transform. This method for the interpretation of a dynamic light scattering signal directly provides data for particle size distributions with more accuracy. FPS is delivered as a Lorentz function.

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The difference between static and dynamic light scattering

Apart from dynamic light scattering (DLS) the static light scattering (SLS) method is often used for quality control to characterize polymers, nanoparticles, biopolymers or proteins. As a laser scattering technology SLS has advantages as well as disadvantages compared to dynamic light scattering. Basically, static light scattering results from the interaction of light and particles. In this case, a laser beam is sent through a dispersion and is scattered by the particles in the batch. For static light scattering the scattered light is measured by a number of detectors (multi-angle light scattering, MALS) over a large angle range. Finally, the particle size distribution can be calculated with the resulting pattern of the scattered light. Here, the influence of particle sizes on the scattered light is decisive, as the scattering angle increases the smaller the particles in dispersion are. This is because the light is scattered in all directions. Also the scattering intensity and the distinct maxima decrease with declining particle size.

The theory of static light scattering

The light scattering particle size analysis is based on Fraunhofer diffraction and Mie theory. While Fraunhofer diffraction explains the relation between particles and forward scattering and is used for particle analysis in the micrometer range, Mie theory is used to measure particles, whose diameter roughly matches the wavelength of the laser beam. Therefore, samples with larger particles (mostly bigger than a micrometer) are interpreted by Fraunhofer diffraction, while the static light scattering for samples with fine particles is calculated with Mie theory. This can only be done if the refractive index and the absorption index of the material are known though. The Fraunhofer approximation, on the other hand, just measures the diffraction ratio. The absorption of the material is not taken into account. If the diameter of a particle is many times smaller than the wavelength of the light, Rayleigh scattering can help for interpretation. However, Rayleigh scattering is just able to approximate Mie theory and loses its accuracy and even its validity when the diameter of the particle increases. Of course, the diameter of the particle can also be significantly bigger than the wavelength of the light. In this case, you can easily approximate Mie theory through the physical phenomenon of the refraction of light.

Determining the molecular weight with multi-angle light scattering (MALS)

A multi-angle light scattering detector can determine the radius of gyration and the molecular weight of a sample. This measurement method is a form of static light scattering and is mainly used as a flow meter technique, less often together with goniometer systems. MALS measures the intensity of the scattered light as a function of the scattering angles. The number of scattering angles inside a MALS detector varies from 2 to 21. The more measuring angles are used, the higher is the quality of the measurement results. MALS and MALLS are often used in polymer chemistry to determine the absolute molecular weight and the radius of gyration of polymer samples. In pharmaceutics multi-angle light scattering detectors are deployed in combination with chromatography technologies; again to determine the molar mass. Furthermore, a MALS detector can be used to determine the size of nanoparticles by means of a field-flow fractionation plant. A multi-angle light scattering instrument is even able to measure aggregates and nanoparticles.

Advantages and disadvantages of DLS and SLS - Dynamic light scattering and static light scattering compared

Although static light scattering covers a broad measuring range, it delivers the best results in the measuring range between 100 nm and 1 mm. For the measurement of finer particles dynamic light scattering is the better choice, as the intensity of the scattered light as well as the angle dependence strongly decrease. Although it is possible to analyze finer samples with short-wave light sources the results from DLS are far more accurate. Static light scattering stands out for its large measurement flexibility but has issues with metallic nanoparticles because of the swinging electrons, which cause a shift of the scattered light (bigger wavelengths) and therefore distort the results. If you measure particle sizes and particle size distributions with DLS instead, this phenomenon has no influence on the measurement results. But that’s only one side of the story, as samples need more complex preparation for DLS measurements as the media needs dilution, filtration or dispersing aids. Finally, there is a fundamental difference in the output of the size distribution between DLS and SLS.

Dynamic light scattering (DLS) Static light scattering (SLS)
Measurement range
0.3 nm to 10 µm
10 nm to 100 nm
Ideal measuring range
Smaller than 100 nm
Bigger than 30 nm
Typical samples
Proteins, liposomes, metal oxides, colloidal metals, viruses, nanoparticles, polymers, hydrogels, aggregates
Suspensions, emulsions, granulates, fine powders
Measurement results
Determines the hydrodynamic diameter
Determines the radius of gyration
Output of the size distribution
Intensity-based
Volume-based
Time for analysis
About two minutes
About one minute
Aquisition of the measurement data
In the range of microseconds
5-10 measurement results per second

DLS

SLS

Applications of dynamic light scattering

DLS is ideal for the determination of some big molecules amidst a big number of small particles. Particularly in the field of pharmaceutical technologies the DLS method is often used to verify whether a dissolved protein exists as a pure monomer. If this is not the case, the compatibility and efficiency of a pharmaceutical will suffer. Despite its limits, dynamic light scattering is the established method to determine the size and the size distribution of nanoparticles in other industries such as biochemistry or biotechnology as well. It is used to analyze peptides, proteins, liposomes, viruses, gene vectors, nucleic acid, polymers, synthetic particles, micelles, hydrogels and many more. Furthermore, dynamic light scattering is used to develop vaccines, gene therapies and monoclonal antibodies. Molecular biologists examine liposomes and extracellular vesicles with DLS. In analytical chemistry and environmental sciences dynamic light scattering is used to characterize polymers or to examine nanoplastics, toxic particles, water pollution or tracer particles in the environment. By measurement of the zeta potential, DLS can also evaluate the quality of dispersions.

Zeta potential measurement with dynamic light scattering

Some DLS instruments also have the capability to determine the zeta potential of a sample. To do this, the mobility of charged particles is elicited using the laser Doppler electrophoresis technique. The charged particles are sent through an electric field where their velocity is measured. This is possible when charged particles (particles, colloids, droplets) are located in suspensions. In this case ions of the suspension medium are deposited on the particle surface and build an electric double layer. As the surface charge of the particles attracts the ions of the medium the double layer always moves with the particle around which it formed – along the shear plane. This electric potential is called zeta potential and is stated in millivolt. If the zeta potential is strongly positive or negative, a strong electrostatic interaction occurs (agglomerates may form). If the zeta potential instead is close to zero, hardly any repellent force occurs (coagulation is possible). Using this information, you can relatively accurately predict the stability of a dispersion.

The limits of dynamic light scattering

DLS is still seen as the standard method for the determination of nanoparticle sizes and nanoparticle size distributions, although it comes up against many insurmountable barriers. First of all a DLS measurement procedure must be carried out under static conditions to make sure that the particle movement is solely caused by Brownian motion. Thus dynamic light scattering is unsuitable for measurement during the production process, as the liquids are constantly moving. Furthermore, measurement results by DLS do not deliver actual D-values, but a Z-average value which is not able to accurately represent the individual particle populations. In addition, dynamic light scattering cannot detect smaller particles hidden behind bigger ones. As soon as a sample is opaque or strongly diluted, DLS measurement results are not particularly informative. Another problem arises with polydisperse samples as the different particle sizes cannot be characterized properly by DLS. Ultra-low particle concentrations cannot be detected by dynamic light scattering. Finally, DLS results can be distorted by the smallest change of temperature or viscosity.

Disadvantages and limitations of dynamic light scattering in a nutshell

Dynamic Light Scattering - Frau mit Schutzmaske, Laborkittel und Handschuhen schaut in Mikroskop

Our patented OF2i method has huge advantages compared to dynamic light scattering

Although dynamic light scattering is an established measurement technique for laboratory particle analysis, it is strongly limited when analyzing polydisperse liquids or very low particle concentrations. Especially in research and development as well as online process control OF2i offers many benefits. The OptoFluidic Force Induction method delivers much faster measurement results, analyzes particles in real-time and continuously and provides live measurement data. Furthermore, OF2i can be integrated directly into the manufacturing process (BRAVE B-Continuous). DLS is unsuited for continuous analysis of particles during production.

DLS OF2i
Speed of measurement
Measurement is dependent on Brownian motion and therefore takes time (up to 2 minutes)
Measurement results are not dependent on Brownian motion and are available immediately
Sample preparation and cleaning routines
Diluted samples can be measured directly, other samples must be diluted first. Manual cleaning of the measurement chambers is time-consuming.
Diluted samples can be measured directly, other samples must be diluted. Up to 9 samples can be measured before an automated routine cleans the measurement cell.
Integrity of measurement results
Only delivers snapshots of a sample and average values of particle populations. Also determines the zeta potential and the polydispersity index.
Delivers a 3D result curve which shows what happens inside the sample second by second. Results are based on a statistically relevant number of single particles and are available as a size chart (D-values). Zeta potential cannot be measured.
Continuous measurement
DLS is unsuited for online particle measurements.
Delivers continuous and time-resolved measurement results. With sufficient sample quantities OF2i can measure for several hours and is available as a PAT sensor for 24/7 production control (BRAVE B-Continuous).
Accuracy
Delivers an average value for particle sizes of diluted dispersions if polydispersity is low as well as reproducible results for spherical particles if the particle size distribution range is narrow.
Measurement results are representative for the whole particle population as they are based on single-particle sensitivity. OF2i delivers great results for polydisperse substances.
Boundaries of measurability
Does not deliver reliable results for low particle concentrations or polydispersity. Opaque or sedimented samples cannot be measured accurately.
Delivers reliable results for complex, polydisperse systems, ultra-low particle concentrations as well as for ultra-low sample volumes.
Integration into the production process
Not well-suited for use in flowing media.
Can easily be integrated into the production process as an online PAT sensor.

Six reasons why OF2i is a better alternative compared to DLS

I.

OF2i is able to measure each individual particle, even really small ones hiding behind the “juggernaut” particles, as it measures up to 4000 particles per minute. These small particles cannot be detected by dynamic light scattering.

II.

While with DLS you just get an average value (Z-average) that does not deliver accurate data about individual particle populations, OF2i measures with single-particle sensitivity and therefore delivers statistically relevant results and actual D-values. With OF2i you get a far better understanding of your samples.

III.

OF2i gives you insights into your sample behavior like never before - over seconds, minutes or several hours. All results are displayed on one measuring report.

IV.

Polydispersity does not influence OF2i measurement results.

V.

OF2i can even measure ultra-low particle concentrations (up to a few particles per milliliter) as well as ultra-low sample volumes of 20 µL.

VI.

OF2i monitors your sample’s behavior during aggregation, dissociation, condensate formation or dissolution, as it happens.

References

Berne, B.J.; Pecora, R. Dynamic Light Scattering. Courier Dover Publications (2000) ISBN 0-486-41155-9

Block, I.; Scheffold, F. (2010). “Modulated 3D cross-correlation light scattering: Improving turbid sample characterization”. Review of Scientific Instruments. 81 (12): 123107–123107–7. arXiv:1008.0615. Bibcode:2010RScI…81l3107B. doi:10.1063/1.3518961. PMID 21198014. S2CID 9240166.

Chu, B. (1 January 1970). “Laser Light Scattering”. Annual Review of Physical Chemistry. 21 (1): 145–174. Bibcode:1970ARPC…21..145C. doi:10.1146/annurev.pc.21.100170.001045.

Goodman, J (1976). “Some fundamental properties of speckle”. J. Opt. Soc. Am. 66 (11): 1145–1150. Bibcode:1976JOSA…66.1145G. doi:10.1364/josa.66.001145.

Graciani, Guillaume; King, John T.; Amblard, François (30 August 2022). “Cavity-Amplified Scattering Spectroscopy Reveals the Dynamics of Proteins and Nanoparticles in Quasi-transparent and Miniature Samples”. ACS Nano. 16 (10): 16796–16805. arXiv:2111.09616. doi:10.1021/acsnano.2c06471. ISSN 1936-0851. PMID 36039927. S2CID 244345602.

ISO 22412:2017. Particle Size Analysis – Dynamic Light Scattering (DLS). International Organization for Standardization.

Pecora., R. (1964). “Doppler Shifts in Light Scattering from Pure Liquids and Polymer Solutions”. The Journal of Chemical Physics. 40 (6): 1604. Bibcode:1964JChPh..40.1604P. doi:10.1063/1.1725368.

Schaetzel, K. (1991). “Suppression of multiple-scattering by photon cross-correlation techniques” (PDF). J. Mod. Opt. 38: 1849. Bibcode:1990JPCM….2..393S. doi:10.1088/0953-8984/2/S/062. S2CID 250745836. Retrieved 7 April 2014.

Urban, C.; Schurtenberger, P. (1998). “Characterization of turbid colloidal suspensions using light scattering techniques combined with cross-correlation methods”. J. Colloid Interface Sci. 207 (1): 150–158. Bibcode:1998JCIS..207..150U. doi:10.1006/jcis.1998.5769. PMID 9778402.

OECD Guidelines for the Testing of Chemicals, Section 1; Test No. 125: Nanomaterial Particle Size and Size Distribution of Nanomaterials