# Light scattering

Light scattering is a physical phenomenon in which light deviates from a straight-line trajectory when it interacts with particles, macromolecules, or colloids; measurement techniques analyze the scattered light in order to determine particle size and molar mass in solution.<sup>[32](https://technav.ieee.org/topic/optical-scattering/)</sup> The main branches are static light scattering (SLS), which measures time-averaged intensity to yield molar mass and radius of gyration; dynamic light scattering (DLS), which measures intensity fluctuations from [Brownian motion](https://www.edgepedia.org/brownian-motion) to yield hydrodynamic size; and [multi-angle light scattering](https://www.edgepedia.org/multi-angle-light-scattering) (MALS), which measures intensity at several angles simultaneously, most often coupled to size-exclusion chromatography (SEC).<sup>[1](https://arxiv.org/pdf/1810.00611)</sup> DLS is also called quasi-elastic light scattering (QELS) and photon correlation spectroscopy (PCS).<sup>[2](https://webstore.ansi.org/standards/iso/iso224122017)</sup>

| Key fact | Value |
|---|---|
| SLS outputs | Weight-average molar mass, radius of gyration, second virial coefficient on an absolute scale<sup>[3](https://www.fz-juelich.de/en/ibi/ibi-4/downloads/lang_002/@@download/file/Lang_002.pdf)</sup> |
| DLS outputs | Intensity-weighted harmonic mean (z-average) hydrodynamic diameter<sup>[4](https://cdn.standards.iteh.ai/samples/iso/iso-22412-2025/cbd726a36eff42918667d750258f7777/iso-22412-2025.pdf)</sup> |
| DLS size range | Roughly 1 nm to 1000 nm, from small proteins to viruses and bacteria<sup>[1](https://arxiv.org/pdf/1810.00611)</sup> |
| MALS ranges (DAWN-class detector) | Molar mass 1 Da to 1 GDa; radius of gyration 10 to 500 nm; 18-angle temperature-controlled flow cell<sup>[5](https://ostr.ccr.cancer.gov/wp-content/uploads/2023/07/MALS-FFF-1.pdf)</sup> |
| DLS accuracy | Relative expanded uncertainty of the z-average ≤3% on narrow-distribution 100 nm materials; higher for polydisperse samples<sup>[6](https://cdn.standards.iteh.ai/samples/73972/3eec0af3b4c647bb8691ffac8d6019a8/ISO-TR-22814-2020.pdf)</sup> |
| Practical MALS limit | Approximately 10 kDa for proteins; 10–20 ng of BSA suffices for good data<sup>[5](https://ostr.ccr.cancer.gov/wp-content/uploads/2023/07/MALS-FFF-1.pdf)</sup> |

## How it works

Static (elastic) scattering measures the time-averaged intensity of light scattered at each angle. For dilute solutions the excess Rayleigh ratio, the solution scattering minus the solvent scattering, carries the molar mass \( M \), the radius of gyration \( R_{G} \), and the second virial coefficient \( A_{2} \).<sup>[7](http://www.chem.cmu.edu/groups/Berry/scattering98.pdf)</sup> Scattered intensity is directly proportional to the product of weight-average molar mass and concentration, \( LS \sim M_{w} \cdot c \), which is what makes absolute mass determination possible.<sup>[8](https://medicine.yale.edu/keck/biophysics/technologies/lightscatter/theory/)</sup>

The applicable theory depends on particle size. Particles with diameter below one tenth of the laser wavelength (about 60 nm for a 633 nm He-Ne laser) are Rayleigh scatterers with isotropic intensity proportional to the sixth power of diameter, \( I \propto d^{6} \).<sup>[9](https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/DLS%20concept.pdf)</sup> The Rayleigh-Gans-Debye (RGD) approximation is usually appropriate for dilute polymer solutions and adequate for dilute dispersions of small particles; the Mie-Lorentz theory is used for large spherical particles.<sup>[10](http://www.chem.cmu.edu/groups/Berry/scattering15.pdf)</sup>

Dynamic (quasi-elastic) scattering instead records fluctuations of the scattered intensity caused by Brownian motion. The principal measured quantity in dynamic work is the photon count autocorrelation function \( G_{2}(\tau; q) \), computed for a scattering vector modulus \( q = (4\pi n/\lambda)\sin(\vartheta/2) \).<sup>[7](http://www.chem.cmu.edu/groups/Berry/scattering98.pdf)</sup> Under homodyne, random Gaussian conditions, \( G_{2} \) relates to the field autocorrelation through the Siegert relationship.<sup>[11](https://www.eng.uc.edu/~beaucag/Classes/Characterization/DLS/PaulRussoLSU2012DLS_Minicourse.pdf)</sup>

## How it is done

In SLS, a laser passes through the sample and intensity is measured at a fixed angle, typically 90°, which gives the best signal-to-noise ratio for weakly scattering samples. A concentration series of at least four concentrations plus pure solvent is measured, and commercial instruments are calibrated against a reference solvent of known Rayleigh ratio, most often toluene or benzene.<sup>[12](https://wiki.anton-paar.com/en/molecular-mass-measurement-using-static-light-scattering/)</sup> For particles larger than roughly \( \lambda_{0}/20 \), the angular form factor cannot be neglected and multi-angle measurement is required.<sup>[12](https://wiki.anton-paar.com/en/molecular-mass-measurement-using-static-light-scattering/)</sup>

The Debye-Zimm equation, \( K \cdot c/\Delta R = 1/(M_{w}P(\theta)) + 2A_{2} \cdot c \), with \( K = 4\pi^{2}(dn/dc)^{2}n_{0}^{2}/(N_{A} \cdot \lambda_{0}^{4}) \), is the theoretical basis of SLS.<sup>[12](https://wiki.anton-paar.com/en/molecular-mass-measurement-using-static-light-scattering/)</sup> In the Zimm plot, \( K^{*} \cdot c/R(Q) \) is plotted against \( \sin^{2}(Q/2) \) and concentration; the intercept gives \( M_{w} \), the angular slope extrapolated to \( c = 0 \) gives \( R_{g} \), the concentration slope extrapolated to \( \theta = 0 \) gives \( A_{2} \), and the two extrapolations share a common intercept.<sup>[8](https://medicine.yale.edu/keck/biophysics/technologies/lightscatter/theory/)</sup><sup> • </sup><sup>[1](https://arxiv.org/pdf/1810.00611)</sup>

In DLS, the decay of \( G_{2}(\tau) \) yields the translational diffusion coefficient, converted to hydrodynamic radius by the Stokes-Einstein relation, \( R_{H} = k \cdot T/(6\pi\eta_{s} \cdot D) \).<sup>[7](http://www.chem.cmu.edu/groups/Berry/scattering98.pdf)</sup> Because \( D \) is concentration-dependent, it should be measured at several concentrations and extrapolated to infinite dilution, then converted to standard conditions (water, 20 °C).<sup>[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC5425802/)</sup> The cumulants analysis, defined in ISO 13321 (1996) and ISO 22412 (2008), gives the z-average size and a polydispersity index from 0 to 1; the CONTIN constrained regularization algorithm represents the correlation function as an integral over a distribution of decay rates for multimodal samples.<sup>[9](https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/DLS%20concept.pdf)</sup><sup> • </sup><sup>[1](https://arxiv.org/pdf/1810.00611)</sup>

In SEC-MALS, the chromatograph separates the sample and the MALS detector records \( R(\theta) \) across each peak; at the low eluent concentrations in the detector, \( M_{w} \) can be determined from the light scattering detector alone if the calibration constant, \( dn/dc \), and injected mass are known.<sup>[7](http://www.chem.cmu.edu/groups/Berry/scattering98.pdf)</sup> For proteins, \( dn/dc \) is approximately 0.19 mL/g and nearly independent of amino acid composition.<sup>[8](https://medicine.yale.edu/keck/biophysics/technologies/lightscatter/theory/)</sup>

## Origin

The theoretical line runs from the scattering equation for an ideal gas, through the fluctuation theory of fluids and the theory of scattering by large particles; the standard reference on absorption and scattering by small particles is the book by Craig F. Bohren and Donald R. Huffman, first published in 1983.<sup>[3](https://www.fz-juelich.de/en/ibi/ibi-4/downloads/lang_002/@@download/file/Lang_002.pdf)</sup><sup> • </sup><sup>[14](https://link.springer.com/rwe/10.1007/978-1-4020-4465-6_7)</sup><sup> • </sup><sup>[15](https://doi.org/10.1002/9783527618156)</sup> A 1948 paper by [Bruno H. Zimm](https://www.edgepedia.org/bruno-h-zimm) in The Journal of Chemical Physics described a photoelectric apparatus for measuring the angular dependence of light scattering from solutions, with calculation methods for determining the average extension of the scattering molecules, demonstrated on polystyrene fractions.<sup>[16](https://doi.org/10.1063/1.1746740)</sup>

For dynamic scattering, H. Z. Cummins, N. Knable, and Y. Yeh reported the observation of diffusion broadening of Rayleigh scattered light in [Physical Review Letters](https://www.edgepedia.org/physical-review-letters) in 1964.<sup>[17](https://doi.org/10.1103/physrevlett.12.150)</sup> The method of cumulants for polydispersity analysis was presented by Dennis E. Koppel in The Journal of Chemical Physics in 1972,<sup>[18](https://doi.org/10.1063/1.1678153)</sup> and the exponential sampling method by Nicole Ostrowsky and colleagues in Optica Acta in 1981.<sup>[19](https://doi.org/10.1080/713820704)</sup> Philip J. Wyatt's 1993 Analytica Chimica Acta paper, "Light scattering and the absolute characterization of macromolecules," is cited as a modern classic on online and offline MALS.<sup>[20](https://doi.org/10.1016/0003-2670%2893%2980373-s)</sup><sup> • </sup><sup>[21](https://www.nature.com/articles/s43586-025-00410-1)</sup> Commercial multi-angle systems generating the scattering function from flowing solutions have been available since 1986, spanning three to eighteen or more detection angles.<sup>[22](https://pmc.ncbi.nlm.nih.gov/articles/PMC8406046/)</sup> RT-MALS as a process analytical technology for real-time molecular weight in downstream process control was introduced by Bhumit A. Patel and colleagues in mAbs in 2018,<sup>[23](https://doi.org/10.1080/19420862.2018.1505178)</sup> and the machine-learning light scattering method for aerosol sizing was reported by Jin Zeng and Jingjing Xia in ACS Sensors in 2024.<sup>[24](https://doi.org/10.1021/acssensors.3c02009)</sup>

## Variants

Naming reflects geometry and time domain. SLS denotes the static technique generally; MALS distinguishes multi-detector instruments from earlier single low-angle instruments, and the abbreviation DLS shifted from "differential light scattering" to dynamic light scattering as photon correlation spectroscopy took over.<sup>[22](https://pmc.ncbi.nlm.nih.gov/articles/PMC8406046/)</sup> DLS instruments detect at 90° or in backscatter at 173° or 158°, the latter allowing measurement of highly concentrated samples by avoiding multiple scattering.<sup>[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC5425802/)</sup> Multi-angle DLS (MADLS) combines correlograms from three angles (backscatter, 90°, forward scatter), improving sizing resolution typically from 3:1 to 2:1 and reducing angular dependence.<sup>[25](https://www.malvernpanalytical.com/br/learn/knowledge-center/whitepapers/wp180926improvedcomponentresolutionmadls)</sup> Other named variants include diffusing wave spectroscopy (DWS), two-color cross-correlation DLS, depolarized DLS, and gated cross-correlation DLS.<sup>[11](https://www.eng.uc.edu/~beaucag/Classes/Characterization/DLS/PaulRussoLSU2012DLS_Minicourse.pdf)</sup> Hyphenated forms add separation: SEC-MALS, field-flow fractionation MALS (FFF-MALS), and μMALS, a micro-MALS design introduced by Vincent H. Hsieh and Philip J. Wyatt in [Scientific Reports](https://www.edgepedia.org/scientific-reports) in 2017 that makes MALS compatible with SE-UHPLC peaks.<sup>[26](https://www.nature.com/articles/s41598-017-09051-1)</sup> RT-MALS integrates MALS in-line with process equipment for real-time molar mass and size.<sup>[23](https://doi.org/10.1080/19420862.2018.1505178)</sup> The third edition of the DLS standard, ISO 22412:2025, added multi-angle dynamic light scattering (MADLS), imaging DLS (IDLS and UIDLS), and polarization-separated backscatter photon cross-correlation spectroscopy, replacing the 2017 edition.<sup>[4](https://cdn.standards.iteh.ai/samples/iso/iso-22412-2025/cbd726a36eff42918667d750258f7777/iso-22412-2025.pdf)</sup>

## Applications

SEC-MALS is principally used for determining the molar mass of natural and synthetic macromolecules and colloidal suspensions.<sup>[21](https://www.nature.com/articles/s43586-025-00410-1)</sup> The second virial coefficient predicts aggregation tendency: a negative \( A_{2} \) suggests aggregation is likely, while a positive value reflects good particle-solvent interactions and stability.<sup>[12](https://wiki.anton-paar.com/en/molecular-mass-measurement-using-static-light-scattering/)</sup> DLS provides size estimates within a few minutes on commercial instruments and is widely used for nanoparticles, emulsions, and fine bubbles in liquids.<sup>[4](https://cdn.standards.iteh.ai/samples/iso/iso-22412-2025/cbd726a36eff42918667d750258f7777/iso-22412-2025.pdf)</sup> RT-MALS provides molar mass from 1000 g/mol to 1 billion g/mol and radius from 10 nm to 250 nm; for AAV gene vectors it combines MALS with UV absorbance at 260 and 280 nm to determine capsid and genome molar masses.<sup>[27](https://www.wyatt.com/solutions/techniques/real-time-mals.html)</sup>

## Limitations and alternatives

Dust is a major contamination problem; the dispersant should be filtered, with syringe filters from 1 µm down to 20 nm pore size.<sup>[9](https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/DLS%20concept.pdf)</sup> At high concentrations, particle-particle interactions and multiple scattering can produce apparent sizes that differ between concentrated and dilute suspensions.<sup>[4](https://cdn.standards.iteh.ai/samples/iso/iso-22412-2025/cbd726a36eff42918667d750258f7777/iso-22412-2025.pdf)</sup> Because intensity scales as \( d^{6} \), analysis is heavily weighted toward larger particles, so even a small number of agglomerates overshadows the scattering of primary particles and aggregation distorts the size distribution.<sup>[28](https://www.tandfonline.com/doi/pdf/10.1080/14686996.2018.1517587)</sup><sup> • </sup><sup>[29](https://pubs.rsc.org/en/content/articlepdf/2016/ay/c6ay00419a)</sup> Converting intensity distributions to volume or number basis is deprecated because of the inherent errors, except for comparative purposes.<sup>[6](https://cdn.standards.iteh.ai/samples/73972/3eec0af3b4c647bb8691ffac8d6019a8/ISO-TR-22814-2020.pdf)</sup> Angle dependence is a fundamental artifact in polydisperse DLS: higher-\( q \) instruments under-sample large particles and report smaller effective hydrodynamic diameters than lower-\( q \) instruments on identical samples.<sup>[30](https://iopscience.iop.org/article/10.1088/1361-6501/ac42b2)</sup> [Resolution](https://www.edgepedia.org/resolution) limits are reported differently: one review states DLS cannot resolve populations whose diffusion coefficients differ by less than a factor of ten,<sup>[1](https://arxiv.org/pdf/1810.00611)</sup> while a comparative perspective states DLS requires a greater than three-fold size difference for peak resolution.<sup>[28](https://www.tandfonline.com/doi/pdf/10.1080/14686996.2018.1517587)</sup>

Among alternatives, nanoparticle tracking analysis (NTA) produces number-based, particle-by-particle distributions including concentration, and resolves peaks differing by less than 50% in size, but DLS detects smaller particles, below the NTA lower detection limit, and offers ISO-standard mean size and PDI for quality control.<sup>[28](https://www.tandfonline.com/doi/pdf/10.1080/14686996.2018.1517587)</sup><sup> • </sup><sup>[31](https://www.malvernpanalytical.com/en/learn/knowledge-center/whitepapers/wp161122nta-dlscomplementarynanocharacterization)</sup> For separation before scattering, SEC size limits depend on column pore sizes, generally well below 300 nm, whereas [asymmetric flow field-flow fractionation](https://www.edgepedia.org/asymmetric-flow-field-flow-fractionation) can separate samples often exceeding 1000 nm with superior resolution for high molar mass samples.<sup>[22](https://pmc.ncbi.nlm.nih.gov/articles/PMC8406046/)</sup> Where MALS gives absolute molar mass, DLS can only estimate molecular mass from an empirical power law based on globular proteins, which is erroneous for non-spherical particles.<sup>[1](https://arxiv.org/pdf/1810.00611)</sup>

## References

1. [Analytical light scattering methods in molecular and structural biology](https://arxiv.org/pdf/1810.00611)
2. [ISO 22412:2017 — Particle size analysis — Dynamic light scattering (DLS)](https://webstore.ansi.org/standards/iso/iso224122017)
3. [Scattering Methods: Basic Principles and Application to Polymer and Colloidal Solutions (Forschungszentrum Jülich lecture notes)](https://www.fz-juelich.de/en/ibi/ibi-4/downloads/lang_002/@@download/file/Lang_002.pdf)
4. [ISO 22412:2025, Particle size analysis, Dynamic light scattering (DLS)](https://cdn.standards.iteh.ai/samples/iso/iso-22412-2025/cbd726a36eff42918667d750258f7777/iso-22412-2025.pdf)
5. [NCI CCR facility specification: MALS and FFF/SEC-MALS](https://ostr.ccr.cancer.gov/wp-content/uploads/2023/07/MALS-FFF-1.pdf)
6. [ISO/TR 22814:2020, Guidance for good practice in DLS](https://cdn.standards.iteh.ai/samples/73972/3eec0af3b4c647bb8691ffac8d6019a8/ISO-TR-22814-2020.pdf)
7. [Static and Dynamic Light Scattering (G. C. Berry, Encyclopedia of Analytical Chemistry)](http://www.chem.cmu.edu/groups/Berry/scattering98.pdf)
8. [Light Scatter Theory (Yale Keck Biophysical Resource)](https://medicine.yale.edu/keck/biophysics/technologies/lightscatter/theory/)
9. [Dynamic Light Scattering Training (Malvern Zetasizer training notes)](https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/DLS%20concept.pdf)
10. [Classical (time-averaged) light scattering methods (Berry, Encyclopedia of Analytical Chemistry, longer version)](http://www.chem.cmu.edu/groups/Berry/scattering15.pdf)
11. [DLS Minicourse (Paul Russo, LSU)](https://www.eng.uc.edu/~beaucag/Classes/Characterization/DLS/PaulRussoLSU2012DLS_Minicourse.pdf)
12. [Molecular mass measurement using static light scattering (Anton Paar Wiki)](https://wiki.anton-paar.com/en/molecular-mass-measurement-using-static-light-scattering/)
13. [Dynamic light scattering: a practical guide and applications in biomedical sciences](https://pmc.ncbi.nlm.nih.gov/articles/PMC5425802/)
14. [Dynamic Light Scattering (Springer Encyclopedia chapter)](https://link.springer.com/rwe/10.1007/978-1-4020-4465-6_7)
15. [Craig F. Bohren, Donald R. Huffman (1998). Absorption and Scattering of Light by Small Particles. .](https://doi.org/10.1002/9783527618156)
16. [Bruno H. Zimm (1948). Apparatus and Methods for Measurement and Interpretation of the Angular Variation of Light Scattering; Preliminary Results on Polystyrene Solutions. The Journal of Chemical Physics.](https://doi.org/10.1063/1.1746740)
17. [H. Z. Cummins, N. Knable, Y. Yeh (1964). Observation of Diffusion Broadening of Rayleigh Scattered Light. Physical Review Letters.](https://doi.org/10.1103/physrevlett.12.150)
18. [Dennis E. Koppel (1972). Analysis of Macromolecular Polydispersity in Intensity Correlation Spectroscopy: The Method of Cumulants. The Journal of Chemical Physics.](https://doi.org/10.1063/1.1678153)
19. [Nicole Ostrowsky and colleagues (1981). Exponential Sampling Method for Light Scattering Polydispersity Analysis. Optica Acta International Journal of Optics.](https://doi.org/10.1080/713820704)
20. [Light scattering and the absolute characterization of macromolecules (Analytica Chimica Acta, 1993)](https://doi.org/10.1016/0003-2670%2893%2980373-s)
21. [Size-exclusion chromatography with multi-angle static light scattering (Nature Reviews Methods Primers, 2025)](https://www.nature.com/articles/s43586-025-00410-1)
22. [Differential light scattering and the measurement of molecules and nanoparticles: A review](https://pmc.ncbi.nlm.nih.gov/articles/PMC8406046/)
23. [Bhumit A. Patel and colleagues (2018). Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control. mAbs.](https://doi.org/10.1080/19420862.2018.1505178)
24. [Jin Zeng, Jingjing Xia (2024). Light Scattering Method for Aerosol Sizing Based on Machine Learning. ACS Sensors.](https://doi.org/10.1021/acssensors.3c02009)
25. [Improved component resolution with Multi-Angle DLS (MADLS)](https://www.malvernpanalytical.com/br/learn/knowledge-center/whitepapers/wp180926improvedcomponentresolutionmadls)
26. [Measuring proteins with greater speed and resolution while reducing sample size (μMALS/SE-UHPLC-MALS, Scientific Reports)](https://www.nature.com/articles/s41598-017-09051-1)
27. [Real-Time MALS (RT-MALS) for Absolute Molar Mass and Size Measurements](https://www.wyatt.com/solutions/techniques/real-time-mals.html)
28. [Characterisation of particles in solution – a perspective on light scattering and comparative techniques](https://www.tandfonline.com/doi/pdf/10.1080/14686996.2018.1517587)
29. [A comparison of techniques for size measurement of nanoparticles in cell culture medium (Analytical Methods, RSC)](https://pubs.rsc.org/en/content/articlepdf/2016/ay/c6ay00419a)
30. [Angle-dependent effects in DLS measurements of polydisperse particles (Meas. Sci. Technol.)](https://iopscience.iop.org/article/10.1088/1361-6501/ac42b2)
31. [Synergy of complementary nanomaterial characterization techniques (DLS vs NTA, Malvern Panalytical)](https://www.malvernpanalytical.com/en/learn/knowledge-center/whitepapers/wp161122nta-dlscomplementarynanocharacterization)
32. [Optical scattering, IEEE Technology Navigator](https://technav.ieee.org/topic/optical-scattering/)

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*Topic: Encyclopedia › Physical world and mathematics › Physics › Physics methods, practice, and community › Flow and particle diagnostics*

*Initially written Sep 29, 2026 · Edited: Oct 15, 2026*

*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*

License: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license