Dynamic Light Scattering method development using the Zetasizer Advance Series

This technical note discusses DLS method development with the Zetasizer Advance series and highlighted the various factors that need to be considered. These include cell selection, instrument verification, sample preparation, measurement set up and results analysis. 


The technique of Dynamic Light Scattering (DLS) is commonly used to measure particle size and is available in the Zetasizer Advance series of instruments (Figure 1).

DLS is a non-invasive technique suitable for the size characterization of nanoparticles. The technique measures the time-dependent fluctuations in the intensity of scattered light that occur due to the random movement of the particles or molecules undergoing Brownian motion. The velocity of this Brownian motion is measured and is called the translational diffusion coefficient (D) which can be converted into a hydrodynamic diameter (DH) using the Stokes-Einstein equation [1,2].

A summary video of the DLS technique can be viewed here.

[Figure 1 AN230209-DLS-method-development-zetasizer.jpg] Figure 1 AN230209-DLS-method-development-zetasizer.jpg

Figure 1 : Schematic diagram summarizing dynamic light scattering. A focussed laser is passed into a cell containing a sample and the scattered light produced is detected using a photon counting device. The time dependent fluctuations in the intensity of the scattered light are autocorrelated to derive a correlation function. A suitable algorithm is applied to determine a particle size distribution.

This technical note discusses method development and highlights various factors that need to be considered, from instrument verification, sample preparation, measurement set up and result analysis.

Instrument Verification 

Dynamic light scattering is an absolute technique that uses first principles in its measurement protocol i.e. it cannot be calibrated but should be verified that it is working correctly by measuring a sample with a known particle size value. The frequency of instrument verification should be defined by the user.

Polystyrene latex spheres are monodisperse and spherical and are commonly used to verify correct instrument performance. The sphere is the only three-dimensional shape whose size can be unambiguously described by a single figure (i.e. diameter). Polystyrene latex samples have other benefits such as having a density similar to water, so particles less than 1 micron will remain in suspension during measurement. Dispersions can be stored at room temperature and have storage lifetimes of months or years.

A wide range of monodisperse polystyrene lattices are available from a variety of manufacturers. However, not all are supplied with an individual calibration certificate. Some size standards are supplied with their own calibration certificate, measured by transmission electron microscopy (TEM) and are traceable to the National Institute of Standards and Technology (NIST) [3]. The certificate provided also includes a hydrodynamic diameter measured by dynamic light scattering (DLS). The result quoted on the standard bottle is the certified TEM result. The DLS result (i.e. the hydrodynamic size) is not a certified value. The samples should be prepared in 10mM NaCl [2] to suppress the electrical double layer. Dilution of the standard in deionized water will give an extended double layer and result in an artificially increased size which may be out of specification.

The pass/fail criteria for sizing standards are defined in ISO22412 [2]. In summary, the mean value of the first 5 repeat measurements must fall within the range specified on the Certified Reference Material (CRM) certificate (hydrodynamic diameter). The polydispersity index (PdI) value must be less than 0.1 for each of the repeat measurements. In addition, there must be no measurement bias as assessed by verifying that the difference between the mean value measured and the certified mean sample size stated on the CRM certificate (TEM size value) should be less than the combined uncertainty from the CRM stated mean size and the measured mean uncertainty, as described on page 13 of ISO22412 [2]. The relative standard deviation of the first 5 repeat measurements must be <2%.

Cell Selection

The Zetasizer Advance range of instruments has various cuvettes/cells options available for the measurement of particle size (Table 1). The choice of cell will be dependent upon the application (Table 2).  

Table 1 : Summary of cuvette types available for measuring particle size using the Zetasizer Advanced Series.
Cell Size MADLS Particle concentration


Disposable 10 x 10 mm plastic cuvette


Low volume disposable sizing cell


Low volume disposable cuvette


Low volume batch quartz cuvette


Glass cuvette with round aperture


Glass cuvette with square aperture


Disposable folded capillary cell


High concentration zeta potential cell


Dip cell

Zeta potential cells are also capable of measuring particle size for certain Zetasizer Advance models. 

Table 2 : Applications of aqueous and non-aqueous samples and recommended cell types
Application Aqueous Non-Aqueous
Particle Size DTS0012, PCS1115, ZSU1002, ZEN0040, ZEN2112, 
PCS8501, DTS1070, ZEN1010, ZEN1002
ZEN2112, PCS8501, PCS1115
MADLS/Particle Concentration DTS0012, PCS1115, ZEN2112, PCS8501
ZEN2112, PCS1115
Thermal Trends PCS1115, ZEN2112, PCS8501
ZEN2112, PCS8501, PCS1115
Low Volume Samples ZSU1002, ZEN0040, ZEN2112
Backscatter (NIBS) – Variable Measurement Position DTS0012, PCS1115, PCS8501
PCS8501, PCS1115
Side Scatter PCS1115, ZSU1002, ZEN2112, PCS8501
ZEN2112, PCS8501, PCS1115
Forward Scatter DTS0012, PCS1115, ZEN2112, PCS8501, DTS1070
ZEN2112, PCS8501, PCS1115

Sample Preparation

Sample Concentration

Each sample has its own ideal concentration range for optimal measurements. The minimum and maximum required sample concentration should be experimentally determined.

Data Quality Guidance in the Zetasizer Advance series helps determine whether the sample concentration is appropriate. If the sample concentration is too low, there may not be enough light scattered to make a measurement. This is unlikely to occur with the Zetasizer except in extreme circumstances. 

If the sample is too concentrated, then light scattered by one particle will itself be scattered by another (this is known as multiple scattering). Non-Invasive Backscatter (NIBS) optics are designed to minimise multiple scattering issues [7]. Consistency of the measurement position between samples can confirm that the concentrations are the same or comparable. 

The upper limit of the concentration is also governed by the point at which the particles no longer freely diffuse, because of particle interactions. The size of the particles is an important factor in determining the onset of restricted diffusion and particle-particle interactions.

Sample Dilution

If a sample must be diluted, it should be done carefully to preserve the existing state of the particle surface. Results obtained from DLS should be independent of sample concentration [2].  

Measurement Set Up

This section discusses the setup of DLS measurements in ZS Xplorer software. 

To set up a measurement, the following steps need to be completed (Figure 2) and these parameters are discussed in Table 3:

Sample Parameters 

  • Enter a sample name
  • Select a cell
  • Select material and dispersant
  • Add steps to the method
  • Modify the parameters of each if necessary

[Figure 2 AN230209-DLS-method-development-zetasizer.jpg] Figure 2 AN230209-DLS-method-development-zetasizer.jpg

Figure 2: Sample details and method builder

Table 3: Sample details that are required to be entered
Setting Description
Name This is the name the sample will have in the results.
Parameters Any custom parameter to the sample can be added to this box (e.g. batch number) which can be used to help filter results later on.
Cell The cell type to be used for the measurement. For Size measurements, one of the cells/cuvettes listed in Table 1 needs to be selected. Apart from those listed, the zeta potential cells can also be selected for Size measurements.
Material/ Dispersant The material and dispersant used in the sample should be selected from the dropdown lists and specified here. Custom materials and dispersants can also be added.
Project Select which project the results are to be added to or create a new project.

Instrument Settings 

The measurement properties, data processing options and post-analysis settings are shown in Figure 3 and discussed in Table 4 with a discussion of the size analysis models available in Table 5.

[Figure 3 AN230209-DLS-method-development-zetasizer.jpg] Figure 3 AN230209-DLS-method-development-zetasizer.jpg

Figure 3: Measurement properties, data processing options and post-analysis settings

Table 4: Measurement properties
Setting Description
Temperature (°C) The temperature at which the measurement is to be performed. The upper limit is affected by the cell type and dispersant used. The Zetasizer Advance series has a temperature range of 0oC to 120oC.
Equilibration time (s) The length of time given for the sample to thermally stabilize once it has reached the specified temperature. 120s is the default, but this can be adjusted as required.
Analysis mode Selects the most appropriate analysis model to process the sampled data. The analysis models available are discussed in further detail in Table 5.
Size display limit mode A custom upper and lower size limit can be selected by using “manual” from the measurement properties/data processing pane.
Size threshold mode A custom upper and lower size threshold can be selected by using “manual” from the measurement properties/data processing pane.
Auto size averaging Creates an average result after the measurement has completed.

Table 5 : Size analysis models
Analysis Model Description
General Purpose The default processing type for size measurements. General purpose should be suitable for most samples other than the special cases mentioned in the next two options.
Multiple Narrow Modes This analysis model should be used when you know you have more than one peak, and the peaks are narrow. This analysis method gives a higher resolution than the 'General Purpose' model and will resolve peaks more effectively.
L-Curve Analysis This analysis method optimizes the distribution result to give the highest possible resolution while maintaining minimal noise. This process is suitable for low scattering samples such as proteins that may produce correlation functions that contain noise.

Advanced Settings

In this box (Figure 4), additional measurement settings can be altered, and their influences are discussed in detail in Table 6 below.  For a rapid, routine measurement, there might be no need to alter these settings.

[Figure 4 AN230209-DLS-method-development-zetasizer.jpg] Figure 4 AN230209-DLS-method-development-zetasizer.jpg

Figure 4: Advanced settings

Table 6 : The additional method settings 
Setting Comments
Angle of detection The measurement angle used for the measurement should be reported as different angles can give different results for certain samples. Choose from Forward scatter, Backscatter, and Side scatter. For some cells this is limited or fixed.
Positioning method It is recommended to use the default setting, Measure at optimal position, during method development so that the best measurement position can be found. When the optimum measurement position is known, use Measure at fixed position to specify a fixed value. The measurement position is measured from the front of the cuvette. Smaller values are closer to the front of the cell. For some cells, the measurement position cannot be changed.

Select the attenuator level to use. This can be found automatically or fixed at a specific level. It is recommended to use the default setting, Automatic, during method development so that the optimum level can be found. The attenuation range is shown below; the transmission value is the percentage of laser light that enters the sample cuvette.

Attenuator Index 1 2 3 4 5 6 7 8 9 10 11
% Transmission 0.0003 0.003 0.01 0.03 0.1 0.3 1 3 10 30 100

The optimum count rate should be between 300 and 500kcps where possible. If the count rate is below 100kcps when using attenuator 11 for a size measurement, a longer sub run length may improve data quality. The mean count rate should be automatically determined by the software. Appropriate values for each sample can be obtained from studies performed during R & D. There should be an excess level of scattered light to ensure the successful measurement of the sample.
Measurement process This sets the data capture method to either Automatic or Manual. If Manual is selected, the duration and number of runs to complete the measurement can be specified.
Use pause after sub runs If yes is selected, the length of the pause after each sub run can be specified.
Optical filter Choose from Fluorescence filter, Horizontal polarization, Vertical polarization, or No filter. These options are only available in backscatter detection mode.
Pause between repeats Adds a pause between repeat measurements of a specified length allowing the sample to re-equilibrate between measurements if required.

DLS users often enquire as to which instrument parameters are important when transferring a measurement method from Research & Development to Quality Control for the same material. Table 6 lists the key parameters that need to be considered. Note that these could vary when the measurements are transferred from Research and Development to Quality Control. 

Whenever the sample is being measured for a comparative evaluation, as in a Quality Control environment, it is advised that the parameters mentioned above i.e. Angle of Detection, Positioning Method, Attenuation and Measurement Process be ‘fixed’ in a Method. 

Result Analysis

Various parameters and reports are available in ZS Xplorer which will aid the interpretation of the results.

The precision (repeatability) of the size results obtained is a key parameter in the analysis of the data. For monodisperse samples with diameters between 50nm and 200nm, the repeatability of the average particle size should be lower than 2 % [2]. 

For samples where the repeatability of the results is poor, improved results could be achieved by averaging the records. This should increase the quality of the underlying raw data and subsequently improve result repeatability. In ZS Xplorer software, automatic averaging can be set up as part of the measurement (Figure 3) or, results can be averaged once they have completed by either selecting the required records, right-mouse clicking and selecting the Create Average Result option (Figure 5) or clicking on the Create Average Result button (Figure 6).

[Figure 5 AN230209-DLS-method-development-zetasizer.jpg] Figure 5 AN230209-DLS-method-development-zetasizer.jpg

Figure 5: Results can be averaged by selecting them, right-mouse clicking and selecting the Create Average Result option

[Figure 6 AN230209-DLS-method-development-zetasizer.jpg] Figure 6 AN230209-DLS-method-development-zetasizer.jpg

Figure 6: Results can be averaged by selecting them and clicking on the Create Average Result button


This technical note has discussed DLS method development with the Zetasizer Advance series and highlighted the various factors that need to be considered. These include cell selection, instrument verification, sample preparation, measurement set up and results analysis. 


[1] R. Pecora (1985) Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy. Plenum Press, New York.

[2] International Standard ISO22412 (2017) Particle Size Analysis: Dynamic Light Scattering (DLS). International Organization for Standardization (ISO).

[3] National Institute of Standards and Technology (www.nist.gov).

[4] R.S. Chow and K. Takamura (1988) J. Colloid. Int. Sci, 125, 266.

[5] E. Jakeman, E.R. Pike and S. Swain, Journal of Physics A (1971), 4, 517-534.

[6] M. Kaszuba, M.T. Connah, F.K. McNeil-Watson and U. Nobbmann, Particle and Particle Systems Characterization (2007), 24(3):159-162.


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