ISO 22412:2025

Particle size analysis - Dynamic light scattering (DLS)

This document specifies the application of dynamic light scattering (DLS) to the following: - measurement of average hydrodynamic particle size; - measurement of the size distribution of mainly sub micrometre-sized particles, emulsions or fine bubbles dispersed in liquids. This document is applicable to the measurement of a broad range of dilute and concentrated suspensions.

Analyse granulométrique — Dispersion lumineuse dynamique (DLD)

General Information

• Status: Published
• Publication Date: 04-Sep-2025
• ICS: 19.120 - Particle size analysis. Sieving
• Technical Committee: ISO/TC 24/SC 4 - Particle characterization
• Drafting Committee: ISO/TC 24/SC 4 - Particle characterization
• Current Stage: 6060 - International Standard published
• Start Date: 05-Sep-2025
• Due Date: 17-Aug-2025
• Completion Date: 05-Sep-2025

Relations

• Revises: ISO 22412:2017 - Particle size analysis - Dynamic light scattering (DLS)
• Effective Date: 20-Aug-2022

Overview

ISO 22412:2025 - Particle size analysis - Dynamic light scattering (DLS) specifies how to apply dynamic light scattering for measuring average hydrodynamic particle size and size distributions of mainly sub‑micrometre particles, emulsions and fine bubbles dispersed in liquids. The third edition extends applicability to both dilute and concentrated suspensions and adds multi‑angle and imaging DLS methods. The standard covers instrument requirements, sample preparation, measurement procedures, data evaluation and quality control.

Key technical topics and requirements

• Scope and definitions: clear terms for hydrodynamic diameter, polydispersity index (PI), scattering volume, count rate, reference materials and qualification concepts.
• Principle and apparatus: fundamentals of DLS measurement geometry, lasers and detectors; differences between homodyne/heterodyne modes and single vs multi‑angle setups.
• Sample preparation: guidance on concentration limits, checks for concentration suitability and recommendations for handling dilute and concentrated suspensions (Annex C).
• Measurement procedures: stepwise measurement planning, acquisition modes (autocorrelation, cross‑correlation, frequency analysis, spatial correlation) and multi‑angle/online options (Annex B).
• Data evaluation methods: correlation analysis (cumulants method), distribution algorithms (NNLS, CONTIN and others), frequency analysis, and advanced approaches such as MADLS (multi‑angle DLS), imaging DLS (IDLS, UIDLS) and polarization‑separated backscatter cross‑correlation.
• Quality assurance: system qualification, use of reference materials/CRMs, quality control checks, method precision and measurement uncertainty (Clause 10).
• Reporting and artefact guidance: required elements for test reports and annexed guidance on artefacts, interpretation and measurement planning (Annexes D and E).

Practical applications

ISO 22412:2025 is applicable where reliable sub‑micrometre particle sizing is required, including:

• Pharmaceutical formulations (suspensions, liposomes, protein aggregates)
• Nanomaterials and colloid research (R&D and characterization)
• Food and cosmetic emulsions and quality control
• Environmental and process monitoring (fine bubbles, wastewater colloids, online process control)
• Instrument validation and commercial DLS system development

Who should use this standard

• Analytical laboratories and quality control teams performing particle size analysis
• Research scientists in colloid, nanoscience and formulation development
• Instrument manufacturers and service providers for DLS systems
• Regulatory and accreditation bodies assessing measurement traceability and uncertainty

Related standards

• ISO 9276 (particle size distribution presentation)
• ISO 26824 (terminology)
• ISO 17034, ISO 33405, ISO 33401 (reference material and certification guidance)
• ISO/IEC Guide 99 (metrological traceability)

Using ISO 22412:2025 ensures standardized, reproducible DLS measurements and supports robust interpretation of hydrodynamic size and distribution data across laboratory and industrial contexts.

Standards Content (Sample)

International
Standard
ISO 22412
Third edition
Particle size analysis — Dynamic
2025-09
light scattering (DLS)
Analyse granulométrique — Dispersion lumineuse dynamique (DLD)
Reference number
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and units. 3
5 Principle . 4
6 Apparatus . 5
7 Test sample preparation . 7
7.1 General .7
7.2 Concentration limits .7
7.3 Checks for concentration suitability .8
8 Measurement procedure . 8
9 Evaluation of results .10
9.1 General .10
9.2 Correlation analysis .11
9.2.1 Cumulants method .11
9.2.2 Distribution calculation algorithms .11
9.3 Frequency analysis . 12
9.4 Multi angle dynamic light scattering (MADLS) . 12
9.4.1 Measurement of an angular-independent particle size distribution . 12
9.4.2 General angular and concentration dependence .14
9.4.3 Measurement of particles with optical anisotropy .14
9.5 Imaging dynamic light scattering (DLS) .14
9.5.1 Image-based dynamic light scattering (IDLS) .14
9.5.2 Ultrafast image-based dynamic light scattering (UIDLS) . 15
10 System qualification and quality control .15
10.1 System qualification . 15
10.2 Quality control of measurement results .16
10.3 Method precision and measurement uncertainty .16
11 Test report . 17
Annex A (informative) Theoretical background . 19
Annex B (informative) Online measurements .33
Annex C (informative) Recommendations for sample preparation .35
Annex D (informative) Guidance on measurement planning, data interpretation and quality
control . 41
Annex E (informative) Guidance on potential measurement artefacts and on ways to minimize
their influence .58
Bibliography . 61

iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
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Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 24, Particle characterization including sieving,
Subcommittee SC 4, Particle characterization.
This third edition cancels and replaces the second edition (ISO 22412:2017), which has been technically
revised.
The main changes are as follows:
— inclusion of multi-angle dynamic light scattering (MADLS);
— inclusion of imaging dynamic light scattering (DLS);
— addition of Clause A.5 on polarisation-separated backscatter photon cross-correlation spectroscopy;
— extension of Annex B on online measurements;
— incorporation of the content from ISO TR 22814 into Annexes C and D.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
Introduction
Particle size analysis in the sub micrometre size range is performed on a routine basis using the dynamic
light scattering (DLS) technique, which probes the hydrodynamic mobility of the particles. The technique
is successful because it provides estimates of the average particle size and size distribution within a few
minutes, and because user-friendly commercial instruments are available. Nevertheless, proper use of the
instrument and interpretation of the result involve certain precautions.
The principle of DLS for a concentrated suspension is the same as for a dilute suspension. However, specific
requirements for the instrument setup and specification of test sample preparation are specified for
concentrated suspensions. At high concentrations, particle-particle interactions and multiple light scattering
can become dominant and can result in apparent particle sizes that differ between concentrated and dilute
suspensions.
DLS is also referred to as “quasi-elastic light scattering (QELS)” and “photon correlation spectroscopy (PCS),”
although PCS is actually one of the measurement methods.
Several methods have been developed for DLS. These methods can be classified in several ways:
a) by the difference in raw data acquisition (autocorrelation, cross-correlation and frequency analysis,
spatial correlation);
b) by the difference in optical setup (homodyne mode versus heterodyne mode);
c) by the angle of observation.
In addition, instruments show differences with respect to the type of laser source and often allow application
of different data analysis algorithms, e.g. cumulants, non-negative least squares (NNLS), CONTIN, etc.

v
International Standard ISO 22412:2025(en)
Particle size analysis — Dynamic light scattering (DLS)
1 Scope
This document specifies the application of dynamic light scattering (DLS) to the following:
— measurement of average hydrodynamic particle size;
— measurement of the size distribution of mainly sub micrometre-sized particles, emulsions or fine bubbles
dispersed in liquids.
This document is applicable to the measurement of a broad range of dilute and concentrated suspensions.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
[SOURCE: ISO 26824:2022, 1.1, modified — Note 3 to entry has been deleted.]
3.2
average hydrodynamic diameter
x
DLS
hydrodynamic diameter that reflects the central value of the underlying particle (3.1) size distribution
Note 1 to entry: The average particle diameter is either directly determined without calculation of the particle size
distribution, or calculated from the computed intensity-, volume- or number-weighted particle size distribution or
from its fitted (transformed) density function. The exact nature of the average particle diameter depends on the
evaluation algorithm.
Note 2 to entry: The cumulants method yields a scattered light intensity-weighted harmonic mean particle diameter,
which is sometimes also referred to as the “z-average diameter.”
Note 3 to entry: Arithmetic, geometric and harmonic mean values can be calculated from the particle size distribution
according to ISO 9276-2.
Note 4 to entry: Mean values calculated from density functions (linear abscissa) and transformed density functions
(logarithmic abscissa) can differ significantly (see ISO 9276-1).

Note 5 to entry: x also depends on the particle shape, polydispersity and the scattering vector (and thus on the
DLS
angle of observation, laser wavelength and refractive index of the suspension medium).
Note 6 to entry: The hydrodynamic radius may also be specified.
3.3
polydispersity index
PI
dimensionless measure of the broadness of the size distribution
3.4
scattering volume
volume defined by the intersection of the incident laser beam and the scattered light intercepted by the
detector
3.5
scattered intensity
intensity of the light scattered by the particles (3.1) in the scattering volume (3.4)
3.6
count rate
I
s
number of photon pulses per unit time
Note 1 to entry: In frequency analysis, the term photocurrent captures a similar concept. The current at the
photodetector is proportional to the scattered intensity (3.5) as measured by a detector.
3.7
validation
proof with reference material (3.8) that a measurement procedure is acceptable for all elements of its scope
Note 1 to entry: Evaluation of trueness requires a certified reference material (3.9).
3.8
reference material
RM
material, sufficiently homogeneous and stable with respect to one or more specified properties, which has
been established to be fit for its intended use in a measurement process
Note 1 to entry: Reference material is a generic term.
Note 2 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 3 to entry: Uses may include the calibration of a measurement system, assessment of a measurement procedure,
assigning values to other materials, and quality control.
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition, but restricts the term “measurement” to apply
to quantitative values. However, Note 3 of the definition in ISO/IEC Guide 99:2007 specifically includes qualitative
properties, called “nominal properties”.
[SOURCE: ISO 17034:2016, 3.3]
3.9
certified reference material
CRM
reference material (3.8) characterized by a metrologically valid procedure for one or more specified
properties, accompanied by a certificate that provides the value of the specified property, its associated
uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or
sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence.

Note 2 to entry: Metrologically valid procedures for the production and certification of reference materials are given
in, among others, ISO 33405.
Note 3 to entry: ISO 33401 gives guidance on the contents of reference material certificates.
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition.
[SOURCE: ISO 17034:2016, 3.2]
3.10
qualification
proof with reference material (3.8) that an instrument is operating in agreement with its specifications
4 Symbols and units
For the purposes of this document, the following symbols and units apply.
C(Γ) normalized distribution function of decay rates or charac- dimensionless
teristic frequencies
D translational diffusion coefficient metres squared per m /s
T
second
D collective diffusion coefficient metres squared per m /s
c
second
D self-diffusion coefficient metres squared per m /s
s
second
f frequency, f = ω/(2 π) Hertz Hz
(1)
g (τ) normalized electric field correlation function dimensionless
(2)
G (τ) scattered intensity correlation function arbitrary units
G(Γ ) normalized distribution function of the individual decay arbitrary units
j
rate Γ
j
I scattered intensity, count rate, photocurrent arbitrary units
s
I intensity of the incident light arbitrary units
M number of steps in the histogram dimensionless
n refractive index of the suspension medium dimensionless
P(ω) power spectrum arbitrary units
PI polydispersity index dimensionless
ΔQ scattered light intensity-weighted amount of particles in dimensionless
int,i
size fraction i,
i.e. x < x < = x
i−1 i
x hydrodynamic diameter of a particle (within this docu- nanometres nm
ment)
x average hydrodynamic diameter nanometres nm
DLS
−1
Γ scattered light intensity-weighted average value of the reciprocal seconds s
distribution function of the decay rate or characteristic
frequency
−1
Γ maximum decay rate (histogram method) reciprocal seconds s
max
−1
Γ minimum decay rate (histogram method) reciprocal seconds s
min
η viscosity of the suspension medium Milli Pascal seconds mPa·s
θ scattering angle degrees °
λ wavelength of the laser light in vacuum nanometres nm
−2
μ second cumulant of the distribution function of decay reciprocal square  s
rates or characteristic frequencies seconds
ρ particle density grams per cubic  g/cm
centimetre
τ correlation time seconds s
−1
q modulus of the scattering wave vector reciprocal  nm
nanometres
φ particle volume fraction dimensionless
ω angular frequency radian per seconds rad/s
5 Principle
Particles suspended in a fluid are in constant Brownian motion as the result of the interaction with the
[1]
molecules of the suspending fluid. In the Stokes-Einstein theory of Brownian motion, particle motion of
smooth spheres at very low concentration is determined by the suspending fluid viscosity and temperature,
as well as the size of the particles. Thus, from a measurement of the particle motion in a fluid of known
temperature and viscosity, the particle size can be determined.
[2],[3],[4],[5],[6]
The DLS technique probes the particle motion optically. The suspended particles are illuminated
with a coherent monochromatic light source. The light scattered from the moving suspended particles has
a time-dependent phase imparted to it from the time-dependent position. The time-dependent phase of the
scattered light can be considered either as a time-dependent phase shift or as a spectral frequency shift from
the central frequency of the light source. Measured over time, random particle motion forms a distribution
of optical phase shifts or spectral frequency shifts. These shifts are determined by comparison either with
all scattered light (homodyne or self-beating mode) or by using a portion of the incident light as reference
(heterodyne mode). Regardless of the setup, the optical signals received from the particles are related to the
scattering efficiency of the particles and are thus scattered intensity-weighted.
Sedimentation of particles, dependent on their density, sets an upper limit to the particle size that can be
assessed by the technique; typically, the upper limit is much less than 10 μm.
For very small particles, the impact of the molecules of the dispersing liquid on the particles becomes
[7]
relevant and thus sets a lower size limit to the validity of the Stokes-Einstein equation. The bias is usually
smaller than 1 % for particles with diameters larger than 10 nm, but it will increase for particles with
diameters of less than 10 nm.
DLS was developed for static suspensions. If orthogonal flow and observation axes are adopted, flowing
samples may, under some circumstances, be measured if the procedure is properly validated (see Annex B).
Different modes of diffusion, particle-particle interaction, multiple scattering and fluorescence can
significantly influence the apparent particle diameter calculated from a DLS experiment. Annexes D and E
should be consulted.
6 Apparatus
A typical apparatus consists of the following components:
6.1 Laser, emitting polarized coherent monochromatic light. Any kind of lasers may be used, e.g. gas lasers
(He-Ne laser, Ar-ion laser), solid-state lasers, diode-pumped solid-state lasers and laser diodes.
6.2 Optics, lenses and equipment used to focus the incident laser light into a scattering volume and to detect
scattered light. Optical fibres are often used as a part of the detection system and for light-delivering optics.
The use of a coherent optical reference allows using interference between the scattered light and the
reference to measure the frequency shift of the scattered light. Three methods of referencing are commonly
used and are illustrated in Figures 1 a) and b). A cross-correlation setup using two simultaneous homodyne
experiments is shown in 1 c).
— In homodyne detection (also referred to as “self-beating detection”), the mixing at the optical detector of
all of the collected scattered light provides the reference for frequency- or phase-difference measurement.
See Figure 1 a).
— In heterodyne detection, the scattered light is mixed with a portion of the incident light. The unshifted
incident light provides the reference for the frequency- or phase-difference measurement. See Figure 1 b).
NOTE In DLS, “heterodyne” is understood as mixing of scattered light with unscattered light from the same
source. This convention differs from, for example, the use in optical interferometry.
— In a cross-correlation setup, two homodyne scattering measurements are performed simultaneously in
such a way that the two scattering vectors and scattering volumes are the same, but the corresponding
wave vectors are not coincidental. These two laser beams produce two correlated fluctuation patterns.
The correlation intercept is reduced because both detectors collect light from the other scattering
experiment. This cross scattering is suppressed by state-of-the-art instruments (for a description
of polarisation-separated backscatter photon cross-correlation spectroscopy (PsB PCCS) see A.5).
The contribution of the multiply scattered light to the detector signals, however, does not contribute
to the cross-correlation function, i.e. does not contribute to the PSD, but to an enhanced background.
[Figure 1 c)]
a) Homodyne b) Heterodyne c) Cross-correlation
Key
1 laser
2 sample
3, 3’, 3’’ detector(s): one or several (not limited to three) detectors can be used
4 correlator, spectrum analyser or computer data processing
5 reflection area
6 beam splitter and optics
Figure 1 — Typical optical arrangements

6.3 Test sample holder, allowing fluctuations of the sample temperature to be controlled to within ±0,3 °C.
While precise knowledge of the sample temperature is required for evaluation, it is not necessary to regulate
the temperature to any defined value. However, setting a specific temperature is necessary in some cases to
prevent temperature driven effects like convection, demixing, coagulation, etc.
6.4 Photodetector, with an output that is proportionally related to the intensity of the collected scattered
light. A photomultiplier tube or an (avalanche) photodiode or a charge-coupled device (CCD)/ complimentary
metal-oxide-semiconductor (CMOS) is typically used. In imaging DLS, the detector is a CMOS chip that
records spatially distinct signals. Detectors can be placed at any angle, or, like in multi-angle DLS, in more
than one angle. Data collection can be performed in a linear or logarithmic manner. In the image DLS and
ultrafast image DLS methods, data collection can be performed continuously for hundreds and thousands of
images, or only one pair of images.
The position of the detector determines the scattering angle of the measurement, which can have an influence
on the measurement result. At particle diameters smaller than about λ/10, mainly isotropic Rayleigh light
scattering is observed, whereas particle diameters > λ/10 (Mie light scattering) result in a size dependent
pattern of minima and maxima.
Detection at backscattering angles (typically about 150° to about 175°) can be used to accommodate
different geometries where the 90° (side scatter) can be blocked. Often, backscatter detection means that
the path length of light through the sample is shorter and can be less affected by multiple scattering.
The short path length also means that the light intensity from highly-diluted, weak scatterers can be
insufficient for a reliable result.
Detection at a scattering angle of about 90° is often recommended for weak scatterers as this setup blocks
the flare of laser light at the cuvette wall.
Detection at forward scattering angles (typically about 15°) is especially sensitive to large particles, which
scatter more light in the forward direction, and is therefore well suited to monitor few large particles or to
monitor agglomeration. However, forward scatter can suffer from the most artefacts due to cleanliness or
contaminated systems.
6.5 Signal processing unit, capable of taking the time-dependent scattered light intensity signal and
outputting the autocorrelation function or autocorrelation coefficient, cross-correlation function or power
spectrum of the input signal. This correlation can be performed by either hardware or software correlators,
or both, operating linearly, logarithmically or in a mixed mode.
The resulting output from either mode contains a distribution of characteristic frequencies or time-
dependent phases representative of the particle size of the suspended particles. Photon detection has a
probability distribution of photon arrival times, which means that a fluctuating signal is obtained even if the
intensity of the incident light is constant. The intensity of the photons arriving at varying time intervals is
superimposed on this already fluctuating signal. In correlation analysis, the uncorrelated signal is constant,
whereas the signal associated with the diffusing particles decays exponentially. In spectrum analysis, the
uncorrelated signal is akin to a DC or zero frequency term, which is not recorded. The time-dependent
component is analysed to determine the particle-size distribution using the theory of DLS.
6.6 Computation unit, capable of signal processing to obtain either the particle size or particle size
distribution, or both. Some computation units also function as the signal-processing unit.
— Evaluation via the autocorrelation function allows determination of a mean diameter without
determination of the particle size distribution, but determination of the distribution is also possible.
— Evaluation via the frequency distribution determines the particle size distribution using the power
spectrum of the signal.
— Evaluation via photon cross-correlation allows quantification and minimization of the effects of multiple
scattering, thus extending the useful concentration range towards higher concentrations (however, the
effect of particle-particle interaction cannot be eliminated).

6.7 Instrument location, placed in a clean environment, free from excessive electrical noise and
mechanical vibration and out of direct sunlight. If organic liquids are used as the suspension medium, the
area shall be well ventilated. The instrument shall be placed on a rigid table or bench to avoid the necessity
for frequent realignment of the optical system.
WARNING — DLS instruments are equipped with a low- or medium-power laser whose radiation can
cause permanent eye damage. Never look into the direct path of the laser beam or its reflections.
Ensure highly reflecting surfaces are not in the path of the laser beam when the laser is on. It is
the responsibility of the user of this document to determine the applicability of local regulations for
laser radiation safety.
7 Test sample preparation
7.1 General
Test samples should consist of well-dispersed particles in a liquid medium. Dispersion procedures like
sonication, filtration, etc. can influence the result and therefore shall be reported. The suspension liquid shall:
a) be sufficiently transparent (non-absorbing) and non-fluorescent at the laser wavelength to create a
clear signal;
b) be free of particulate contamination;
c) not dissolve, swell or coagulate the particulate material;
d) have a known refractive index that is sufficiently different from that of the particulate materials to
create a clear signal;
e) have a known value of viscosity within ±2 % over the operational range of temperature to be used;
NOTE As x is directly proportional to η, the uncertainty of x will always be larger than the
DLS DLS
uncertainty of η.
f) meet the guidelines of the instrument for low background scattering.
This can be checked by measuring the count rate for the suspending medium alone and the dark count
with no sample or solvent present. The former should be at least one order of magnitude lower than the
sample, and the latter should be within the recommended range for the instrument.
Inadequate suppression of the double layer can have a significant influence on the hydrodynamic diameter.
A medium with ionic strength high enough to suppress the electric or diffuse double layer can improve
agreement between results obtained by DLS and electron microscopy. A conductivity of 1 mS/cm is usually
sufficient to achieve this between the hydrodynamic diameter and that obtained by microscopy techniques,
especially for small particles.
Water is often used as a suspension medium. The use of freshly deionised and filtered (pore size 0,2 µm)
water is recommended. A trace of ionic additive (e.g. NaCl at a concentration of 10 mmol/l = 0,6 g/l) may be
added to such samples to reduce the double-layer thickness. However, precaution must be made that such
ionic strength adjustment will not make sample unstable or that the additive does not react with the sample
(e.g. Cl with Ag ions).
7.2 Concentration limits
The lower concentration limit of the working range of DLS is determined, amongst other factors like particle
size, detector sensitivity, etc., by the number of particles that are present in the scattering volume.
The scattered light intensity (e.g. expressed as count rate or I ) of the sample containing the dispersed
s
particles should ideally be ≥10 times the signal obtained by the suspension medium alone. Scattered intensity
ratios below 10, either caused by low particle mass fractions or by very broad particle size distributions,
will result in a higher variation of results and poorer precision.

The maximum concentration of dispersed particles that can be measured without the concentration
influencing the particle size reported is determined by particle-particle interaction and multiple scattering.
This concentration limit should be determined empirically by dilution (see Annex C).
7.3 Checks for concentration suitability
Different instruments adopt differing optical observation angles and optical arrangements. The observations
and checks given are for the general case, but the specific instrument operational advice should also be
considered.
The following observations and checks are recommended.
a) Visually inspect the dispersed sample prior to placing it into the instrument. At low concentrations, the
sample will look almost transparent. At higher concentrations, a milky or opaque appearance is seen.
b) Ensure that the sample is placed in the instrument prior to performing the measurement, allowing the
sample to equalize its temperature. Check the count rate or signal level. Check the count rate or signal
level is appropriate, per the instrument manufacturer’s recommendations.
c) For instruments using correlation analysis in autocorrelation mode, conduct a measurement using
appropriate correlator settings and examine the intercept value, which should be above the value
specified by the vendor of the instrument. A low value of the intercept value corresponds to the factor
B in Formula A.3 and can result from either a poorly aligned optical system, multiple scattering or from
very weakly scattering samples requiring the detector aperture be increased, resulting in a multiple
coherence area detection. For larger particles, the measurement volume can need to be increased to
accommodate an adequate number of particles. This can also reduce the intercept value. A low value of
the intercept can also be caused by sample absorbance or fluorescence. All of these factors can reduce
the intercept value, requiring further tests to establish the reason.
A low intercept can also result from low sample concentration (at or below the limit of quantitation),
inappropriate optical settings (laser power, aperture, attenuation, etc.), or incorrect loading of the
sample. Autocorrelation functions that do not decay smoothly to baseline may indicate contamination
with large particles, back reflections, or fluorescence.
d) Measurements performed at different concentrations should give the same results within their
measurement uncertainties. A decreasing particle size with increasing concentration indicates a
significant amount of multiple scattering, a change of viscosity of the suspension caused by different
viscosities of diluent and original suspension and/or collective motion of particles which can result
from intermolecular (or particle-particle) interactions. Measurement at different concentrations is also
used to extrapolate the particle size to infinite dilution [see Clause 8 k)].
−5 −4
In many applications, a volume fraction (φ) of dispersed particulate material in the range 10 to 10 fulfils
the requirements for particle sizes below about 500 nm. For cross-correlation or backscatter methods,
higher concentrations can be achieved dependent on the sample. For either polydisperse or larger particles,
or both, it is potentially not possible to find a concentration that satisfies all requirements without either
increasing the coherence aperture of the receiver or increasing the diameter of the incident laser beam
in order to increase the measurement volume. If this is the case, then the intercept values obtained will
potentially not meet the criterion set out in c). For particle sizes above 1 µm, the requirements c) and d) can
only be fulfilled in exceptional cases.
All sample preparation steps (suspension medium, particulate concentration, dispersion procedure) should
be recorded. Recommendations for sample preparation are given in Annex C.
8 Measurement procedure
A typical measurement consists of the following steps:
a) Switch the instrument on and allow it to warm up. Typically, about 15 min to 30 min is required to
stabilize the laser intensity and to bring the sample holder to an equilibrium at the desired temperature.

NOTE 1 Monitoring the temperature rather than controlling it is sufficient for many applications, provided a
known value of viscosity is available for the reported monitored temperature.
b) A measurement cell filled only with suspension medium should be checked to ensure a low count rate
or signal level, without radical fluctuations, which can indicate particle contamination. A high count
rate or signal level can indicate cell flare or dirty cell walls. For instruments that provide background
subtraction, measure and store the background signal for the dispersion medium being used.
c) Visually inspect the sample for the presence of optically visible particles, flocs, fibres and other possible
contaminants. If these are present, repeat the sample preparation.
d) The measurement cell can be disposable (e.g. PMMA, polystyrene) or re-usable (e.g. optical-quality
glass or quartz). Compared to disposable plastic cells, glass cells have as main advantage that their use
can result in a better signal to noise ratio. The walls of plastic cuvettes are easily scratched and do not
provide the optical quality of glass or quartz. Therefore, disposable cuvettes should not be used with
weak scattering samples, and should be cleaned only by either rinsing with suspending medium or by
using a particle-free air stream to remove loose dust on the cell walls, or both.
The material of the measurement cell must be chemically compatible with the dispersion medium and
the particles. Clean and prepare measurement cells according to manufacturer's instructions. Additional
recommendations for cell cleaning are given in Annex C.4.
The pore size of the filter used for filtering the water or solvent should be appropriate to the application.
Ideally, a membrane with a pore size smaller than the smallest particle to be measured should be used.
Take care not to touch the windows of the measurement cell with bare hands or to wipe the cell surface
with any potentially abrasive material (including optical paper or tissue). Transfer a required amount
of sample to a suitable and clean measurement cell. Ensure that no air bubbles are entrapped in the test
sample for non-air bubble samples. Ensure that no bubbles are attached to the walls of the cell. Place
the test sample in the instrument or place the measurement probe into the sample. Allow temperature
equilibrium to be established. The temperature value should be known to better than ±0,5 °C with
minimal fluctuation of the value during the measurement.
Uncertainties in particle size determined in aqueous suspensions will be approximately 2 % per degree
Celsius at ambient temperature if the test sample has not reached thermal equilibrium.
NOTE 2 For a temperature change of 3 °C, it can take about 10 min for the liquid in the measurement volume of
a measuring cell holding a 1 ml of sample to thermally equilibrate.
e) Record test sample identification, date and time of the measurement and measurement duration,
number of individual measurements, measurement temperature, temperature fluctuations during
measurement, refractive index and viscosity of the suspension medium, particle concentration or
dilution, wavelength of laser and scattering angle, as well as the particle concentration, if known.
f) Check the average scattered intensity of the sample.
For homodyne optical arrangements, it is preferred that the average scattering intensity be controlled
by adjusting the light output power, using neutral density filters, or by minimizing the detector aperture,
to maintain detection coherence, while adjusting the receiver sensitivity within the limits specified by
the manufacturer.
For homodyne optical arrangements, it is preferred that the average scattering intensity be controlled
within the limits specified by the manufacturer. The scattering signal from the test sample should be
≥10 times the signal from the dispersing medium alone.
For heterodyne optical arrangements, the reference signal should be substantially greater than the test
sample scattering signal (a ratio reference signal:scattering signal of 10:1 should be aimed for). It is
preferred that the reference signal can be blocked so that the scattering signal can be assessed as being
greater than the suspension signal alone.
g) Measurements should not be continued if the light signal intensity contains isolated bursts of high count
rates, which can indicate contamination of the test sample.

h) Measurements should not be continued if the correlation function does not decline monotonically or if
the power spectrum is not of Lorentz type.
i) Record the average particle diameter, x , and polydispersity index (PI), for each of the measurements
DLS
performed using the cumulants method.
j) If a systematic concentration dependence of the average particle size is observed, the results of an
extrapolation to infinite dilution (or the results obtained at the lowest acceptable concentration) shall
be reported. The dilution shall be performed with particle-free suspension medium, containing the
same concentration of salts, surfactants, pH, etc., in order to not alter the particle-solution interactions.
Although the checks described here will minimize biasing effects due to multiple scattering, particle
interactions can potentially, in particular for particles below 100 nm (diameter) at volume fractions
above 0,01, bias the estimation of the average diameter. Therefore, for unknown dispersed systems, it is
recommended that measurements are performed on at least two concentrations varying by a factor of
at least two.
k) Check at the end of the measurement that no significant sedimentation has occurred in the test
sample, either by visually checking for sediment or by inspecting the results of multiple, sequential
measurements for trends. If sedimentation is found, then it should be decided whether it is small
enough so that
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