ISO/TR 22814:2020

TECHNICAL ISO/TR
REPORT 22814
First edition
2020-04
Good practice for dynamic light
scattering (DLS) measurements
Bonnes pratiques pour l'analyse de la dispersion lumineuse
dynamique (DLD)
Reference number
ISO/TR 22814:2020(E)
©
ISO 2020

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ISO/TR 22814:2020(E)

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ISO/TR 22814:2020(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Instrument types . 1
4.1 General . 1
4.2 Information prior to analysis . . 2
4.2.1 Sample information . 2
4.2.2 Desired outcome of analysis . 3
4.3 Appropriateness of samples for DLS analysis . 5
4.4 Sample preparation . 5
4.4.1 General. 5
4.4.2 Dispersion . 6
4.4.3 Filtering of sedimenting particle systems . 6
4.4.4 Dissolution and expansion . 7
4.4.5 Colour of samples . 7
4.4.6 Dilution. 8
4.4.7 Generation of air bubbles . 9
4.4.8 Measurement cell (only relevant when using cuvettes in a homodyne setup) . 9
4.5 Instrument verification. 9
4.5.1 General. 9
4.5.2 Operation qualification . 9
4.6 Data quality and interpretation: Correlation analysis .10
4.6.1 Interpretation of correlograms .10
4.6.2 Interpretation of particle size distribution .12
4.6.3 Conversion from intensity to volume or number-based results.12
4.6.4 Influence of the observed scattering angle .13
4.6.5 How to judge good data quality .13
4.7 Data quality and interpretation: Frequency power spectrum analysis .14
4.7.1 Frequency power spectrum .14
4.7.2 Precision and run time .15
4.7.3 Sample quality .16
4.8 Method robustness .18
4.8.1 Robustness .18
4.8.2 Ruggedness .18
4.8.3 Investigation of these parameters .18
Bibliography .21
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ISO/TR 22814:2020(E)

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 documents 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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
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iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 24, Particle characterization including
sieving, Subcommittee SC 4, Particle characterization.
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.
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ISO/TR 22814:2020(E)

Introduction
Dynamic light scattering (DLS) is a widely used technique for the characterization of particles with
equivalent hydrodynamic diameters below a few micrometres. Modern instruments allow users with
minimal training or background to use this technique. The downside is that not all users are familiar
with the potential pitfalls, limitations and proper interpretation of results for DLS.
Therefore, this document has been developed as a guidance for good practice in DLS and complements
ISO 22412:2017.
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TECHNICAL REPORT ISO/TR 22814:2020(E)
Good practice for dynamic light scattering (DLS)
measurements
1 Scope
This document provides practical guidance for performing and interpreting measurements using
dynamic light scattering (DLS) that goes beyond the treatment of measurement artefacts in
ISO 22412:2017.
This document is intended to help users with experiments planning, in particular with respect to
obtaining the necessary information on the sample and deciding whether DLS is the most appropriate
method. It provides information on how to prepare samples in an appropriate way, verify the proper
functioning of the instrument and interpret the data correctly, including ways to assess data quality.
This document focuses on the practical steps required to obtain DLS results of good quality, rather
than on theoretical considerations, and covers not only the measurement of solid particles, but also
emulsions and bubbles.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 Instrument types
4.1 General
A discussion on what constitutes good practices requires knowledge of the instrument type being
considered. Different optical configurations require different adjustment to control the optical
layout: different signal processing techniques require different techniques to allow for background
conditions; different analysis techniques require different conditioning parameters of the processed
signal. Two commonly applied variants are homodyne detection with correlation function processing
(see ISO 22412:2017, 9.2) and heterodyne detection with frequency spectrum processing (see
ISO 22412:2017, 9.3.
Additionally, good practice, as it relates to instrument type, also depends on the scattering angle used
for the measurement. For instance large spurious particles generally scatter more power into forward
angles than higher angles, so that samples measured in forward-scatter typically require significantly
more care regarding the cleanliness of the cuvette used, prior to filling with sample, the filtering of
the sample between the particle size distribution (PSD) of interest and unwanted large size fractions
and the dispensing to waste of the first few drops of sample from a syringe filter to remove filter spoil.
Additionally, the single-scattering relaxation time is known to be well approximated by higher order
scattering from concentrated samples as the scattering angle approaches 180 °, thereby allowing the
characterization in backscatter of concentrated samples, so long as bulk scattering losses through
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ISO/TR 22814:2020(E)

concentrated media can be avoided. Losses are mitigated in many commercial instruments by moving
the optical detection point closer to the cuvette wall using opto-, mechanical or opto-mechanical means.
4.2 Information prior to analysis
4.2.1 Sample information
The customer or submitter of a sample for DLS analysis provides, as available, all information
relevant to the measurement of the sample. Absence of information does not preclude analysis, but
availability of information aids the analyst with respect to sample preparation, measurement design
and interpretation of results. In general, the more information is available about a sample, the more
likely the analysis will be successful and the results meaningful for the customer. Availability of this
information can also reduce the uncertainty for the overall measurement result.
The following questions are answered where possible.
a) Questions related to the analysis step in DLS.
1) What is the primary composition of the sample?
The composition will determine the scattering properties and the complex refractive index, as
well as colloidal stability.
2) What is the crystallographic phase (if known)?
3) What is the density of the sample? Has a Stokes’ law calculation been carried out to show the
settling rate for particles of different sizes?
4) Is the sample coated (e.g. is there a polymeric coating, ligand or surfactant that modifies the
surface functionality and stability)?
5) What is the colloidal stability (sedimentation/agglomeration/dissolution…)?
If the colloid sediments or agglomerates, measurement results will change with time. This does
not necessarily invalidate the results, but it is important to know whether such changes are
expected and what information the user wants to obtain from the analysis (see 4.6.5.1, 4.6.5.2
and 4.7.3).
6) What is the complex refractive index of the particles?
The complex refractive index consists of a real and imaginary part. The former defines the
light scattering behaviour, the latter defines the light absorption behaviour. The real and the
imaginary part of the refractive index of the particle are necessary for converting intensity-
weighted to volume-weighted results.
b) Questions related to sample preparation.
1) What is the dispersing medium?
2) If the sample requires dilution, a diluent of similar chemical composition, ionic strength, pH
and that contains the same other additives is chosen.
3) What is the refractive index of the medium?
The refractive index of the medium is required for analysis. If unknown, it can be looked up in
tables or measured.
4) Does the medium contain surfactants necessary to maintain stability? If so, the surfactant and
its concentration are identified.
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5) What is the viscosity of the dispersion medium?
The viscosity of the dispersion medium is required for analysis. The viscosity of common fluids
is available in tabulated forms. If this is not available, it can be measured.
6) Can the sample be filtered to remove large scatterers such as dust or residual aggregates?
This depends on the size of the principal component and desired information from analysis
of sample.
7) If the sample is in suspension, is it clear? Does it contain sediment?
8) Are specific ingredients or procedures required for preparation of the sample?
9) Does the medium show non-Newtonian behaviour?
c) Questions related to the choice of the appropriate particle concentration.
1) What is the mass concentration of the sample (e.g. 0,01 mg/ml)?
d) Questions related to the selection of the appropriate evaluation algorithm.
Is the sample polydisperse (e.g. does it contain multiple size populations or a very broad size range,
is it agglomerated) and what is the anticipated size distribution?
Providing any available information about degree and nature of polydispersity can help to set up
the analysis.
The size distribution is in most cases the purpose of the measurement, but an expectation can help
not only to set up the experiment, but also allows checking the plausibility of the result.
Many modern instruments are capable of characterising multi-modal samples with distribution-
based analyses such as non-negative least squares. This is often the first step, prior to use of
cumulants to provide a z-average size and polydispersity if and only if the sample is monodisperse.
Laser diffraction may be considered for large aggregates; however, the user needs to be aware that
laser diffraction reports the hard sphere size in comparison to dynamic light scattering which
reports the hydrodynamic diameter.
e) Questions related to the identification of potential artefacts.
1) Are there other (non-principal) scattering components in the sample (e.g. proteins, a second
solid phase, micelles)?
Providing the known sizes of secondary components helps in the interpretation of results.
2) Are the principal particles highly asymmetric (e.g. rod-like)?
f) Questions related to sample storage before analysis.
1) Are special conditions necessary for sample storage before analysis (e.g. refrigerated, in dark,
exclusion of CO uptake etc.)?
2
2) Is the sample material subject to dissolution?
4.2.2 Desired outcome of analysis
In addition to providing the analyst with basic information about the sample, it is equally important
for the customer or submitter to stipulate the purpose and desired outcome of the analysis. This will
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determine the level of effort expended and will aid the analyst in the experimental design. The following
questions are most relevant.
a) In which context will the results be used?
1) Applications in quality control (QC) typically require less stringent analysis than applications
in research and development or product characterization. Often the goal is detection of change
rather than accurate determination of size/size distribution. In these cases, factors that lead
to a constant bias (non-Newtonian media, inaccurate knowledge of the refraction indices etc.)
will not affect the conclusion drawn from e.g. a time series.
2) Applications in research and development or product characterization may require higher
levels of scrutiny depending on the application need.
b) Is DLS able to deliver results at the required uncertainty level?
On a material with a narrow size distribution and a median diameter of 100 nm, relative expanded
uncertainties of the z-average of ≤3 % are achievable (see ISO 22412:2017, 10.1). For more
polydisperse materials, higher uncertainties are expected. More data on uncertainties are given in
e.g. References [4],[5], and [6]. Depending on how the result is used, DLS may not be able to deliver
results with the required accuracy.
c) Should a mean size and polydispersity index value be reported?
1) This typically involves cumulants analysis, which delivers robust results for a monomodal
Gaussian size distribution. It is not applicable to highly polydisperse systems or samples with a
more complex distribution.
NOTE The scattering intensity into all scattering angles from particles of diameter < 1/10th of the
wavelength of the illuminating light beam is well-approximated, to within a few per cent, as proportional
the 6th power of the particle radius.
2) The polydispersity index can be indicative of sample quality and hence for the suitability of
DLS to measure the sample in question.
3) Cumulants may be useful for QC applications in particular, but see caveat in 1) above.
4) The use of software that generates Gaussian distributions from a mean value and a
polydispersity index is deprecated, as the generated distributions may not correlate to the
actual particle size distribution of the sample.
d) Is a size distribution rather than just a mean/modal size required?
1) The basic distribution analysis yields a scattered intensity-weighted hydrodynamic size
distribution.
2) To convert the intensity-weighted distribution to volume or number basis, the complex
refractive index for the sample material is required. However, it is deprecated to convert from
intensity to volume and especially to number basis due to the inherent errors involved in this
process, except for comparative purposes.
3) Due to low resolution of DLS, decentiles (e.g. x , x ) can carry high uncertainties, especially
10 20
those away from the median diameter. Therefore, use of decentiles is not recommended.
NOTE The mean size of the distribution can differ from that obtained by cumulants analysis.
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4.3 Appropriateness of samples for DLS analysis
a) If sedimentation is clearly observed over a time period relevant to the measurement, then the
sample is not appropriate for DLS analysis.
Sedimentation will manifest itself as a trend towards smaller particle sizes over time, so a simple
check for excessive sedimentation is to re-measure the same suspension after some period of time
has elapsed.
Potential solutions are filtration to remove the sedimenting fraction or the choice of other
techniques, for example laser diffraction.
b) If the sample contains a substantial amount of very coarse particles, then it may not be appropriate
for DLS.
Very coarse particles can be removed by filtration unless these very coarse particles are of interest
in the analysis.
c) If the medium is shear thinning or generally non-Newtonian, and the zero-shear viscosity is not
known or cannot be measured, then the sample is probably not appropriate for DLS analysis.
If the purpose of the analysis is a comparison of different samples of the same composition (for
example comparison of different batches or observation of a sample over time) rather than the
determination of an absolute size, these effects are not relevant, as they affect all samples in the
same way.
d) If the sample is too highly concentrated, multiple scattering, particle-particle interactions and
restricted diffusion can influence the result (see ISO 22412:2017, B.2). Measurement of such samples
may require dilution or specialized equipment. For example, certain instrument configurations can
be used to minimize multiple scattering. Measurement at different dilutions is the method of choice
to detect these effects.
NOTE ISO/TR 19997 describes methods for diluting a sample with the dispersing liquid.
e) In some instances, the particles can be excited by the incident light causing fluorescence that
interferes with DLS measurements. As is the case for absorption [see f) and 4.4.5], also fluorescence
is wavelength dependent.
When fluorescence is a problem, two approaches can be used to avoid or minimize its influence.
One is using a different wavelength (a longer wavelength than the original) that does not generate
fluorescence. The other is the installation of a narrow bandpass filter that blocks the fluorescent
interference from reaching the detector.
f) If the sample is darkly coloured, this may interfere with DLS analysis due to absorption of laser
light (see 4.4.5).
Potential solutions include dilution of the sample, measurement at a wavelength in which the
medium does not absorb light or measurement in backscattering mode.
4.4 Sample preparation
4.4.1 General
Sample preparation is conducted with due consideration of the purpose of the measurement. This
process also considers the light scattering properties of the sample and the way the signal is detected
and processed. For instance, in a typical DLS measurement, scattered light is detected from a very small
well-defined volume within the suspension. The intensity is, as stated previously, highly dependent on
the particle size, and the rate of intensity fluctuations results from interference phenomena due to light
scattered by many particles simultaneously. The following are important considerations for sample
preparation.
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4.4.2 Dispersion
The sample particles are uniformly dispersed in a suspension medium during the measurement in order
to perform the measurement with high accuracy and reproducibility. As the measurement volume,
which is called “scattering volume”, is very small in a DLS measurement, the uniformity of a sample
suspension strongly influences the determination of particle size.
It can be difficult to disperse particles, especially inorganic particles, down to constituent particles
without additives or the application of mechanical or ultrasonic energy. To improve the dispersibility of
particles, one or more dispersants can be added to the sample suspension, and/or ultrasonic treatment
can be used if it does not alter the sample properties. The dispersants, which may be inorganic species,
surfactants or polymers, adsorb to the particle surface to increase surface charge and/or provide steric
repulsion (this adsorption also increases the apparent particle size by a few nanometres). The principal
dissociative groups of dispersants are phosphoric acid, carboxylic acid, sulfonic acid, and amine. The
concentration of dispersant is higher than the amount needed to stabilize the suspension but below
the critical micelle formation concentration. Typical dispersants for aqueous suspension are shown in
Table 1. The appropriate dispersant depends on the condition of sample suspensions such as particle
size, concentration and shape. For further details, see Reference [2].
In addition, pH adjustment can increase the surface charge of particles. The pH of a sample suspension
can be adjusted by adding an acid or alkali to make it lower or higher than the isoelectric point of sample
particles. For a general discussion on this issue, see Reference [3]. The acid or alkali added to a sample
suspension have ions in common to a sample and do not interact with particles.
One needs to filter solvents and other dilution media so as to reach a particle size that is equal to
or lower than the expected particle size of the sample. For example, if 20 nm to 70 nm particles are
to be measured, then filtration of the dispersion medium through a 0,02 μm filter is recommended.
Measurement of the dilution medium (filtered or not) by DLS can ensure that it does not contain
particles that interfere with the measurement of the sample.
NOTE Some filter materials can shed particles and contaminate the sample or can interact with the
dispersion medium.
Table 1 — Typical dispersants for aqueous suspension
Category Anion/Cation Dispersant
Inorganic compound Anion Polyphosphoric acid
Surfactant Anion Alkylsulfonic acid
Cation Quaternary amine
Polymer Anion Polycarboxylic acid
Polyacrylic acid
Naphthalenesulfonic acid
Non-ionic Polyethylene glycol
4.4.3 Filtering of sedimenting particle systems
When the average particle size tends to decrease with repeated measurements, sedimentation within
the sample is suspected and the accuracy of the measurement might be compromised. If the sedimenting
fraction does not contain the target particles, it can be removed by filtration with an appropriate pore
size and filter material or by centrifugation.
NOTE Filtration significantly changes the particles available for analysis by removing an unknown fraction
of oversize particles/aggregates/agglomerates and potentially also particles of interest. Depending on the use of
the results, filtration can be inappropriate.
A filter is chosen that is not changed (in the worst case: dissolved) by the medium, which means that
often different filter types are used for aqueous and organic media. When a protein solution of low
concentration is filtered, the use of a
...