Good practice for dynamic light scattering (DLS) measurements

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.

Bonnes pratiques pour l'analyse de la dispersion lumineuse dynamique (DLD)

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Publication Date
13-Apr-2020
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6060 - International Standard published
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18-Mar-2020
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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 2020

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Published in Switzerland
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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

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

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.

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 © ISO 2020 – All rights reserved
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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) 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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ISO/TR 22814:2020(E)

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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ISO/TR 22814:2020(E)
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
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