ISO 19430:2016
(Main)Particle size analysis — Particle tracking analysis (PTA) method
Particle size analysis — Particle tracking analysis (PTA) method
ISO 19430:2016 describes the evaluation of the number?based particle size distribution in liquid dispersions (solid, liquid or gaseous particles suspended in liquids) using the particle tracking analysis method for diffusion velocity measurements.
Analyse granulométrique — Méthode d'analyse de suivi de particule (PTA)
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INTERNATIONAL ISO
STANDARD 19430
First edition
2016-12-15
Particle size analysis — Particle
tracking analysis (PTA) method
Analyse granulométrique — Méthode d’analyse de suivi de
particule (PTA)
Reference number
ISO 19430:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO 19430:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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ii © ISO 2016 – All rights reserved
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ISO 19430:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 4
5 Principles . 4
5.1 General . 4
5.2 Key physical parameters. 5
5.3 Detection limits . 5
5.3.1 Lower size limit . . 5
5.3.2 Upper size limit . 6
5.3.3 Sample and sampling volume . 6
5.3.4 Maximum particle number concentration . 6
5.3.5 Minimum particle number concentration . 7
5.4 Measurement precision and uncertainties . 7
5.4.1 General. 7
5.4.2 Measurement precision . 7
5.4.3 Size range . 8
5.4.4 Counting efficiency . 8
5.4.5 Sizing accuracy . 9
5.4.6 Size resolution . . 9
6 Apparatus .10
7 Procedure.11
7.1 General .11
7.2 Sample preparation .12
7.3 Instrument set-up and initialisation .12
7.4 Measurement .13
7.4.1 Sample delivery . .13
7.4.2 Sample illumination .13
7.4.3 Particle imaging and tracking .14
7.4.4 Track analysis .14
7.5 Results evaluation .14
7.5.1 General.14
7.5.2 Particle size evaluation .14
7.5.3 Distribution analysis.14
7.5.4 Data analysis and reporting .14
8 System qualification and quality control .15
8.1 General .15
8.2 System installation requirements .15
8.3 System maintenance .15
8.4 System operation .15
8.5 System qualification .16
9 Data recording .17
10 Test report .17
Annex A (informative) Theory.20
Annex B (informative) Apparatus settings and best practice .23
Bibliography .25
© ISO 2016 – All rights reserved iii
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ISO 19430:2016(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 on 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 the following URL: www.iso.org/iso/foreword.html
The committee responsible for this document is Technical Committee ISO/TC 24, Particle
characterization including sieving, Subcommittee SC 4, Particle characterization.
iv © ISO 2016 – All rights reserved
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ISO 19430:2016(E)
Introduction
Regulatory, scientific and commercial requirements for nanomaterial characterization or
characterization of particulate suspensions where particle sizing and counting is required provide a
strong case for further development of techniques such as Particle Tracking Analysis (PTA), also known
[14]
as Nanoparticle Tracking Analysis (NTA) . Due to the fact that the term PTA covers a larger size
1)
range and is more generic , the term PTA is used throughout this document to refer to NTA and PTA.
For all aims and purposes, the term PTA also means NTA in this document.
PTA is based on measuring the diffusion movement of particles in a suspension by means of laser
illumination, imaging of scattered light, particle identification and localization, and individual particle
2)
tracking . In this case, suspension is an even dispersion of particles, gas bubbles or other liquid
droplets. The hydrodynamic diameter of the individual particles, droplets or bubbles is related to
Brownian motion parameters via the Stokes–Einstein equation.
In recent years the academic community working in fields such as liposomes and other drug
delivery vehicles, nanotoxicology, viruses, exosomes, protein aggregation, inkjet inks, pigment
particles, cosmetics, foodstuffs, fuel additives and fine bubbles began using the PTA technology for
[10]
characterization. An ASTM standard guide (E2834–12) was developed to give guidance to the
measurement of particle size distribution by means of Nanoparticle Tracking Analysis. The present
document aims to broaden the scope of the specification and to introduce system tests for PTA
operation.
This document outlines the theory and basic principles of the particle tracking analysis method along
with its limitations and advantages. It also describes commonly used instrument configurations and
measurement procedures as well as system qualifications and data reporting. One of the key aspects
is the meaning of the data and its interpretation. It should be noted that the key measurand obtained
from PTA measurement is the number-based particle size distribution where the size is taken to mean
the hydrodynamic diameter (3.11) of the particles in the sample. This size can be different from other
[6] [4].
sizes obtained with different techniques such as dynamic light scattering or electron microscopy
1) NTA is the most recognised abbreviation for the technique described in this document. However the Particle
Tracking Analysis (PTA) includes NTA in its size range of measurements.
2) For the purpose of this document “tracking” will mean “following in terms of particle x and y position” and the
“track” will mean “the path of that particle defined by such x and y coordinates of each step”
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INTERNATIONAL STANDARD ISO 19430:2016(E)
Particle size analysis — Particle tracking analysis (PTA)
method
1 Scope
This document describes the evaluation of the number–based particle size distribution in liquid
dispersions (solid, liquid or gaseous particles suspended in liquids) using the particle tracking analysis
method for diffusion velocity measurements.
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 terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.2
nano-object
material with one, two or three external dimensions in the nanoscale (3.1)
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3.3
nanoparticle
nano-object (3.2) with all three external dimensions in the nanoscale (3.1)
Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically
by more than three times), the terms nanofibre or nanoplate are intended to be used instead of the term
nanoparticle.
[SOURCE: ISO/TS 80004-4:2011, 2.4]
3.4
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
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ISO 19430:2016(E)
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general particle definition applies to nano-objects (3.2).
[SOURCE: ISO/TS 80004-6:2013, 2.9]
3.5
agglomerate
collection of weakly bound particles or aggregates or mixtures of the two where the resulting external
surface area is similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces, or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-4:2011, 2.8]
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area may
be significantly smaller than the sum of calculated surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent bonds, or those
resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-4:2011, 2.7]
3.7
particle size
linear dimension of a particle (3.4) determined by a specified measurement method and under specified
measurement conditions
Note 1 to entry: Different methods of analysis are based on the measurement of different physical properties.
Independent of the particle property actually measured, the particle size can be reported as a linear dimension,
e.g. as an equivalent spherical diameter.
[SOURCE: ISO/TS 80004-6:2013, 3.1.1]
3.8
particle size distribution
distribution of particles (3.4) as a function of particle size (3.7)
Note 1 to entry: Particle size distribution may be expressed as cumulative distribution or a distribution density
(distribution of the fraction of material in a size class, divided by the width of that class).
[SOURCE: ISO/TS 80004-6:2013, 3.1.2]
3.9
equivalent diameter
diameter of a sphere that produces a response by a given particle-sizing method, that is equivalent to
the response produced by the particle being measured
Note 1 to entry: The physical property to which the equivalent diameter refers is indicated using a suitable
subscript [ISO 9276-1:1998].
Note 2 to entry: For discrete-particle-counting, light-scattering instruments, an equivalent optical diameter is used.
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ISO 19430:2016(E)
Note 3 to entry: Other material constants like density of the particle are used for the calculation of the equivalent
diameter like Stokes diameter or sedimentation equivalent diameter. The material constants, used for the
calculation, should be reported additionally.
Note 4 to entry: For inertial instruments, the aerodynamic diameter is used. Aerodynamic diameter is the
−3
diameter of a sphere of density 1 000 kg m that has the same settling velocity as the irregular particle.
[SOURCE: ISO/TS 80004-6:2013, 3.1.5]
3.10
light scattering
change in propagation of light at the interface of two media having different optical properties
[SOURCE: ISO 13320:2009, 3.1.17]
3.11
hydrodynamic diameter
equivalent spherical diameter of a particle in a liquid having the same diffusion coefficient as the real
particle in that liquid
[SOURCE: ISO/TS 80004-6:2013, 3.2.6]
3.12
particle tracking analysis
PTA
method where particles undergoing Brownian motion in a liquid suspension are illuminated by a laser
and the change in position of individual particles is used to determine particle size
Note 1 to entry: Analysis of the time-dependent particle position yields translational diffusion coefficient and
hence the particle size as hydrodynamic diameter using the Stokes-Einstein relationship.
Note 2 to entry: Nanoparticle Tracking Analysis (NTA) is often used to describe PTA. NTA is a subset of PTA since
PTA covers larger range of particle sizes than nanoscale (3.1).
[SOURCE: ISO/TS 80004-6:2013, 3.2.8, modified — Nanoparticle tracking analysis has been removed
from the term, and Notes 1 and 2 have been modified.]
3.13
nanomaterial
material with any external dimension in the nanoscale (3.1) or having internal structure or surface
structure in the nanoscale
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.14
diluent
non-volatile homogeneous liquid which is used to decrease the number concentration of particles
(3.4) in a suspension without any deleterious effects such as changing particle total number, state of
aggregation, particle size (3.7) or surface chemistry
3.15
viscosity
measure of the resistance to flow or deformation of a liquid
[SOURCE: ISO 3104:1994]
3.16
percentile
value of a variable below which a certain percentage of observations fall
[SOURCE: ISO 11064-4:2013, 3.7]
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ISO 19430:2016(E)
4 Symbols and abbreviated terms
For the purposes of this document, the following symbols and abbreviated terms apply.
CCD Charge Coupled Device
CMOS Complementary Metal Oxide Semiconductor
CV Coefficient of Variation (standard deviation divided by arithmetic
average)(ISO 27448:2009, 3.11)
CCD Charge Coupled Device
d hydrodynamic diameter metre m
2
translational diffusion coefficient in 1 dimension m /s
D
x
2
translational diffusion coefficient in 2 dimensions m /s
D
xy
2
translational diffusion coefficient in 3 dimensions m /s
D
xyz
η viscosity of the suspension medium pascal second Pa·s
2 −2 −1
k Boltzmann’s constant m kg s K
B
RSD Relative Standard Deviation (ISO/TR 13843:2000, 2.34) %
T absolute temperature kelvin °K
t time second s
2
mean square displacement in 1 dimension metre squared m
2
x
()
2
mean square displacement in 2 dimensions metre squared m
2
xy,
()
2
mean square displacement in 3 dimensions metre squared m
2
xy,,z
()
5 Principles
5.1 General
Determination of particle size distribution by PTA makes use of the Brownian motion and light
scattering properties of particles suspended in liquids. Irradiation of the sample (typically by means
of a laser beam of wavelength in the visible region) leads to light scattering by objects with a refractive
index that is different from that of the surrounding medium. Light scattered from each particle is
collected by magnifying optics and visualized by way of a suitable detector, such as a Charge Coupled
Device (CCD) or Complementary Metal Oxide Semiconductor (CMOS) camera. By recording a series of
sequential images, the instrument’s software tracks positions of particles as a function of time, allowing
analysis of their movement.
3)
[6] [13],
By tracking individual particles, undergoing random Brownian motion from frame to frame,
the average spatial displacement of the particles per unit time can be calculated, and this displacement
3) For the purpose of this document, “frame” will mean a still image obtained from video capturing of the moving
objects in PTA measurement equipment”.
4 © ISO 2016 – All rights reserved
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ISO 19430:2016(E)
[13]
can be related to the hydrodynamic diameter of the particles through the Stokes-Einstein equation .
Although translational Brownian motion is a three-dimensional process, it is possible to use a one-,
two-, or three-dimensional diffusion coefficient to determine particle hydrodynamic diameter. The
relevant formulae are derived in Annex A and can be summarized with three formulae below:
2kTt
2
B
xD==t (1)
()
x
3πηd
4kTt
2
B
xy, ==Dt (2)
()
xy
3πηd
2kTt
2
B
xy,,zD==t (3)
()
xyz
πηd
2
Mean square displacement x can be measured in x and y directions independently to give two
()
2
independent values for particle size [Formula (1)]. In most PTA instruments, xy, is evaluated as
()
shown in Formula (2). It should be noted that in all three cases there is no assumption of two
dimensional movement of particles. All particles are assumed to be moving freely in all three
dimensions while the measurement is sampling the projection of each x, y and z component of that
movement onto the xy observation plane. As described in Annex A, these components (observables)
are independent variables
5.2 Key physical parameters
Formulae (1) to (3) show that as well as the diffusion coefficient, the temperature and the viscosity of
the sample shall be known in order to calculate the hydrodynamic diameter.
5.3 Detection limits
Like any measurement technique, PTA has detection limits in terms of the particle size and the particle
number concentration. These limits are heavily dependent on the particle material, diluent and
polydispersity of the sample.
Depending on the physical properties of the particles, the typical working range of the PTA can be from
about 10 nm to about 2 μm in diameter.
5.3.1 Lower size limit
The lower limit of detection in terms of the particle hydrodynamic diameter is determined (apart
from sensitivity and dynamic range of the camera) by the light scattering from the particles. It is the
combination of refractive indexes of the particle material and the diluent that affect the amount of light
scattering the detection and tracking system. A large difference in refractive indexes results in higher
scattering and therefore lower detection limit for all other parameters being the same.
Better tracking of highly scattering particles results in preferential counting of particles. The accuracy
of counting is covered in 5.4.4.
Sample polydispersity affects the ability to track and therefore analyse different size fractions in the
particle number-size distribution. The underlying effect is linked to the dynamic range of the video
capture and image analysis. In a polydisperse sample large particles scatter a lot more than small
particles making it difficult to detect or track small size particles. All the values in Table 1 are given
for monodisperse samples. In the case of a monodisperse gold spheres in suspension, the lower limit of
detection is typically 15 nm but can range from approximately 10 nm to 20 nm.
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ISO 19430:2016(E)
Below is the table of detection limits for commonly used dispersions (particle-diluent combinations).
Table 1 — Lower limit of detection for monodisperse suspensions of nanoparticles
Particle material Approximate lower detection limit
(Hydrodynamic diameter in nm)
Gold 15
Polystyrene 45
Silica 75
Biological materials 60
Other metals or metal oxides 25
General effects of samples and measurement parameters on the detection limits are described in the
subclauses below. The typical values quoted for room temperature water dispersion are provided in
Table 1. These values are approximate and could vary (as much as 30 %, for example leading to low
detection limit for gold ranging from approximately 10 nm to 20 nm) depending on factors such as
porosity of silica or the type of biological material.
5.3.2 Upper size limit
The upper particle size limit is limited by slowing Brownian motion at larger particle sizes. The motion
of such particles is very slow and long observation periods may be required. Very large particles can
also produce so much scattering that the detection system may not track much smaller particles in the
same polydisperse sample.
In the limit of very large particles (or gas bubbles) the sample may separate with heavy particles
sedimenting (or large bubble creaming). These effects shall be considered at all times for PTA
measurement.
5.3.3 Sample and sampling volume
In a number of applications the knowledge of sample volume and sampling volume involved in a PTA
measurement can be important. Typically used equipment requires approximately 1 ml of sample to be
used for measurement.
The subsampling methods can vary between manufacturers, yet the sampling volume of liquid that is
4)
being investigated within the PTA microscope field of view is often limited to a range of approximately
0,1 nl to 1 nl volume. The sampling volume, for the PTA measurement is limited laterally by the optical
field of view of the system to (typically of the order of) 100 μm by 100 μm area. The particles in that
area are tracked using imaging power of the optics with an approximate focus depth of (the order of)
10 μm which is taken as the sampling volume depth. This results in a sampling volume of 0,1 nl. Larger
sampling volumes may be obtained for optical systems with larger field of view or lower ma
...
DRAFT INTERNATIONAL STANDARD
ISO/DIS 19430
ISO/TC 24/SC 4 Secretariat: JISC
Voting begins on: Voting terminates on:
2015-03-23 2015-06-23
Determination of particle size distribution — Particle
tracking analysis
Détermination de la distribution granulométrique — Suivi de particule unique
ICS: 19.120
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
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STANDARDS MAY ON OCCASION HAVE TO
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POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 19430:2015(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
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ISO/DIS 19430:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2015
All rights reserved. Unless otherwise specified, 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
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2015 – All rights reserved
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ISO/DIS 19430:2015(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative References . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 4
5 Principles . 5
5.1 Key physical parameters. 5
5.2 Detection limits . 5
5.3 Measurement Accuracy . 6
6 Apparatus . 6
7 Procedure. 7
7.1 Sample preparation . 8
7.2 Instrument set-up and Initialisation . 8
7.3 Measurement . 9
7.3.1 Sample Delivery . 9
7.3.2 Sample Illumination . 9
7.3.3 Particle Imaging and Tracking . 9
7.3.4 Track Analysis .10
7.4 Results evaluation .10
7.4.1 Particle Size Evaluation .10
7.4.2 Distribution Analysis .11
7.4.3 Data Analysis and Reporting .11
8 System qualification and quality control .11
8.1 System installation requirements .11
8.2 System Maintenance .11
8.3 System Operation .12
8.4 System Qualification .12
9 Data Recording .13
10 Test report .13
Annex A (informative) Theory.15
Annex B (informative) Apparatus settings and best practice .18
Annex C (informative) Test Reporting .20
Bibliography .21
© ISO 2015 – All rights reserved iii
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ISO/DIS 19430:2015(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of normative document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical
experts in an ISO working group and is accepted for publication if it is approved by more than 50 %
of the members of the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a
technical committee and is accepted for publication if it is approved by 2/3 of the members of the
committee casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for
a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or
ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be
transformed into an International Standard or be withdrawn.
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.
ISO/TS 19430 was prepared by Technical Committee ISO/TC 24/SC 4, Particle characterization
including sieving
iv © ISO 2015 – All rights reserved
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ISO/DIS 19430:2015(E)
Introduction
Regulatory, scientific and commercial requirements for nanomaterial characterization or
characterization of particulate suspensions where particle sizing and counting is required provide a
strong case for further development of techniques such as Particle Tracking Analysis (PTA), also known
[1]
as Nanoparticle Tracking Analysis (NTA). Due to the fact that PTA covers a larger size range and is
1)
more generic the term PTA will be used throughout this document to refer to NTA and PTA. For all
aims and purposes the term PTA will also mean NTA in this document.
PTA is based on measuring the diffusion movement of particles in a suspension by means of laser
illumination, imaging of scattered light, particle identification and localization, and individual particle
tracking. The hydrodynamic diameter of the individual particles is related to Brownian motion
parameters via the Stokes-Einstein equation.
In recent years the academic community working in fields such as liposomes and other drug delivery
vehicles, nanotoxicology, viruses, exosomes, protein aggregation, ink jet inks, pigment particles,
cosmetics, foodstuffs, fuel additives and fine bubbles began using the PTA technology for characterization.
[2]
An ASTM standard guide (E2834–12) was developed to give guidance to the measurement of particle
size distribution by means Nanoparticle Tracking Analysis. The present document aims to broaden the
scope of the specification and to introduce system tests for PTA operation.
This document aims to outline the theory and basic principles of the particle tracking analysis with its
limitations and advantages. It will also describe commonly used apparatus and measurement procedures
as well as system qualifications and data reporting. One of the key aspects is the meaning of the data
and its interpretation. It should be noted that the key measurand obtained from PTA measurement is
the number-based particle size distribution where the size is taken to mean hydrodynamic diameter
(3.12) of the particles in the sample. This size may be different from other sizes obtained with different
[3] [4]
techniques such as dynamic light scattering or electron microscopy.
1) NTA is the most recognised abbreviation for the technique described in this document. However the Particle
Tracking Analysis (PTA) includes the NTA in its size range of measurements.
© ISO 2015 – All rights reserved v
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DRAFT INTERNATIONAL STANDARD ISO/DIS 19430:2015(E)
Determination of particle size distribution — Particle
tracking analysis
1 Scope
This document provides guidance and specification for the determination of the number-based particle
size distribution of suspensions of submicron particles using particle tracking analysis methodology.
2 Normative References
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO/FDIS 13322-1:2014, Particle size analysis — Image analysis methods — Part 1: Static image
analysis methods
ISO 26824:2013, Particle characterization of particulate systems — Vocabulary
3 Terms and definitions
3.1
nanoscale
size range from approximately 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size will typically, but not exclusively, be
exhibited in this size range. For such properties the size limits are considered approximate.
Note 2 to entry: The lower limit in this definition (approximately 1 nm) is introduced to avoid single and small
groups of atoms from being designated as nano-objects (3.2) or elements of nanostructures, which might be
implied by the absence of a lower limit.
[SOURCE: ISO/TS 80004-1:2010, definition 2.1]
3.2
nano-object
material with one, two or three external dimensions in the nanoscale (3.1)
Note 1 to entry: Generic term for all discrete nanoscale objects.
[SOURCE: ISO/TS 80004-1:2010, definition 2.5]
3.3
nanoparticle
nano-object (3.2) with all three external dimensions in the nanoscale (3.1)
Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically
by more than three times), the terms nanofibre or nanoplate are intended to be used instead of the term
nanoparticle.
[SOURCE: ISO/TS 27687:2008, definition 4.1]
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ISO/DIS 19430:2015(E)
3.4
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.
Note 3 to entry: This general particle definition applies to nano-objects (3.2).
[SOURCE: ISO 14644-6:2007, definition 2.102 and ISO/TS 27687:2008, definition 3.1]
3.5
agglomerate
collection of weakly bound particles or aggregates or mixtures of the two where the resulting external
surface area is similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces, or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 27687:2008, definition 3.2]
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area may be
significantly smaller than the sum of calculated surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent bonds, or those
resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 27687:2008, definition 3.3]
3.7
suspension
heterogeneous mixture of materials comprising a liquid and a finely dispersed solid material
[SOURCE: ISO/DIS 4618:2012]
3.8
particle size
linear dimension of a particle (3.4) determined by a specified measurement method and under specified
measurement conditions
[SOURCE: ISO/DIS 26824, definition 1.4]
Note 1 to entry: Different methods of analysis are based on the measurement of different physical properties.
Independent of the particle property actually measured, the particle size can be reported as a linear dimension,
e.g. as an equivalent spherical diameter.
3.9
particle size distribution
distribution of particles (3.4) as a function of particle size
[SOURCE: adapted from ISO 14644-1:1999, definition 2.2.4]
Note 1 to entry: Particle size distribution may be expressed as cumulative distribution or a distribution density
(distribution of the fraction of material in a size class, divided by the width of that class).
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3.10
equivalent diameter
diameter of a sphere that produces a response by a given particle-sizing method, that is equivalent to the
response produced by the particle being measured
Note 1 to entry: The physical property to which the equivalent diameter refers is indicated using a suitable
subscript [ISO 9276-1:1998].
Note 2 to entry: For discrete-particle-counting, light-scattering instruments, an equivalent optical diameter is used.
Note 3 to entry: Other material constants like density of the particle are used for the calculation of the equivalent
diameter like Stokes diameter or sedimentation equivalent diameter. The material constants, used for the
calculation, should be reported additionally.
Note 4 to entry: For inertial instruments, the aerodynamic diameter is used. Aerodynamic diameter is the
−3
diameter of a sphere of density 1 000 kg m that has the same settling velocity as the irregular particle.
[SOURCE: adapted from ISO/TS 27687:2008, A.3.2]
3.11
light scattering
change in propagation of light at the interface of two media having different optical properties
[SOURCE: ISO 13320:2009, definition 3.1.17]
3.12
hydrodynamic diameter
equivalent spherical diameter of a particle in a liquid having the same diffusion coefficient as the real
particle in that liquid
[SOURCE: ISO/TS 80004-6:2013, definition 3.2.6]
3.13
nanoparticle tracking analysis
NTA
particle tracking analysis
PTA
a method where particles undergoing Brownian motion in a liquid suspension are illuminated by a laser
and the change in position of individual particles is used to determine particle size
Note 1 to entry: Analysis of the time-dependent intensity of the scattered light can yield the translational diffusion
coefficient and hence the particle size as hydrodynamic diameter using the Stokes-Einstein relationship.
Note 2 to entry: The analysis is applicable to nanoparticles (3.3) as the size of particles detected is typically in the
range 10 to 2000 nm. The lower limit requires particles with high refractive index and the upper limit is due to
limited Brownian motion and sedimentation.
[SOURCE: ISO/TS 80004-6:2013, definition 3.2.8]
3.14
nanomaterial
material with any external dimension in the nanoscale (2.1) or having internal structure or surface
structure in the nanoscale
[SOURCE: ISO/TS 80004-4:2011(en), 2.3]
3.15
diluent
volatile liquid, single or blended, which, whilst not a solvent, may be used in conjunction with the solvent
without causing any deleterious effects
[SOURCE: ISO 4618:2006(en), 2.78]
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3.16
concentration
amount-of-substance of a component divided by the volume of the system
[SOURCE: ISO 18113-1:2009(en), A.3.10]
Note 1 to entry: The “amount-of-substance” usually refers to mass of substance in metrology.
3.17
viscosity
measure of the resistance to flow or deformation of a liquid [ISO 3104:1994]
[SOURCE: ISO 16165:2001, 2.1.12]
3.18
arithmetic mean
sum of the (population or sample) data divided by the number of values used
[SOURCE: ISO 19003:2006(en), 3.4]
3.19
standard deviation
measure of the dispersion of a series of results around their mean, equal to the positive square root of
the variance and estimated by the positive square root of the mean square
[SOURCE: ISO 4259:2006(en), 3.23]
3.20
percentile
value of a variable below which a certain percentage of observations fall
[SOURCE: ISO 11064-4:2013(en), 3.7]
4 Symbols and abbreviated terms
For the purposes of this document, the following symbols apply.
CCD Charge Coupled Device
CMOS Complementary Metal Oxide Semiconductor
d Hydrodynamic diameter meters m
η viscosity of the suspension medium pascal seconds Pa·s
2 -2 -1
k Boltzmann’s constant m kg s K
B
NTA Nanoparticle (3.3) Tracking Analysis (Definition 3.13)
PTA Particle Tracking Analysis (Definition 3.13)
RSD Relative Standard Deviation (ISO/TR 13843:2000(en), 2.34) %
T Absolute Temperature Kelvin °K
t Time seconds s
2
Mean Square distance in 1 (one) dimen- meters squared m
2
x
()
sion
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2
Mean Square distance in 2 (two) dimen- meters squared m
2
xy,
()
sions
2
Mean Square distance in 3 (three) dimen- meters squared m
2
xy,,z
()
sions
5 Principles
Determination of particle size distribution by PTA makes use of the Brownian motion and light scattering
properties of particles suspended in liquids. Irradiation of the sample (typically by means of a laser
beam of wavelength in the visible region) leads to light scattering by objects with a refractive index
that is different from that of the surrounding medium. Light scattered from each particle is collected by
magnifying optics and visualized by way of a suitable detector, such as a Charge Coupled Device (CCD) or
Complementary Metal Oxide Semiconductor (CMOS) device. By recording a series of sequential images a
computer tracks individual particles in time and space, allowing analysis of their movement.
[5,6]
By tracking individual particles, undergoing random Brownian motion, from frame to frame, the
average spatial displacement of the particles per unit time can be calculated, and this displacement
[5]
can be related to the hydrodynamic diameter of the particles through the Stokes-Einstein Formula.
Although translational Brownian motion is a three-dimensional process it is possible to use a one, two,
or three dimensional diffusion coefficient to determine particle hydrodynamic diameter. The relevant
equations are derived in Annex A and can be summarized with three equations below:
2kTt
2
B
x = (1)
()
3πηd
4kTt
2
B
xy, = (2)
()
3πηd
2kTt
2
B
xy,,z = (3)
()
πηd
5.1 Key physical parameters
It may be seen that as well as diffusion coefficient, the temperature and viscosity of the sample must
be known in order to calculate hydrodynamic diameter. Generally, the temperature of the sample is
measured and the viscosity calculated from this using well tabulated data for solvents, rather than a
direct viscosity measurement being made.
Uncertainty in temperature measurement of the sample relates directly to the calculation of particle
size. Temperature of the system should therefore be stabilized and measured with ± 0.3 K. Incorrect
temperature or liquid viscosity values lead to incorrect particle size values. Experimental procedures
are covered in Section 7.
5.2 Detection limits
The lower limit of detection in terms of particle hydrodynamic diameter is determined by the light
scattering from the particles (diluent and particle refractive indices) as well as on the sample itself
(monodisperse or polydisperse). If an object can be tracked by the imaging system then it is registered
in the data set. Small particles tend to scatter less and are therefore more difficult to track. In case of a
monodisperse gold spheres in suspension the lower limit of detection can be approximately 10 nm. For
biological samples this limit may be > 30 nm since the refractive index of particles is close to that of the
solution. In polydisperse samples the larger particles are identified with greater ease than smaller ones.
The upper particle size limit is defined by decreasing Brownian motion at larger particle sizes. The
motion of such particles is very limited and long observation periods may be required. Very large
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ISO/DIS 19430:2015(E)
particles may also produce so much scattering that the detection system may not track much smaller
particles in the same polydisperse sample.
Both detection limits are sample dependent, but the technique generally covers materials in the size
−6
range from 10 nm to 1 µm (10 m).
5.3 Measurement Accuracy
It is important to note that although PTA usually involves the simultaneous tracking and analysis of
multiple light-scattering particles, the diffusion coefficient and hence the hydrodynamic diameter of
each particle is determined individually before the data are integrated to produce a number-based
particle size distribution.
The accuracy of the measurement of the individual particle hydrodynamic diameter is determined by how
long the particle is tracked for. The longer the particle is tracked, the greater the accuracy of evaluation
of its hydrodynamic diameter. The number of such tracks of different particles determines the level of
[4][7]
representative sampling. Tracking enough particles for an appropriate statistical representation of
the sample is important. More detail about experimental procedures is found in section 7.
It should be noted that most recent algorithms of particle tracking take into account the finite number
of tracks and tracked particles and by varying the threshold for track length are able to minimize the
uncertainty in the resulting particle size distribution.
It should be noted that in the case of particles of differing scattering intensity, the brighter (typically larger
or higher refractive index) particles may be visible over a greater depth that the dimmer (smaller or lower
refractive index) particles. This can result in an over estimation of the proportion of larger particles.
6 Apparatus
PTA equipment will generally comprise a common collection of basic components, with the possibility
of additional peripherals that may be desirable or required for specific experiment types. The core
apparatus is described below.
The sample to be analysed is held in the sample cell. This cell should be inert to the sample, and should
be able to hold the sample at a stable thermal equilibrium. Ideally the temperature of the sample and cell
should be controllable, and as a minimum requirement the temperature of the sample should be measured
with ± 0.3 K precision. The cell should, at least in part, be optically transparent to allow illumination of
the sample by the radiation source and collection of scattered light by the optical assembly.
Figure 1 illustrates a common geometry of the PTA experimental setup. It should be noted that the
orientation shown above implies the particle tracking in xy plane. The arrangement of optical illumination
and detection may also be rotated about x-axis with CCD Camera pointing along y direction. The right
angles between the illumination laser and the optical detection is not a requirement as other angles
allowing dark field imaging of the sample are commonly used.
The irradiation source should be such that the intensity and wavelength of the emitted light are suited
to the sample to be analysed and the image collection and recording apparatus. The beam should be
focused to maximize illumination of in-focus particles, and minimize optical noise generated by the
illumination of out-of-focus particles. The irradiation should be non-destructive and give rise to minimal
localized heating or photophoresis. For certain experimental procedures, such as the visualization of
fluorescently labelled particles against a non-fluorescent scattering background, the radiation should
be monochromatic and the wavelength matched to both the excitation wavelength of the fluorophore
and the optical filters used in signal collection.
Scattered (or emitted) light is collected and delivered to the image capture apparatus through a series
of lenses, filters, and mirrors generally resembling a conventional optical microscope. It is worth noting
that magnification sufficient to image the particles is unnecessary, the only requirement being sufficient
resolution to allow s
...
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