ASTM E2834-12(2018)

Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Nanoparticle Tracking Analysis (NTA)

SIGNIFICANCE AND USE
5.1 NTA is one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size region. This guide describes the NTA technique for direct visualization and measurement of Brownian motion, generally applicable in the particle size range from several nanometers until the onset of sedimentation in the sample. The NTA technique is usually applied to dilute suspensions of solid material in a liquid carrier. It is a first principles method (that is, calibration in the standard understanding of this word, is not involved). The measurement is hydrodynamically based and therefore provides size information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from size diameters determined by other techniques and users of the NTA technique need to be aware of the distinction of the various descriptors of particle diameter before making comparisons between techniques (see 8.7). Notwithstanding the preceding sentence, the technique is routinely applied in industry and academia as both a research and development tool and as a QC method for the characterization of submicron systems.
SCOPE
1.1 This guide deals with the measurement of particle size distribution of suspended particles, from ~10 nm to the onset of sedimentation, sample dependent, using the nanoparticle tracking analysis (NTA) technique. It does not provide a complete measurement methodology for any specific nanomaterial, but provides a general overview and guide as to the methodology that should be followed for good practice, along with potential pitfalls.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Historical
• Publication Date: 31-Dec-2017
• ICS: 07.120 - Nanotechnologies
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.02 - Physical and Chemical Characterization

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E2834 − 12 (Reapproved 2018)
Standard Guide for
Measurement of Particle Size Distribution of Nanomaterials
in Suspension by Nanoparticle Tracking Analysis (NTA)
This standard is issued under the fixed designation E2834; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope E2490Guide for Measurement of Particle Size Distribution
of Nanomaterials in Suspension by Photon Correlation
1.1 This guide deals with the measurement of particle size
Spectroscopy (PCS)
distributionofsuspendedparticles,from~10nmtotheonsetof
2.2 ISO Standards:
sedimentation,sampledependent,usingthenanoparticletrack-
ISO 13320Particle Size Analysis—Laser Diffraction Meth-
ing analysis (NTA) technique. It does not provide a complete
ods
measurement methodology for any specific nanomaterial, but
ISO 13321 Particle Size Analysis—Photon Correlation
provides a general overview and guide as to the methodology
Spectroscopy
that should be followed for good practice, along with potential
ISO 14488Particulate Materials—Sampling And Sample
pitfalls.
Splitting for the Determination of Particulate Properties
1.2 The values stated in SI units are to be regarded as
ISO 22412Particle Size Analysis—Dynamic Light Scatter-
standard. No other units of measurement are included in this
ing (DLS)
standard.
3. Terminology
1.3 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1 Definitions:
responsibility of the user of this standard to establish appro-
3.1.1 diffusion coeffıcient, n—a measure to characterize the
priate safety, health, and environmental practices and deter-
rate a particular molecule or particle moves in a particular
mine the applicability of regulatory limitations prior to use.
medium when driven by random thermal agitation (Brownian
1.4 This international standard was developed in accor-
motion).
dance with internationally recognized principles on standard-
3.1.1.1 Discussion—After measurement, the value is to be
ization established in the Decision on Principles for the
inputted into the Stokes-Einstein equation (Eq 1, see
Development of International Standards, Guides and Recom-
7.2.1.2(3)). Diffusion coefficient units in nanoparticle tracking
mendations issued by the World Trade Organization Technical
analysis (NTA) measurements are typically cm /s, rather than
Barriers to Trade (TBT) Committee.
the correct SI units of m /s.
3.1.2 repeatability, n—in NTA and other particle sizing
2. Referenced Documents
techniques, this usually refers to a measure of the precision of
2.1 ASTM Standards:
repeated consecutive measurements on the same group of
C322Practice for Sampling Ceramic Whiteware Clays
particles under identical conditions and is normally expressed
E456Terminology Relating to Quality and Statistics
as a relative standard deviation (RSD) or coefficient of varia-
E1617Practice for Reporting Particle Size Characterization
tion (CV).
Data
3.1.2.1 Discussion—The repeatability value reflects the sta-
bility (instrumental, but mainly the sample) of the system over
time. Changes in the sample could include dispersion, aggre-
gation and settling.
1 3.1.3 reproducibility, n—in NTA and particle sizing this
This guide is under the jurisdiction of ASTM Committee E56 on Nanotech-
nology and is the direct responsibility of Subcommittee E56.02 on Physical and usually refers to a measure of the deviation of the results
Chemical Characterization.
obtained from the first aliquot to that obtained for the second
Current edition approved Jan. 1, 2018. Published January 2018. Originally
and further aliquots of the same bulk sample (and therefore is
approved in 2012. Last previous edition approved in 2012 as E2834 – 12. DOI:
subject to the homogeneity or heterogeneity of the starting
10.1520/E2834-12R18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
the ASTM website. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2834 − 12 (2018)
material and the sampling method employed). Normally ex- 3.2 Acronyms:
pressed as a relative standard deviation (RSD) or coefficient of 3.2.1 CV—coefficient of variation
variation (CV).
3.2.2 CCD—charge-coupled device
3.1.3.1 Discussion—Inaheterogenousandpolydisperse(for
3.2.3 CMOS—complementary metal–oxide–semiconductor
example, slurry) system, it is often the largest error when
3.2.4 DLS—dynamic light scattering
repeatedsamplesaretaken.Otherdefinitionsofreproducibility
also address the variability among single test results gathered
3.2.5 EMCCD—electron-multiplying charge-coupled de-
from different laboratories when inter-laboratory testing is vice
undertaken, or operator-to-operator, instrument-to-instrument,
3.2.6 NTA—nanoparticle tracking analysis
location-to-location, or even day-to-day. It is to be noted that
3.2.7 PCS—photon correlation spectroscopy
the same group of particles can never be measured in such a
3.2.8 RSD—relative standard deviation
system of tests and therefore reproducibility values may
typically be considerably in excess of repeatability values.
4. Summary of Guide
3.1.4 robustness,n—ameasureofthechangeoftherequired
4.1 Nanoparticle tracking analysis (NTA) is a method for
parameter with deliberate and systematic variations in any or
the direct and real-time visualization and analysis of nanopar-
all of the key parameters that influence it.
ticles in liquids. Particles in suspension are illuminated with a
3.1.4.1 Discussion—For example, dispersion energy input
focused laser beam. Light scattered from each particle is
(that is, ultrasound power and duration) almost certainly will
visible through magnifying optics fitted to a digital camera
affectthereportedresults.VariationinpHislikelytoaffectthe
such as a CCD. The software analyzes the video stream from
degree of agglomeration and so forth. A useful discussion of
thecamera,identifyingandtrackingthemotionofeachparticle
robustness experiment considerations is found in the ICH
with time. Because each particle in the field of view is being
Validation of Analytical Procedures Q2(R1) Guideline (1).
simultaneously but separately tracked and analyzed, the par-
3.1.5 rotational diffusion, n—a process by which the equi-
ticle size distribution profile obtained by NTA is a direct
librium statistical distribution of the overall orientation of
number-based distribution.
molecules or particles is maintained or restored.
4.2 The laser beam is focused such that only particles in the
3.1.6 translational diffusion, n—a process by which the
focal plane of the magnifying optics are illuminated. Particles
equilibrium statistical distribution of molecules or particles in
out of the focal plane are not illuminated and at the size range
space is maintained or restored.
under discussion are not visible to the camera. This yields a
3.1.7 visualization, n—as it relates to the NTA technique,
highsignaltonoiseimage,allowingparticlesassmallas10nm
the particles themselves are not imaged, being below the
to be visualized, depending on sample material. While outside
diffractionlimit.Eachparticleactsasapointscatterer,meaning
the scope of this document, the technique is generally able to
that the imaging system only sees the scattered light from the
measure particles as large as approximately 1 µm.
particle. This allows the position of each particle to be
4.3 Theaveragedistanceeachparticlemovesintheimageis
identified and followed with respect to time. See 7.2.
automatically calculated by the software. From this value, the
3.1.7.1 Discussion—Theintensityandshapeofthescattered
particle diffusion coefficient can be obtained and through the
light pattern for each particle may vary, and some additional
use of the Stokes-Einstein equation, particle size can be
information may be obtained from these differences, at least
determined.
qualitatively, but is outside the scope of this guide.
4.4 This guide discusses the scientific basis for the
3.1.8 percentile, n—a statistical measure of the distribution
technique, size limits, concentration ranges, sampling and
of sizes. The size below which a certain percent of the
sample preparation considerations, condition and analysis
distribution falls. For example, the 10th percentile is the size
selection, data interpretation and comparison to other comple-
below which 10 percent of the particles may be found.
mentary techniques.
Expressed in ISO form as x ,x ,x , and also commonly
10 50 90
expressed as D10, D50, D90. The 50th percentile is the
5. Significance and Use
median.
5.1 NTA is one of the very few techniques that are able to
3.1.9 coeffıcient of variation, n—in statistics, a normalized
deal with the measurement of particle size distribution in the
measureofdispersionofadistribution.Definedasthestandard
nano-size region. This guide describes the NTA technique for
deviation divided by the mean value. (Note: CV = SD/Mean)
direct visualization and measurement of Brownian motion,
3.1.10 relative standard deviation, n—in statistics, the ab- generally applicable in the particle size range from several
solute value of the coefficient of variation, expressed as a nanometers until the onset of sedimentation in the sample.The
percentage. (Note: RSD = 100·SD/Mean) NTAtechniqueisusuallyappliedtodilutesuspensionsofsolid
material in a liquid carrier. It is a first principles method (that
NOTE 1—Other common statistical measures are defined in Terminol-
is,calibrationinthestandardunderstandingofthisword,isnot
ogy E456.
involved). The measurement is hydrodynamically based and
therefore provides size information in the suspending medium
(typically water).Thus the hydrodynamic diameter will almost
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. certainly differ from size diameters determined by other
E2834 − 12 (2018)
techniquesandusersoftheNTAtechniqueneedtobeawareof video data can be examined during and after acquisition. Such
the distinction of the various descriptors of particle diameter examination can provide useful qualitative information about
before making comparisons between techniques (see 8.7). the sample condition, particle concentration, and system op-
Notwithstanding the preceding sentence, the technique is eration. During data acquisition one looks for a consistent
routinely applied in industry and academia as both a research number of particles in the field of view, well-separated
and development tool and as a QC method for the character- particles, a low level of random or background noise, and
ization of submicron systems. particle tracking lengths sufficient for accurate measurements
of each particle. Manufacturers also provide other means of
6. Reagents
assuring the reliability of the data and it is recommended that
these protocols are consulted, as appropriate.
6.1 In general, no reagents specific to the technique are
7.1.2 Given the likelihood that the size standard has been
necessary.However,dispersingandstabilizingagentsoftenare
certified by electron microscopy, care needs to be exercised in
requiredforaspecifictestsampleinordertopreservecolloidal
direct comparison of the results. While electron microscopy is
stability during the measurement.Asuitable diluent is used to
carried out under high-vacuum conditions, NTA performs the
achieve a particle concentration appropriate for the measure-
analysis with particles in suspension. NTA measures the
ment. The apparent hydrodynamic size or diffusion coefficient
diffusion coefficient of the particle and the diameter given by
may undergo change on dilution, as the ionic environment,
the Stokes-Einstein relation (Eq 1) is that of the sphere-
within which the particles are dispersed, changes in nature or
equivalent, hydrodynamic diameter (the particle itself plus the
concentration. This is particularly noticeable when diluting a
molecular-scale layer of solvent associated with the particle
monodisperse latex. A latex that is measured as 60 nm in 1 ×
-3
surface). This solvent layer may be significant relative to the
10 M NaCl can have a hydrodynamic diameter of over 70 nm
-6
size of the standard particles being analyzed, increasing the
in1×10 M NaCl (close to deionized water).
apparentsizeoftheparticles.Forlargerparticles,however,the
6.2 In order to minimize any changes in the system on
effect of this hydrodynamic layer is minimal.
dilution, it is common to use the “mother liquor”. This is the
7.1.3 Note that verification of a system only demonstrates
liquid in which the particles exist in stable form and is usually
that the instrument is performing adequately with the pre-
obtained by centrifuging of the suspension or making up the
scribed standard materials. Practical considerations for real-
same ionic composition of the dispersant liquid if knowledge
world materials (especially “dispersion” if utilized in sample
of these components is available. Many biological materials
preparation or if the distribution is relatively polydisperse)
are measured in a buffer (often phosphate buffered saline),
mean that the method used to measure that real-world material
whichconfersthecorrect(rangeof)conditionsofpHandionic
needs to be carefully evaluated for precision (repeatability).
strength to assure stability of the system. Instability (usually
through inadequate zeta potential—see (2)) can promote ag- 7.2 Measurement:
glomeration leading to settling or sedimentation in a solid-
7.2.1 Introduction:
liquidsystemorcreaminginaliquid-liquidsystem(emulsion).
7.2.1.1 The measurement of particle size distribution in the
Such fundamental changes interfere with the stability of the
nano- (sub 100 nm) region by nanoparticle tracking analysis
suspension and need to be minimized as they affect the quality
depends on the interaction of light with matter and the random
(accuracy and repeatability) of the reported measurements.
orBrownianmotionthataparticleexhibitsinliquidmediumin
These should be investigated in a robustness experiment.
free suspension (3). There must be an inhomogeneity in the
refractive indices of a particle and the medium within which it
7. Procedure
exists in order for light scattering to occur. Without such an
inhomog
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2834 − 12 E2834 − 12 (Reapproved 2018)
Standard Guide for
Measurement of Particle Size Distribution of Nanomaterials
in Suspension by Nanoparticle Tracking Analysis (NTA)
This standard is issued under the fixed designation E2834; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This guide deals with the measurement of particle size distribution of suspended particles, from ~10 nm to the onset of
sedimentation, sample dependent, using the nanoparticle tracking analysis (NTA) technique. It does not provide a complete
measurement methodology for any specific nanomaterial, but provides a general overview and guide as to the methodology that
should be followed for good practice, along with potential pitfalls.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
C322 Practice for Sampling Ceramic Whiteware Clays
E456 Terminology Relating to Quality and Statistics
E1617 Practice for Reporting Particle Size Characterization Data
This guide is under the jurisdiction of ASTM Committee E56 on Nanotechnology and is the direct responsibility of Subcommittee E56.02 on Physical and Chemical
Characterization.
Current edition approved April 1, 2012Jan. 1, 2018. Published May 2012January 2018. Originally approved in 2012. Last previous edition approved in 2012 as E2834
– 12. DOI: 10.1520/E2834-12.10.1520/E2834-12R18.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’sstandard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2834 − 12 (2018)
E2490 Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy
(PCS)
2.2 ISO Standards:
ISO 13320 Particle Size Analysis—Laser Diffraction Methods
ISO 13321 Particle Size Analysis—Photon Correlation Spectroscopy
ISO 14488 Particulate Materials—Sampling And Sample Splitting for the Determination of Particulate Properties
ISO 22412 Particle Size Analysis—Dynamic Light Scattering (DLS)
3. Terminology
3.1 Definitions:
3.1.1 diffusion coeffıcient, n—a measure to characterize the rate a particular molecule or particle moves in a particular medium
when driven by random thermal agitation (Brownian motion).
3.1.1.1 Discussion—
After measurement, the value is to be inputted into the Stokes-Einstein equation (Eq 1, see 7.2.1.2(3)). Diffusion coefficient units
2 2
in nanoparticle tracking analysis (NTA) measurements are typically cm /s, rather than the correct SI units of m /s.
3.1.2 repeatability, n—in NTA and other particle sizing techniques, this usually refers to a measure of the precision of repeated
consecutive measurements on the same group of particles under identical conditions and is normally expressed as a relative
standard deviation (RSD) or coefficient of variation (CV).
3.1.2.1 Discussion—
The repeatability value reflects the stability (instrumental, but mainly the sample) of the system over time. Changes in the sample
could include dispersion, aggregation and settling.
3.1.3 reproducibility, n—in NTA and particle sizing this usually refers to a measure of the deviation of the results obtained from
the first aliquot to that obtained for the second and further aliquots of the same bulk sample (and therefore is subject to the
homogeneity or heterogeneity of the starting material and the sampling method employed). Normally expressed as a relative
standard deviation (RSD) or coefficient of variation (CV).
3.1.3.1 Discussion—
In a heterogenous and polydisperse (for example, slurry) system, it is often the largest error when repeated samples are taken. Other
definitions of reproducibility also address the variability among single test results gathered from different laboratories when
inter-laboratory testing is undertaken, or operator-to-operator, instrument-to-instrument, location-to-location, or even day-to-day.
It is to be noted that the same group of particles can never be measured in such a system of tests and therefore reproducibility values
may typically be considerably in excess of repeatability values.
3.1.4 robustness, n—a measure of the change of the required parameter with deliberate and systematic variations in any or all
of the key parameters that influence it.
3.1.4.1 Discussion—
For example, dispersion energy input (that is, ultrasound power and duration) almost certainly will affect the reported results.
Variation in pH is likely to affect the degree of agglomeration and so forth. A useful discussion of robustness experiment
considerations is found in the ICH Validation of Analytical Procedures Q2(R1) Guideline (1).
3.1.5 rotational diffusion, n—a process by which the equilibrium statistical distribution of the overall orientation of molecules
or particles is maintained or restored.
3.1.6 translational diffusion, n—a process by which the equilibrium statistical distribution of molecules or particles in space is
maintained or restored.
3.1.7 visualization, n—as it relates to the NTA technique, the particles themselves are not imaged, being below the diffraction
limit. Each particle acts as a point scatterer, meaning that the imaging system only sees the scattered light from the particle. This
allows the position of each particle to be identified and followed with respect to time. See 7.2.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
E2834 − 12 (2018)
3.1.7.1 Discussion—
The intensity and shape of the scattered light pattern for each particle may vary, and some additional information may be obtained
from these differences, at least qualitatively, but is outside the scope of this guide.
3.1.8 percentile, n—a statistical measure of the distribution of sizes. The size below which a certain percent of the distribution
falls. For example, the 10th percentile is the size below which 10 percent of the particles may be found. Expressed in ISO form
as x , x , x , and also commonly expressed as D10, D50, D90. The 50th percentile is the median.
10 50 90
3.1.9 coeffıcient of variation, n—in statistics, a normalized measure of dispersion of a distribution. Defined as the standard
deviation divided by the mean value. (Note: CV = SD/Mean)
3.1.10 relative standard deviation, n—in statistics, the absolute value of the coefficient of variation, expressed as a percentage.
(Note: RSD = 100·SD/Mean)
NOTE 1—Other common statistical measures are defined in Terminology E456.
3.2 Acronyms:
3.2.1 CV—coefficient of variation
3.2.2 CCD—charge-coupled device
3.2.3 CMOS—complementary metal–oxide–semiconductor
3.2.4 DLS—dynamic light scattering
3.2.5 EMCCD—electron-multiplying charge-coupled device
3.2.6 NTA—nanoparticle tracking analysis
3.2.7 PCS—photon correlation spectroscopy
3.2.8 RSD—relative standard deviation
4. Summary of Guide
4.1 Nanoparticle tracking analysis (NTA) is a method for the direct and real-time visualization and analysis of nanoparticles in
liquids. Particles in suspension are illuminated with a focused laser beam. Light scattered from each particle is visible through
magnifying optics fitted to a digital camera such as a CCD. The software analyzes the video stream from the camera, identifying
and tracking the motion of each particle with time. Because each particle in the field of view is being simultaneously but separately
tracked and analyzed, the particle size distribution profile obtained by NTA is a direct number-based distribution.
4.2 The laser beam is focused such that only particles in the focal plane of the magnifying optics are illuminated. Particles out
of the focal plane are not illuminated and at the size range under discussion are not visible to the camera. This yields a high signal
to noise image, allowing particles as small as 10 nm to be visualized, depending on sample material. While outside the scope of
this document, the technique is generally able to measure particles as large as approximately 1 μm.
4.3 The average distance each particle moves in the image is automatically calculated by the software. From this value, the
particle diffusion coefficient can be obtained and through the use of the Stokes-Einstein equation, particle size can be determined.
4.4 This Guideguide discusses the scientific basis for the technique, size limits, concentration ranges, sampling and sample
preparation considerations, condition and analysis selection, data interpretation and comparison to other complementary
techniques.
5. Significance and Use
5.1 NTA is one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size
region. This Guideguide describes the NTA technique for direct visualization and measurement of Brownian motion, generally
applicable in the particle size range from several nanometers until the onset of sedimentation in the sample. The NTA technique
is usually applied to dilute suspensions of solid material in a liquid carrier. It is a first principles method (that is, calibration in the
standard understanding of this word, is not involved). The measurement is hydrodynamically based and therefore provides size
information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from size
diameters determined by other techniques and users of the NTA technique need to be aware of the distinction of the various
descriptors of particle diameter before making comparisons between techniques (see 8.7). Notwithstanding the preceding sentence,
the technique is routinely applied in industry and academia as both a research and development tool and as a QC method for the
characterization of submicron systems.
6. Reagents
6.1 In general, no reagents specific to the technique are necessary. However, dispersing and stabilizing agents often are required
for a specific test sample in order to preserve colloidal stability during the measurement. A suitable diluent is used to achieve a
particle concentration appropriate for the measurement. The apparent hydrodynamic size or diffusion coefficient may undergo
E2834 − 12 (2018)
change on dilution, as the ionic environment, within which the particles are dispersed, changes in nature or concentration. This is
-3
particularly noticeable when diluting a monodisperse latex. A latex that is measured as 60 nm in 1 × 10 M NaCl can have a
-6
hydrodynamic diameter of over 70 nm in 1 × 10 M NaCl (close to deionized water).
6.2 In order to minimize any changes in the system on dilution, it is common to use the “mother liquor”. This is the liquid in
which the particles exist in stable form and is usually obtained by centrifuging of the suspension or making up the same ionic
composition of the dispersant liquid if knowledge of these components is available. Many biological materials are measured in a
buffer (often phosphate buffered saline), which confers the correct (range of) conditions of pH and ionic strength to assure stability
of the system. Instability (usually through inadequate zeta potential—see (2)) can promote agglomeration leading to settling or
sedimentation in a solid-liquid system or creaming in a liquid-liquid system (emulsion). Such fundamental changes interfere with
the stability of the suspension and need to be minimized as they affect the quality (accuracy and repeatability) of the reported
measurements. These should be investigated in a robustness experiment.
7. Procedure
7.1 Verification:
7.1.1 The instrument to be used in the measurement should be verified for correct performance, within pre-defined quality
control limits, by following protocols issued by the instrument manufacturer. These confirmation tests normally involve the use
-6
of one or more NIST-traceable spherical particle size standards. In the sub-micron (< 1 × 10 m) region, these standards (for
example, NIST, Duke Scientific - now part of ThermoFisher Scientific) tend to be nearly monodisperse (that is, narrow, single
mode distribution, CV < 17 %) and, while confirming the x (size) axis, do not verify the y (or quantity) axis of the size distribution.
Note that NTA is a first principles measurement and thus calibration in the formal sense (adjustment of the instrument to read a
true and known value) need not be undertaken. In the event of a “failure” at the verification stage, then the issues to check involve
quality of the dilution water, state of dispersion and stability of the standard under dilution plus instrumental issues such as thermal
stability, cleanliness and alignment of optical components. The raw video data can be examined during and after acquisition. Such
examination can provide useful qualitative information about the sample condition, particle concentration, and system operation.
During data acquisition one looks for a consistent number of particles in the field of view, well-separated particles, a low level of
random or background noise, and particle tracking lengths sufficient for accurate measurements of each particle. Manufacturers
also provide other means of assuring the reliability of the data and it is recommended that these protoco
...