ASTM E2834-12
(Guide)Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Nanoparticle Tracking Analysis (NTA)
Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Nanoparticle Tracking Analysis (NTA)
SIGNIFICANCE AND USE
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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E2834 − 12
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 ISO 14488Particulate Materials—Sampling And Sample
Splitting for the Determination of Particulate Properties
1.1 This guide deals with the measurement of particle size
ISO 22412Particle Size Analysis—Dynamic Light Scatter-
distributionofsuspendedparticles,from~10nmtotheonsetof
ing (DLS)
sedimentation,sampledependent,usingthenanoparticletrack-
ing analysis (NTA) technique. It does not provide a complete
3. Terminology
measurement methodology for any specific nanomaterial, but
provides a general overview and guide as to the methodology 3.1 Definitions:
that should be followed for good practice, along with potential
3.1.1 diffusion coeffıcient, n—a measure to characterize the
pitfalls. rate a particular molecule or particle moves in a particular
medium when driven by random thermal agitation (Brownian
1.2 The values stated in SI units are to be regarded as
motion).
standard. No other units of measurement are included in this
3.1.1.1 Discussion—After measurement, the value is to be
standard.
inputted into the Stokes-Einstein equation (Eq 1, see
1.3 This standard does not purport to address all of the
7.2.1.2(3)). Diffusion coefficient units in nanoparticle tracking
safety concerns, if any, associated with its use. It is the
analysis (NTA) measurements are typically cm /s, rather than
responsibility of the user of this standard to establish appro- 2
the correct SI units of m /s.
priate safety and health practices and determine the applica-
3.1.2 repeatability, n—in NTA and other particle sizing
bility of regulatory limitations prior to use.
techniques, this usually refers to a measure of the precision of
2. Referenced Documents
repeated consecutive measurements on the same group of
particles under identical conditions and is normally expressed
2.1 ASTM Standards:
as a relative standard deviation (RSD) or coefficient of varia-
C322Practice for Sampling Ceramic Whiteware Clays
tion (CV).
E456Terminology Relating to Quality and Statistics
3.1.2.1 Discussion—The repeatability value reflects the sta-
E1617Practice for Reporting Particle Size Characterization
bility (instrumental, but mainly the sample) of the system over
Data
time. Changes in the sample could include dispersion, aggre-
E2490Guide for Measurement of Particle Size Distribution
gation and settling.
of Nanomaterials in Suspension by Photon Correlation
Spectroscopy (PCS) 3.1.3 reproducibility, n—in NTA and particle sizing this
2.2 ISO Standards: usually refers to a measure of the deviation of the results
ISO 13320Particle Size Analysis—Laser Diffraction Meth- obtained from the first aliquot to that obtained for the second
ods and further aliquots of the same bulk sample (and therefore is
ISO 13321 Particle Size Analysis—Photon Correlation subject to the homogeneity or heterogeneity of the starting
Spectroscopy material and the sampling method employed). Normally ex-
pressed as a relative standard deviation (RSD) or coefficient of
1 variation (CV).
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
3.1.3.1 Discussion—Inaheterogenousandpolydisperse(for
Chemical Characterization.
example, slurry) system, it is often the largest error when
Current edition approved April 1, 2012. Published May 2012. DOI: 10.1520/
repeatedsamplesaretaken.Otherdefinitionsofreproducibility
E2834-12.
also address the variability among single test results gathered
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
from different laboratories when inter-laboratory testing is
Standards volume information, refer to the standard’s Document Summary page on
undertaken, or operator-to-operator, instrument-to-instrument,
the ASTM website.
3 location-to-location, or even day-to-day. It is to be noted that
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
4th Floor, New York, NY 10036, http://www.ansi.org. the same group of particles can never be measured in such a
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2834 − 12
system of tests and therefore reproducibility values may 3.2.8 RSD—relative standard deviation
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
4 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 NTAtechnique 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
3.2 Acronyms:
therefore provides size information in the suspending medium
3.2.1 CV—coefficient of variation
(typically water).Thus the hydrodynamic diameter will almost
3.2.2 CCD—charge-coupled device
certainly differ from size diameters determined by other
3.2.3 CMOS—complementary metal–oxide–semiconductor techniquesandusersoftheNTAtechniqueneedtobeawareof
the distinction of the various descriptors of particle diameter
3.2.4 DLS—dynamic light scattering
before making comparisons between techniques (see 8.7).
3.2.5 EMCCD—electron-multiplying charge-coupled de-
Notwithstanding the preceding sentence, the technique is
vice
routinely applied in industry and academia as both a research
3.2.6 NTA—nanoparticle tracking analysis
and development tool and as a QC method for the character-
3.2.7 PCS—photon correlation spectroscopy ization of submicron systems.
6. Reagents
6.1 In general, no reagents specific to the technique are
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. necessary.However,dispersingandstabilizingagentsoftenare
E2834 − 12
requiredforaspecifictestsampleinordertopreservecolloidal 7.1.2 Given the likelihood that the size standard has been
stability during the measurement.Asuitable diluent is used to certified by electron microscopy, care needs to be exercised in
direct comparison of the results. While electron microscopy is
achieve a particle concentration appropriate for the measure-
ment. The apparent hydrodynamic size or diffusion coefficient carried out under high-vacuum conditions, NTA performs the
analysis with particles in suspension. NTA measures the
may undergo change on dilution, as the ionic environment,
diffusion coefficient of the particle and the diameter given by
within which the particles are dispersed, changes in nature or
the Stokes-Einstein relation (Eq 1) is that of the sphere-
concentration. This is particularly noticeable when diluting a
equivalent, hydrodynamic diameter (the particle itself plus the
monodisperse latex. A latex that is measured as 60 nm in 1 ×
-3
molecular-scale layer of solvent associated with the particle
10 M NaCl can have a hydrodynamic diameter of over 70 nm
-6
surface). This solvent layer may be significant relative to the
in1×10 M NaCl (close to deionized water).
size of the standard particles being analyzed, increasing the
6.2 In order to minimize any changes in the system on
apparentsizeoftheparticles.Forlargerparticles,however,the
dilution, it is common to use the “mother liquor”. This is the
effect of this hydrodynamic layer is minimal.
liquid in which the particles exist in stable form and is usually
7.1.3 Note that verification of a system only demonstrates
obtained by centrifuging of the suspension or making up the
that the instrument is performing adequately with the pre-
same ionic composition of the dispersant liquid if knowledge
scribed standard materials. Practical considerations for real-
of these components is available. Many biological materials
world materials (especially “dispersion” if utilized in sample
are measured in a buffer (often phosphate buffered saline),
preparation or if the distribution is relatively polydisperse)
whichconfersthecorrect(rangeof)conditionsofpHandionic
mean that the method used to measure that real-world material
strength to assure stability of the system. Instability (usually
needs to be carefully evaluated for precision (repeatability).
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
7.1 Verification:
inhomogeneity (for example, in so-called index-matched sys-
7.1.1 The instrument to be used in the measurement should
tems) there is no scattering and the particle is invisible to light
be verified for correct performance, within pre-defined quality
and no measurements can be made by the NTA or any other
control limits, by following protocols issued by the instrument
technique making use of light scattering. A coating or func-
manufacturer. These confirmation tests normally involve the
tionalization of the primary particle may affect this refractive
use of one or more NIST-traceable spherical particle size
index sufficiently to impact the light scattering properties.
-6
standards. In the sub-micron (<1×10 m) region, these
While some physical phenomena used by the NTA measure-
standards (for example, NIST, Duke Scientific - now part of
ment technique are in common with the dynamic light scatter-
ThermoFisher Scientific) tend to be nearly monodisperse (that ing technique (photon correlation spectroscopy), as defined in
is, narrow, single m
...
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