SIST-TS CEN/TS 17273:2019
(Main)Nanotechnologies - Guidance on detection and identification of nano-objects in complex matrices
Nanotechnologies - Guidance on detection and identification of nano-objects in complex matrices
This document sets requirements for sampling and treatment of the complex matrices in order to obtain a liquid dispersion with sufficiently high concentration of the nano-objects of interest.
This document provides guidelines for detection and identification of specific nano-objects in complex matrices, such as liquid environmental compartments, waste water and consumer products (e.g. food, cosmetics). This document requires for the identification a priori knowledge of the nature of the nano-objects like their chemical composition. The selected detection and identification methods are based on a combination of size classification and chemical composition analysis. Identification can also be supported, e.g. by additional morphology characterization. Currently only Field Flow Fractionation, Electron Microscopy and single particle Inductively Coupled Plasma - Mass Spectrometry fulfil this combination condition.
Nanotechnologien - Leitfaden für die Detektion und Identifizierung von Nanoobjekten in komplexen Matrizen
In diesem Dokument werden Anforderungen an Probenahme und Behandlung der komplexen Matrizen festgelegt, damit eine flüssige Dispersion mit ausreichend hoher Konzentration der zu untersuchenden Nanoobjekte erhalten wird.
Dieses Dokument stellt einen Leitfaden für die Detektion und Identifizierung von spezifischen Nanoobjekten in komplexen Matrizen, wie z. B. flüssige Umweltmedien, Abwasser und Konsumergüter (z. B. Lebensmittel, Kosmetik) zur Verfügung. Dieses Dokument erfordert für die Identifizierung a priori-Wissen über die Art der Nanoobjekte, wie z. B. ihre chemische Zusammensetzung. Die gewählten Detektions und Identifizierungs¬verfahren basieren auf einer Kombination aus Größenklassifikation und Analyse der chemischen Zusammensetzung. Die Identifizierung kann z. B. auch durch zusätzliche morphologische Charakterisierung unterstützt werden. Derzeit erfüllen nur Feld Fluss Fraktionierung, Elektronenmikroskopie und Einzelpartikel ICP MS diese Kombinationsbedingung.
Nanotechnologies - Guide pour la détection et l'identification des nano-objets dans des matrices complexes
Le présent document spécifie les exigences relatives à l’échantillonnage et au traitement des matrices complexes afin d’obtenir une dispersion liquide ayant une concentration suffisamment élevée de nano objets présentant un intérêt.
Le présent document fournit des lignes directrices pour la détection et l’identification de nano-objets spécifiques dans les matrices complexes, telles que les compartiments environnementaux liquides, les eaux usées et les biens de consommation (aliments et cosmétiques, par exemple). Pour l’identification, le présent document nécessite de connaître au préalable la nature des nano-objets, telle que leur composition chimique. Les méthodes de détection et d’identification choisies sont fondées sur une combinaison de classification de taille et d’analyse de composition chimique. La caractérisation de la morphologie peut, par exemple, également venir à l’appui de l’identification. À l’heure actuelle, seules les techniques de fractionnement d’écoulement de champ (FFF), de microscopie électronique (ME) et de spectroscopie de masse à plasma à couplage inductif à particule unique (spICP-MS) remplissent cette condition de combinaison.
Nanotehnologija - Navodilo za odkrivanje in identifikacijo nanopredmetov v kompleksnih matrikah
Ta dokument določa zahteve za vzorčenje in obdelavo kompleksnih matrik za pridobivanje tekoče disperzije z dovolj visoko koncentracijo nanopredmetov, ki nas zanimajo.
Ta dokument vsebuje smernice za odkrivanje in identifikacijo določenih nanopredmetov v kompleksnih matricah, kot so vsebniki s tekočinami, odpadna voda in potrošniški izdelki (npr. hrana, kozmetika). Ta dokument zahteva identifikacijo vnaprejšnjega poznavanja narave nanopredmetov, kot je njihova kemična sestava. Izbrane metode odkrivanja in identifikacije temeljijo na kombinaciji klasifikacije velikosti in analize kemijske sestave. Identifikacijo lahko podpiramo na primer tudi z dodatno morfološko karakterizacijo. Trenutno izpolnjujejo ta pogoj kombinacije samo frakcioniranje v tokovnem polju, elektronska mikroskopija in masna spektrometrija z induktivno sklopljeno plazmo.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 17273:2019
01-marec-2019
Nanotehnologija - Navodilo za odkrivanje in identifikacijo nanopredmetov v
kompleksnih matrikah
Nanotechnologies - Guidance on detection and identification of nano-objects in complex
matrices
Nanotechnologien - Leitfaden für die Detektion und Identifizierung von Nanoobjekten in
komplexen Matrizen
Nanotechnologies - Guide pour la détection et l'identification des nano-objets dans des
matrices complexes
Ta slovenski standard je istoveten z: CEN/TS 17273:2018
ICS:
07.120 Nanotehnologije Nanotechnologies
SIST-TS CEN/TS 17273:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 17273:2019
CEN/TS 17273
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
December 2018
TECHNISCHE SPEZIFIKATION
ICS 07.120
English Version
Nanotechnologies - Guidance on detection and
identification of nano-objects in complex matrices
Nanotechnologies - Document d'orientation pour la Nanotechnologien - Leitfaden für die Detektion und
détection et l'identification des nano-objets dans les Identifizierung von Nanoobjekten in komplexen
matrices complexes Matrizen
This Technical Specification (CEN/TS) was approved by CEN on 28 September 2018 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17273:2018 E
worldwide for CEN national Members.
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Contents Page
European foreword . 4
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 8
4 Symbols and abbreviations . 9
5 Possible tasks and measuring techniques . 11
5.1 Examples for detection and identification tasks in complex matrices . 11
5.2 Overview of measurement techniques . 11
6 Guidance on sample preparation, particle detection and identification of nano-
objects in complex matrices. 12
6.1 Approach for “Detection and Identification of a relevant population of nano-objects
based on a priori knowledge” (guidance chart) . 12
6.2 Information about the targeted nano-objects . 15
6.3 Information about the sample matrix . 16
6.4 Sample Preparation Process Quality assessment . 16
6.4.1 Suspension, dispersion with recovery evaluation and property assessment . 16
6.4.2 Initial size distribution measurement and mass concentration measurement of all
isolated nano-objects . 16
6.5 Measurement of the targeted nano-objects and evaluation . 18
7 Selected measuring techniques for the detection and identification of nano-
objects . 18
7.1 Field-Flow-Fractionation (FFF) technique. 18
7.1.1 General. 18
7.1.2 Fractionation principle and theory — Fractionation principle . 19
7.1.3 Detection principle . 20
7.1.4 Performance . 21
7.1.5 Sample preparation . 22
7.1.6 Analysis report and interpretation of results . 23
7.2 Electron Microscopy (EM) technique . 23
7.2.1 General. 23
7.2.2 Measuring principle of Electron Microscopy . 24
7.2.3 Performance of Electron Microscopy . 25
7.2.4 Specimen preparation for Electron Microscopy . 26
7.2.5 Interpretation of Electron Microscopy results . 27
7.3 Single particle Inductively Coupled Plasma Mass Spetrometry (spICP-MS) . 28
7.3.1 Measuring principle . 28
7.3.2 Performance . 28
7.3.3 Sample preparation . 30
7.3.4 Interpretation of results . 30
8 List of reporting requirements on sample preparation, detection and identification
of nano-objects in complex matrixes . 32
8.1 General reporting . 32
8.2 Sample preparation reporting, explained in Clause 6 . 32
8.3 Measurement reporting, explained in Clause 7 . 33
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Annex A (informative) Indicative ranges of size and concentration of selected measuring
techniques . 34
Annex B (normative) Theory of F4 separations, precautions when separating broad particle
size distributions and sample preparation . 36
B.1 Theory of F4 separations . 36
B.2 F4 calibration using F4 theory or external references . 37
B.3 Approaches to prepare complex samples for FFF. 39
Annex C (informative) Example: Analysis of the release of particles from the coating of
silver-coloured pearls by a combination of descriptive TEM analysis, electron
diffraction, analytical TEM and quantitative TEM . 41
C.1 Approach and methodology . 41
C.2 Results . 42
Annex D (informative) Example: Single particle ICP-MS for sizing and quantitative
determination of nano-silver in chicken meat. 45
D.1 Introduction . 45
D.2 Method Description . 45
D.2.1 Materials and Methods . 45
D.2.2 Sample Preparation . 45
D.2.3 Instrumental Analysis . 45
D.2.4 Data Processing . 46
D.3 Results and Discussion . 46
D.3.1 Sample enzymatic digestion: soft conditions to preserve particle characteristics . 46
D.3.2 Study Design and Results of Validation . 47
D.3.3 Repeatability, reproducibility, and trueness . 47
D.3.4 Linearity and LOD/LOQ, Robustness, specificity/selectivity . 48
Annex E (informative) Overview of alternative detection methods. 50
E.1 General . 50
E.2 Particle Tracking Analysis (PTA) . 51
E.3 Tracer methods by using stable isotopic labelled nano-objects . 52
E.4 HyperSpectral Imaging System (HSIS) by scattering in a dark-field background . 53
E.5 Size evaluation by UV-vis spectroscopy . 53
E.6 Sizing nano-objects in liquids using differential mobility analysing system (DMAS) 53
E.7 Laser-Induced Breakdown Detection (LIBD) . 54
E.8 Hydrodynamic chromatography ICP-MS (HDC-ICP-MS) and size exclusion ICP-MS
(SEC-ICP-MS) . 55
Bibliography . 56
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European foreword
This document (CEN/TS 17273:2018) has been prepared by Technical Committee CEN/TC 352
“Nanotechnologies”, the secretariat of which is held by AFNOR.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
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Introduction
Nanotechnology is a rapidly developing field of science and technology that focuses on processes and
materials at the nanoscale size (particle dimensions that are approximately 1 nm to 100 nm). It is a highly
multidisciplinary field with a wide range of materials and applications, e.g. health care, information and
communication technologies, energy production and storage, materials science/chemical engineering,
manufacturing, environmental protection, consumer products (e.g. food, cosmetics, etc.). Therefore, the
resulting products containing nanoscaled materials are very diverse and different in their properties.
CEN/TS 17010 provides guidelines for the identification of measurands to characterize nano-objects, and
their agglomerates and aggregates and to assess specific properties relevant to the performance of
materials that contain them. This document describes the measurands for characterizing nano-objects
based on popular current techniques for characterizing nano-objects. Due to variable matrix
interferences, a method-specific sample preparation protocol to separate particles of interest from their
respective matrices is mandatory.
The production and use of nanomaterials may lead, among others, to an increasing release of nano-
objects into the environment e.g. by liquid waste and production streams. To ensure sustainable use and
development of nanotechnology there is a need for control and monitoring of nanomaterial systems
according to their application (e.g. risk assessment). For that reason, it is essential to identify useful
measurement techniques for the detection and characterization of nano-objects in so-called complex
matrices, such as natural liquids, waste water, food and cosmetics [1]. It is important that specific
characteristics of the nano-objects are known to be able to identify them.
There are numerous techniques for fractionation of nano-objects based on, e.g. Centrifugal Liquid
Sedimentation (CLS) or flow based separation methods, such as Field-Flow Fractionation (FFF),
hydrodynamic chromatography (HDC) and size exclusion chromatography (SEC). Generally, particle size
distributions are obtained by the measurement of the particle concentration from the different size
fractions.
Imaging techniques such as Electron Microscopy (EM) after appropriate sample preparation allow the
detection/imaging of single particles according to several features, e.g. projection area, longest or
shortest external dimension.
In case of counting techniques, after a high and known dilution of a particle stream only single particles
are present in the detection zone and, e.g. particle volume or particle projection area dependent signals
can be measured and related to the particle numbers by light scattering counting methods.
When many different particles are present in the detection zone, ensemble techniques such as static or
dynamic scattering techniques can be used, provided that the size polydispersity of the particles is
limited. Dynamic light scattering (DLS) analyses, for example, generate signal spectra with size
dependent components. DLS can deconvolute these spectra into primary intensity-weighted particle size
distributions, but only for relatively simple sample systems, with well-separated particle size modes and
with the help of advanced algorithms.
The well-established particle size analysis techniques mentioned so far do not cover the chemical
identification of the nano-objects. This document addresses the detection of nano-objects in complex
liquid matrices which might contain an elevated level of inorganic salts, organic contaminants and larger
organic and inorganic particles as well as natural background nano-objects. Therefore, it is important
that for each particle two measurands are combined: not only the size is needed (for classification as a
nano-object) but also the elemental composition (to discriminate the target particles with an a priori
known elemental composition or morphology, from the matrix and background particles). The aim of this
document is to guide the users how to combine size measurement with chemical identification for each
particle.
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The document proposes the usage of 3 main characterization methods:
— Field Flow Fractionation combined with multiple detection systems delivering size related
information and additionally material identification;
— Electron Microscopy equipped with Energy Dispersive X-ray Spectroscopy (EDX) to determine the
elemental composition of the particles, additionally to their geometrical measures;
— Single particle Inductively Coupled Plasma – Mass Spectrometry as an elemental specific detection
system gives as well size related information.
For the identification of nano-objects, this document requires a priori knowledge of their nature, e.g. their
elemental composition.
All proposed methods currently do not allow in situ but only ex situ characterization.
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1 Scope
This document sets requirements for sampling and treatment of the complex matrices in order to obtain
a liquid dispersion with sufficiently high concentration of the nano-objects of interest.
This document provides guidelines for detection and identification of specific nano-objects in complex
matrices, such as liquid environmental compartments, waste water and consumer products (e.g. food,
cosmetics). This document requires for the identification a priori knowledge of the nature of the nano-
objects like their chemical composition. The selected detection and identification methods are based on
a combination of size classification and chemical composition analysis. Identification can also be
supported, e.g. by additional morphology characterization. Currently only Field Flow Fractionation,
Electron Microscopy and single particle Inductively Coupled Plasma – Mass Spectrometry fulfil this
combination condition.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
CEN/TS 17010:2016, Nanotechnologies — Guidance on measurands for characterising nano-objects and
materials that contain them
EN ISO/IEC 17025:2017, General requirements for the competence of testing and calibration laboratories
(ISO/IEC 17025:2017)
CEN ISO/TS 80004-1:2015, Nanotechnologies — Vocabulary — Part 1: Core terms (ISO/TS 80004-1:2015)
CEN ISO/TS 80004-2:2017, Nanotechnologies — Vocabulary — Part 2: Nano-objects (ISO/TS 80004-
2:2015)
ISO 9276-2, Representation of results of particle size analysis — Part 2: Calculation of average particle
sizes/diameters and moments from particle size distributions
ISO 9276-3, Representation of results of particle size analysis — Part 3: Adjustment of an experimental
curve to a reference model
ISO 9276-4, Representation of results of particle size analysis — Part 4: Characterization of a classification
process
ISO 9276-5, Representation of results of particle size analysis — Part 5: Methods of calculation relating to
particle size analyses using logarithmic normal probability distribution
ISO 9276-6, Representation of results of particle size analysis — Part 6: Descriptive and quantitative
representation of particle shape and morphology
ISO 13322-1, Particle size analysis — Image analysis methods — Part 1: Static image analysis methods
ISO 14488, Particulate materials — Sampling and sample splitting for the determination of particulate
properties
ISO/DIS 19749, Nanotechnologies — Measurements of particle size and shape distributions by scanning
electron microscopy
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ISO/TS 21362, Nanotechnologies — Analysis of nano-objects using asymmetrical-flow and centrifugal field-
flow fractionation
ISO/DIS 21363, Nanotechnologies — Measurements of particle size and shape distributions by transmission
electron microscopy
3 Terms and definitions
For the purposes of this document, the terms and definitions given in CEN ISO/TS 80004-2:2017 and
CEN ISO/TS 80004-1:2015 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size are predominantly exhibited in this
length range.
[SOURCE: CEN ISO/TS 80004-1:2015, 2.1]
3.2
nano-object
discrete piece of 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: CEN ISO/TS 80004-1:2015, 2.5]
3.3
reference material
RM
material, sufficiently homogeneous and stable with respect to one or more specified properties, which
has been established to be fit for its intended use in a measurement process
Note 1 to entry: RM is a generic term.
Note 2 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 3 to entry: Uses may include the calibration of a measurement system, assessment of a measurement
procedure, assigning values to other materials, and quality control.
Note 4 to entry: ISO/IEC Guide 99:2007 (VIM) has an analogous definition (5.13), but restricts the term
“measurement” to apply to quantitative values. However, Note 3 of ISO/IEC Guide 99:2007, 5.13, specifically
includes qualitative properties, called “nominal properties”.
[SOURCE: ISO Guide 30:2015, 2.1.1]
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3.4
certified reference material
CRM
reference material (RM) characterized by a metrologically valid procedure for one or more specified
properties, accompanied by an RM certificate that provides the value of the specified property, its
associated uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or
sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence.
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs are given in, among
others, EN ISO 17034 and ISO Guide 35.
Note 3 to entry: ISO Guide 31 gives guidance on the contents of RM certificates.
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition (5.14).
[SOURCE: ISO Guide 30:2015, 2.1.2, modified — One reference at the end of Note 2 to entry was altered.]
4 Symbols and abbreviations
For the purposes of this document, the following symbols and abbreviations apply.
Symbol Quantity SI Unit
−3
c Nano-object number concentration m
n
2 −1
D Diffusion coefficient m s
l Mean thickness of the particle cloud m
f Friction coefficient dimensionless
−1
k Boltzmann’s constant J⋅K
R Retention ratio (F4) dimensionless
m/z
mass-to-charge ratio dimensionless
r Radius of gyration m
g
r Hydrodynamic radius m
h
T Temperature K
t Elution time (F4) s
elution
t Channel void time of the carrier flow s
0
3
V0 Channel volume/channel void volume m
3 −1
• Cross-flow rate m ⋅s
V
c
3
V Elution volume (F4) m
elution
3
Vvoid Channel void volume of the carrier flow m
w Channel height m
λ Retention parameter (F4) dimensionless
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Abbreviation Term
AAS Atomic Absorption Spectrometry
AF4 Asymmetric Flow-Field Flow Fractionation
BF Bright Field
BSA Bovine Serum Albumine
BSE Back Scattered Electrons
CCD Charge-Coupled Device
CLS Centrifugal Liquid Sedimentation
DLS Dynamic Light Scattering
DRI Differential Refractive Index
ECD Equivalent circular diameter
EDX Energy-Dispersive X-ray Spectrometry
EELS Electron Energy Loss Spectrometry
EM Electron Microscopy
FFF Field Flow Fractionation
F4 Flow-Field Flow Fractionation
HAADF High-Angle Annular Dark-Field
HDC Hydrodynamic Chromatography
hF5 Hollow-Fibre Flow-Field Flow Fractionation
ICP-MS Inductively Coupled Plasma – Mass Spectrometry
ICP-TofMS Inductively Coupled Plasma – Time of flight Mass Spectrometry
ICP-OES Inductively Coupled Plasma – Optical Emission Spectrometry
LC Liquid Chromatography
LIBD Laser-Induced Breakdown Detection
LOD Limit of detection
LOQ Limit of quantification
MALS Multi-Angle Light Scattering
MWCO Molecular Weight Cut-Off
NOAA Nano-objects and their aggregates and agglomerates
MS Mass Spectrometry
PTA Particle tracking analysis
RSD Relative Standard Deviation for Reproducibility
R
RSD Relative Standard Deviation for Repeatability
r
SDS Sodium Dodecyl Sulphate
SE Secondary Electrons
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Abbreviation Term
SEC Size Exclusion Chromatography
SEM Scanning Electron Microscopy
spICP-MS single particle Inductively Coupled Plasma – Mass Spectrometry
STEM Scanning transmission electron microscopy
TEM Transmission Electron Microscopy
TOC Total organic carbon
UPW Ultrapure water
UV-vis Ultraviolet and visible light
XRF X-ray fluorescence
5 Possible tasks and measuring techniques
5.1 Examples for detection and identification tasks in complex matrices
As risk assessment of nano-objects requires, among others, characterization of the nano-objects and their
aggregates in several environmental compartments, appropriate analytical techniques should be applied
enabling to determine the size-distribution and nano-object concentration.
According to the scope of this document already known properties, such as elementary composition
and/or morphology of these manufactured nano-objects should be used to distinguish them from natural
background nano-objects. Quantitative analytical methods are also required to determine nano-objects
at environmental concentrations and enable both effect and exposure assessments.
It should be ensured that these methods are sufficiently sensitive in terms of minimal particle size and
material concentration. An illustration of size and concentration ranges for several sample materials is
given in Annex A.
While studying product safety or risk evaluation, several examples of detection of (nano)materials in
complex matrices have been investigated, e.g. silica in tomato soup, titanium dioxide in sun-lotion, silver
nanoparticles in waste water, pigment and filler nanoparticles in coatings or polymer composites as well
as carbon nanotubes in composites.
Several general approaches to prepare a complex sample for FFF analysis are summa
...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17273:2018
01-september-2018
Nanotehnologija - Navodilo za odkrivanje in identifikacijo nanopredmetov v
kompleksnih matrikah
Nanotechnologies - Guidance on detection and identification of nano-objects in complex
matrices
Nanotechnologien - Leitfaden für die Detektion und Identifizierung von Nanoobjekten in
komplexen Matrizen
Nanotechnologies - Guide pour la détection et l'identification des nano-objets dans des
matrices complexes
Ta slovenski standard je istoveten z: FprCEN/TS 17273
ICS:
07.120 Nanotehnologije Nanotechnologies
kSIST-TS FprCEN/TS 17273:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 17273:2018
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17273
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
June 2018
ICS 07.120
English Version
Nanotechnologies - Guidance on detection and
identification of nano-objects in complex matrices
Nanotechnologies - Guide pour la détection et Nanotechnologien - Leitfaden für die Detektion und
l'identification des nano-objets dans des matrices Identifizierung von Nanoobjekten in komplexen
complexes Matrizen
This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 352.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17273:2018 E
worldwide for CEN national Members.
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FprCEN/TS 17273:2018 (E)
Contents Page
European foreword . 3
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Symbols and abbreviations . 8
5 Possible tasks and measuring techniques . 10
5.1 Examples for detection and identification tasks in complex matrices . 10
5.2 Overview of measurement techniques . 10
6 Guidance on sample preparation, particle detection and identification of nano-
objects in complex matrices. 11
6.1 Approach for “Detection and Identification of a relevant population of nano-objects
based on a priori knowledge” (guidance chart) . 11
6.2 Information about the targeted nano-objects . 13
6.3 Information about the sample matrix . 14
6.4 Sample Preparation Process Quality assessment . 14
6.5 Measurement of the targeted nano-objects and evaluation . 16
7 Selected measuring techniques for the detection and identification of nano-
objects . 16
7.1 Field-Flow-Fractionation (FFF) technique. 16
7.2 Electron Microscopy (EM) technique . 21
7.3 Single particle Inductively Coupled Plasma Mass Spetrometry (spICP-MS) . 26
8 List of reporting requirements on sample preparation, detection and identification
of nano-objects in complex matrixes . 30
8.1 General reporting . 30
8.2 Sample preparation reporting, explained in Clause 6 . 31
8.3 Measurement reporting, explained in Clause 7 . 31
Annex A (informative) Indicative ranges of size and concentration of selected measuring
techniques . 32
Annex B (normative) Theory of F4 separations, precautions when separating broad
particle size distributions and sample preparation . 34
Annex C (informative) Example: Analysis of the release of particles from the coating of
silver-coloured pearls by a combination of descriptive TEM analysis, electron
diffraction, analytical TEM and quantitative TEM . 39
Annex D (informative) Example: Single particle ICP-MS for sizing and quantitative
determination of nano-silver in chicken meat . 43
Annex E (informative) Overview of alternative detection methods . 48
Bibliography . 53
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European foreword
This document (FprCEN/TS 17273:2018) has been prepared by Technical Committee CEN/TC 352
"Nanotechnologies", the secretariat of which is held by AFNOR.
This document is currently submitted to the Vote on TS.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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Introduction
Nanotechnology is a rapidly developing field of science and technology that focuses on processes and
materials at the nanoscale size (particle dimensions that are approximately 1 nm to 100 nm). It is a highly
multidisciplinary field with a wide range of materials and applications, e.g. health care, information and
communication technologies, energy production and storage, materials science/chemical engineering,
manufacturing, environmental protection, consumer products (e.g. food, cosmetics, etc.). Therefore, the
resulting products containing nanoscaled materials are very diverse and different in their properties.
CEN/TS 17010:2016 provides guidelines for the identification of measurands to characterize
nano-objects, and their agglomerates and aggregates and to assess specific properties relevant to the
performance of materials that contain them. This document describes the measurands for characterizing
nano-objects based on popular current techniques for characterizing nano-objects. Due to variable matrix
interferences, a method-specific sample preparation protocol to separate particles of interest from their
respective matrices is mandatory.
The production and use of nanomaterials may lead, among others, to an increasing release of
nano-objects into the environment e.g. by liquid waste and production streams. To ensure sustainable
use and development of nanotechnology there is a need for control and monitoring of nanomaterial
systems according to their application (e.g. risk assessment). For that reason, it is necessary to identify
useful measurement techniques for the detection and characterization of nano-objects in so-called
complex matrices, such as natural liquids, waste water, food and cosmetics.[1] Specific characteristics of
the nano-objects have to be known to be able to identify them.
There are numerous techniques for fractionation of nano-objects based on, e.g. Centrifugal Liquid
Sedimentation (CLS) or flow based separation methods, such as Field-Flow Fractionation (FFF),
hydrodynamic chromatography (HDC) and size exclusion chromatography (SEC). Generally, particle size
distributions are obtained by the measurement of the particle concentration from the different size
fractions.
Imaging techniques such as Electron Microscopy (EM) after appropriate sample preparation allow the
detection/imaging of single particles according to several features, e.g. projection area, longest or
shortest external dimension.
In case of counting techniques, after a high and known dilution of a particle stream only single particles
are present in the detection zone and, e.g. particle volume or particle projection area dependent signals
can be measured and related to the particle numbers by light scattering counting methods.
When many different particles are present in the detection zone, ensemble techniques such as static or
dynamic scattering techniques can be used, provided that the size polydispersity of the particles is
limited. Dynamic light scattering (DLS) analyses, for example, generate signal spectra with size
dependent components. DLS can deconvolute these spectra into primary intensity-weighted particle size
distributions, but only for relatively simple sample systems, with well-separated particle size modes and
with the help of advanced algorithms.
The well-established particle size analysis techniques mentioned so far do not cover the chemical
identification of the nano-objects. This technical specification addresses the detection of nano-objects in
complex liquid matrices which might contain an elevated level of inorganic salts, organic contaminants
and larger organic and inorganic particles as well as natural background nano-objects. Therefore, for each
particle two measurands have to be combined: not only the size is needed (for classification as a
nano-object) but also the elemental composition (to discriminate the target particles with an a priori
known elemental composition or morphology, from the matrix and background particles). The aim of this
Technical Specification is to guide the users how to combine size measurement with chemical
identification for each particle.
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The Technical Specification proposes the usage of 3 main characterization methods:
— Field Flow Fractionation combined with multiple detection systems delivering size related
information and additionally material identification;
— Electron Microscopy equipped with Energy Dispersive X-ray Spectroscopy (EDX) to determine the
elemental composition of the particles, additionally to their geometrical measures;
— Single particle Inductively Coupled Plasma – Mass Spectrometry as an elemental specific detection
system gives as well size related information.
For the identification of nano-objects, this Technical Specification requires a priori knowledge of their
nature, e.g. their elemental composition.
All proposed methods currently do not allow in situ but only ex situ characterization.
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1 Scope
This document sets requirements for sampling and treatment of the complex matrices in order to obtain
a liquid dispersion with sufficiently high concentration of the nano-objects of interest.
This document provides guidelines for detection and identification of specific nano-objects in complex
matrices, such as liquid environmental compartments, waste water and consumer products (e.g. food,
cosmetics). This document requires for the identification a priori knowledge of the nature of the nano-
objects like their chemical composition. The selected detection and identification methods are based on
a combination of size classification and chemical composition analysis. Identification can also be
supported, e.g. by additional morphology characterization. Currently only Field Flow Fractionation,
Electron Microscopy and single particle Inductively Coupled Plasma – Mass Spectrometry fulfil this
combination condition.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
CEN ISO/TS 80004-1:2015, Nanotechnologies – Vocabulary - Part 1: Core terms (ISO/TS 80004-1:2015)
CEN ISO/TS 80004-2:2017, Nanotechnologies - Vocabulary - Part 2: Nano-objects (ISO/TS 80004-2:2015)
ISO 14488:2007, Particulate materials - Sampling and sample splitting for the determination of particulate
properties
ISO/PRF TS 21362:2018, Nanotechnologies - Analysis of nano-objects using asymmetrical-flow and
centrifugal field-flow fractionation
CEN/TS 17010:2016, Nanotechnologies - Guidance on measurands for characterising nano-objects and
materials that contain them
ISO/IEC 17025:2017, General requirements for the competence of testing and calibration laboratories
3 Terms and definitions
For the purposes of this document, the terms and definitions given in CEN ISO/TS 80004-2:2017 and
CEN ISO/TS 80004-1:2015 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size are predominantly exhibited in this
length range.
[SOURCE: CEN ISO/TS 80004-1:2015, 2.1]
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3.2
nano-object
discrete piece of 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: CEN ISO/TS 80004-1:2015, 2.5]
3.3
reference material
RM
material, sufficiently homogeneous and stable with respect to one or more specified properties, which
has been established to be fit for its intended use in a measurement process
Note 1 to entry: RM is a generic term.
Note 2 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 3 to entry: Uses may include the calibration of a measurement system, assessment of a measurement
procedure, assigning values to other materials, and quality control.
Note 4 to entry: ISO/IEC Guide 99:2007 (VIM) has an analogous definition (5.13), but restricts the term
“measurement” to apply to quantitative values. However, Note 3 of ISO/IEC Guide 99:2007, 5.13, specifically
includes qualitative properties, called “nominal properties”.
[SOURCE: ISO Guide 30:2015, 2.1.1]
3.4
certified reference material
CRM
reference material (RM) characterized by a metrologically valid procedure for one or more specified
properties, accompanied by an RM certificate that provides the value of the specified property, its
associated uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or
sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence.
Note 2 to entry: Metrologically valid procedures for the production and certification of RMs are given in, among
others, EN ISO 17034:2016 and ISO Guide 35.
Note 3 to entry: ISO Guide 31:2015 gives guidance on the contents of RM certificates.
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition (5.14).
[SOURCE: Corrected from ISO Guide 30:2015, 2.1.2]
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4 Symbols and abbreviations
For the purposes of this document, the following symbols and abbreviations apply.
Symbol Quantity SI Unit
-3
cn Nano-object number concentration m
2 -1
D Diffusion coefficient m s
l Mean thickness of the particle cloud m
f Friction coefficient dimensionless
−1
k Boltzmann’s constant J⋅K
R Retention ratio (F4) dimensionless
m/z
mass-to-charge ratio g
r Radius of gyration m
g
r Hydrodynamic radius m
h
T Temperature K
t Elution time (F4) s
elution
t Channel void time of the carrier flow s
0
3
V Channel volume/channel void volume m
0
•
3 -1
Cross-flow rate m .s
V
c
3
V Elution volume (F4) m
elution
3
V Channel void volume of the carrier flow m
void
w Channel height m
λ Retention parameter (F4) dimensionless
Abbreviation Term
AAS Atomic Absorption Spectrometry
AF4 Asymmetric Flow-Field Flow Fractionation
BF Bright Field
BSA Bovine Serum Albumine
BSE Back Scattered Electrons
CCD Charge-Coupled Device
CLS Centrifugal Liquid Sedimentation
DLS Dynamic Light Scattering
DRI Differential Refractive Index
ECD Equivalent circular diameter
EDX Energy-Dispersive X-ray Spectrometry
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Abbreviation Term
EELS Electron Energy Loss Spectrometry
EM Electron Microscopy
FFF Field Flow Fractionation
F4 Flow-Field Flow Fractionation
HAADF High-Angle Annular Dark-Field
HDC Hydrodynamic Chromatography
hF5 Hollow-Fibre Flow-Field Flow Fractionation
ICP-MS Inductively Coupled Plasma – Mass Spectrometry
ICP-TofMS Inductively Coupled Plasma – Time of flight Mass Spectrometry
ICP-OES Inductively Coupled Plasma – Optical Emission Spectrometry
LC Liquid Chromatography
LIBD Laser-Induced Breakdown Detection
LOD Limit of detection
LOQ Limit of quantification
MALS Multi-Angle Light Scattering
MWCO Molecular Weight Cut-Off
NOAA Nano-objects and their aggregates and agglomerates
MS Mass Spectrometry
PTA Particle tracking analysis
RSD Relative Standard Deviation for Reproducibility
R
RDS Relative Standard Deviation for Repeatability
r
SDS Sodium Dodecyl Suplhate
SE Secondary Electrons
SEC Size Exclusion Chromatography
SEM Scanning Electron Microscopy
spICP-MS single particle Inductively Coupled Plasma – Mass Spectrometry
STEM Scanning transmission electron microscopy
TEM Transmission Electron Microscopy
TOC Total organic carbon
UPW Ultrapure water
UV-vis Ultra-violet and visible light
XRF X-ray fluorescence
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5 Possible tasks and measuring techniques
5.1 Examples for detection and identification tasks in complex matrices
As risk assessment of nano-objects requires, among others, characterization of the nano-objects and their
aggregates in several environmental compartments, appropriate analytical techniques should be applied
enabling to determine the size-distribution and nano-object concentration.
According to the scope of this Technical Specification already known properties, such as elementary
composition and/or morphology of these manufactured nano-objects should be used to distinguish them
from natural background nano-objects. Quantitative analytical methods are also required to determine
nano-objects at environmental concentrations and enable both effect and exposure assessments.
It should be ensured that these methods are sufficiently sensitive in terms of minimal particle size and
material concentration. An illustration of size and concentration ranges for several sample materials is
given in Annex A.
While studying product safety or risk evaluation, several examples of detection of (nano)materials in
complex matrices have been investigated, e.g. silica in tomato soup, titanium dioxide in sun-lotion, silver
nanoparticles in waste water, pigment and filler nanoparticles in coatings or polymer composites as well
as carbon nanotubes in composites.
Several general approaches to prepare a complex sample for FFF analysis are summarized in Annex B.
The informative Annexes C and D provide examples of analysis of silver nanoparticles in decoration of
pastry (by EM) and in chicken meat (by spICP-MS).
5.2 Overview of measurement techniques
The methods Field-Flow Fractionation (FFF), Electron Microscopy (EM) and single particle Inductively
Coupled Plasma – Mass Spectrometry (spICP-MS) described in this document in Clause 7 are amongst the
most established approaches able to detect and identify nano-objects in a number of complex matrices
(e.g. waste water, environmental compartments). These methods are well known but the applications are
still under development, especially for the detection of newer types of nano-objects in e.g. water and food.
Experience can be gained from fields such as environmental chemistry, food chemistry, natural
nanomaterial research and fundamental colloid chemistry. The methods are based on different physical
phenomena and principles and for the particle size analysis different results can be obtained
(e.g. hydrodynamic diameter, equivalent spherical diameter, length/radius).
FFF is a family of flow-based separation techniques able to physically separate macromolecules and
particles from each other according to their molecular weight and size. FFF is applied to measure the
particle size distribution; specific discrimination of particles due to their chemical composition is only
achieved in conjunction with detection systems following the separation in FFF.
Provided that nano-objects can be representatively transferred from a stable dispersion to a suitable
sample carrier (e.g. TEM-grids), Transmission and Scanning Electron Microscopy (TEM/SEM) can be
applied to visualize these objects based on elastic and inelastic scattering of a parallel or convergent
electron beam. Analysis of the EM images allows estimating the number-based distributions of the
external dimensions of the 2D projections of the nano-objects. Specific nano-objects can be identified
based on characteristic properties such as morphology, crystallographic structure, and, in combination
with methods such as EDX or EELS, elemental composition with high spatial resolution.
Nano-objects in aggregates and agglomerates (NOAA), which are not dispersed by the sample
preparation, can nevertheless in specific cases be detected by EM without physical separation.
spICP-MS can be used for the detection and characterisation of nano-objects with elemental tags visible
to ICP-MS in aqueous suspensions. spICP-MS has the exquisite advantage to allow the determination of
the particle number concentration, an estimation of the particle size and number-based size distribution.
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6 9
Particle number concentrations that can be determined in aqueous suspensions range from 10 to 10
-1
particles l , and therefore sample dilution is often required with the benefit that dilution also reduces the
impact of matrix interference and background signals.
Beside well known techniques also less established alternative methods or methods which are still under
development are described in Annex E. These methods might be applied for special needs as they are not
fully focusing on particle concentration, particle sizes or particle composition.
Some alternative detection methods and methods under development are designed for specific
applications in the scientific field. In some cases the method is specific for special nano-objects which are
easier to detect and trace. For example, special designed isotope-labelled nano-objects are used for
bio-accumulation studies and dynamic environmental studies addressing the need to differentiate
between the targeted nanomaterial content and the elemental background concentrations as well as
allowing testing with environmentally realistic concentrations.
6 Guidance on sample preparation, particle detection and identification of
nano-objects in complex matrices
6.1 Approach for “Detection and Identification of a relevant population of nano-objects
based on a priori knowledge” (guidance chart)
The application of systematic development of sample preparation procedures and end-measurements for
detection, characterization and quantification of the a priori known inorganic nano-objects requires a
stepwise evaluation rather than an "all-in-once" evaluation. This stepwise evaluation as recommended
in order to obtain desired information of particle sizes and concentrations joined with a relevant
(measurement) uncertainty of each step or applied method. Sample preparation will likely be the most
challenging part of the analytical process, despite the usage of robust analytical tools. Therefore, in order
to evaluate each single preparative step and (multi) methodical approach implementing detection,
characterization and quantification of the a priori known inorganic nano-objects, a protocol template can
be used in order to achieve a sub-sequential evaluation for each single step [1].
In order to evaluate each single preparative step and the (multi-)methodical approach to detect, analyse
the size and quantify the amount of the a priori known inorganic nano-objects, a protocol template can
be used.
The user should be aware of the requirements in terms of e.g. particle concentration recovery, size
distribution and requirements concerning sensitivity of the applied measurement method as well as the
interpretation of obtained data during the sample preparation/stabilization process.
Based on the information on the physicochemical properties of the target particles and the matrix, a
suitable sample preparation strategy is selected (Figure 1). Depending on the possible interaction of
target particles with matrix components, a strategy of matrix destruction, physical separation and/or
nano-object detachment and stabilization is chosen.
Stabilization of nanoparticles after digestion/separation is essential to obtain a stable dispersion. This
might require the establishment of a specific pH value and/or addition of dispersing agents. The quality
of the resulting suspension is optimized by maximizing the recovery of the target particles (via
comparison of concentrations of a suitable identifier in the extract and in the original sample) while the
matrix load is expected to be significantly reduced.
Additionally, the particle size distribution of the resulting suspension is measured, to identify the
presence of particles in the micrometre and nanometre range, as well as the zeta potential of the
particulate matter in the sample. Following the optimization of the dispersion (recovery, particle size
range, stability) the best suited instrument(s) is(are) selected based on particle size range, concentration
range and necessary pre-analysis treatment specific for each technique.
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Figure 1 — Guidance chart for sample preparation strategy
The following standards for the measurement methods in Figure 1 can be consulted:
EN ISO 11885:2009, Water quality — Determination of selected elements by inductively coupled plasma
optical emission spectrometry (ICP-OES)
EN ISO 17294-1:2006, Water quality — Application of inductively coupled plasma mass spectrometry
(ICP-MS) — Part 1: General guidelines
ISO 22412:2017, Particle size analysis — Dynamic light scattering (DLS)
ISO 19430:2016, Particle size analysis — Particle tracking analysis (PTA) method
6.2 Information about the targeted nano-objects
As this technical specification assumes a priori knowledge of the targeted nano-objects several
physicochemical parameters should be known in a
...
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