The certification of the equivalent spherical diameters of silica nanoparticles in aqueous solution, ERM®- FD101b : certification report.
Date: 2017-11-03
Institutions: Joint Research Centre
Subjects: approval, materials technology, nanotechnology, organic chemical, physical sciences, research method, research report, technical standard

CERTIFICATION REPORT 

The certification of equivalent diameters of silica 
nanoparticles in aqueous solution: 
ERM®-FD101b

E
U
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8
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6
2

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JRC-IRMM promotes a common and 

provides reference measurements. 

European Commission  
Joint Research Centre 
Directorate F – Health, Consumers and Reference Materials 

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All images © European Union 2017 

JRC105046 

EUR 28362 EN 

ISBN 978-92-79-64637-9
ISSN 1831-9424
doi:10.2787/212519 

Luxembourg: Publications Office of the European Union, 2017 
© European Union, 2017 

Reproduction is authorised provided the source is acknowledged. 

Abstract 

This report describes the production of ERM®-FD101b, silica nanoparticles suspended in an aqueous solution, certified for different equivalent particle 
diameters. The material was produced following ISO Guide 34:2009. 
The certified reference material (CRM) was produced by the Directorate F - Health, Consumers and Reference Materials of the European Commission’s 
Joint Research Centre (JRC) in Geel (Belgium). The CRM was produced from a diluted and pH adjusted commercial colloidal silica slurry. 
Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The minimum 
sample intake for the different methods was determined from the results and information provided by the laboratories that participated in the 
interlaboratory comparison (ILC) exercises of the characterisation study. 
The material was characterised for several equivalent particle diameters based on an interlaboratory comparison amongst laboratories of demonstrated 
competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outlier was eliminated on statistical grounds only. 
Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include 
uncertainty contributions related to possible inhomogeneity and instability and to characterisation. 
The material is intended for quality control and assessment of method performance. The method-defined certified values are regarded as reliable 
estimates of the true values and ERM-FD101b can therefore be used for calibration purposes. The CRM is available in 10 mL pre-scored amber glass 
ampoules each containing about 9 mL of suspension. 
The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.

The certification of equivalent diameters of silica 
nanoparticles in aqueous solution: 
ERM®-FD101b 

Y. Ramaye, V. Kestens, A. Braun, T. Linsinger, A. Held, G. Roebben 

European Commission, Joint Research Centre 

Directorate F – Health, Consumers and Reference Materials 
Geel, Belgium 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Disclaimer 

Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the 
experimental  procedure.  In  no  case  does  such  identification  imply  recommendation  or  endorsement  by  the 
European Commission, nor does it imply that the material or equipment is necessarily the best available for the 
purpose.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Summary 

This report describes the production of ERM®-FD101b, silica nanoparticles suspended in an 
aqueous  solution,  certified  for  different  equivalent  particle  diameters.  The  material  was 
produced following ISO Guide 34:2009 [1]. 

The  certified  reference  material  (CRM)  was  produced  by  the  Directorate  F  -  Health, 
Consumers and Reference Materials of the European Commission’s Joint Research Centre 
(JRC)  in  Geel  (Belgium).  The  CRM  was  produced  from  a  diluted  and  pH  adjusted 
commercial colloidal silica slurry. 

Between  unit-homogeneity  was  quantified  and  stability  during  dispatch  and  storage  were 
assessed  in  accordance  with  ISO  Guide  35:2006  [2].  The  minimum  sample  intake  for  the 
different  methods  was  determined  from  the  results  and  information  provided  by  the 
laboratories  that  participated  in  the  interlaboratory  comparison  (ILC)  exercises  of  the 
characterisation study. 

The  material  was  characterised  for  several  equivalent  particle  diameters  based  on  an 
interlaboratory comparison amongst laboratories of demonstrated competence and adhering 
to ISO/IEC 17025 [3]. Technically invalid results were removed but no outlier was eliminated 
on statistical grounds only. 

Uncertainties  of  the  certified  values  were  calculated  in  accordance  with  the  Guide  to  the 
Expression  of  Uncertainty  in  Measurement  (GUM)  [4]  and  include  uncertainty  contributions 
related to possible inhomogeneity and instability and to characterisation. 

The  material  is  intended  for  quality  control  and  assessment  of  method  performance.  The 
method-defined  certified  values  are  regarded  as  reliable  estimates  of  the  true  values  and 
ERM-FD101b can therefore be used for calibration purposes. The CRM is available in 10 mL 
pre-scored amber glass ampoules each containing about 9 mL of suspension. 
The  CRM  was  accepted  as  European  Reference  Material  (ERM®)  after  peer  evaluation  by 
the partners of the European Reference Materials consortium. 

1 

 
The following certified values were assigned: 

SILICA NANOPARTICLES IN AQUEOUS SOLUTION 

Hydrodynamic diameter 
from DLS 1) 
(cumulants method) 
Hydrodynamic diameter  
from DLS 2) 
(distribution calculation 
algorithms) 

Hydrodynamic diameter  
from PTA 3) 

Stokes diameter 
from CLS 4)  
(turbidimetry) 

Area-equivalent diameter 
from EM 5) 

Size distribution parameter:  
Weighting / Averaging 

Scattered light intensity-weighted / 
harmonic mean 

Scattered light intensity-weighted / 
mean (arithmetic, harmonic, 
geometric) and modal 
Number-weighted / modal 
Number-weighted / arithmetic mean 
Number-weighted / median 

Light extinction-weighted / modal 

Number-weighted / modal 

Number-weighted / median 

Mean particle diameter  
from SAXS 6) 
(model fitting) 

Scattered X-ray intensity-weighted / 
modal 
Volume-weighted / modal 

Number-weighted / modal 

Certified 
value 7)  
[nm] 

89.5 

Uncertainty 8) 

 [nm] 

2.3 

93 

82 
87 
82 

87 

83.7 

83.5 

82.5 

81.7 

80.9 

4 

4 
4 
4 

8 

2.2 

2.2 

1.8 

1.8 

1.7 

1)  As  obtained  with  dynamic  light  scattering  (DLS)  according  to  ISO  22412:2008  applying  the  cumulants 
method described in ISO 13321:1996 at a sample temperature of 25 °C. 
2)  As  obtained  with  dynamic  light  scattering  (DLS)  applying  distribution  calculation  algorithms  such  as  non-
negative  least  square  (NNLS)  and  CONTIN  for  data  analysis,  using  various  averaging  approaches  and  a 
sample temperature of 25 °C. 
3) As obtained with particle tracking analysis (PTA) according to ISO 19430:2016 at a sample temperature of 
25 °C. 
4)  As  obtained  with  centrifugal  liquid  sedimentation  (CLS)  according  to  ISO  13318-1:2001,  using  an 
effective particle density of 2.0 g/cm3 at sample temperatures between 25 °C and 36 °C.  
5) As obtained with transmission and scanning electron microscopy (EM), counting only particles with an 
equivalent diameter larger than 60 nm. 
6)  As  obtained  with  small-angle  X-ray  scattering  (SAXS)  according  to  ISO  17867:2015  at  sample 
temperatures  between  23 °C  and  25 °C,  using  'model  fitting'  assuming  homogeneous  spheres  and  a 
Gaussian size distribution. 
7) Unweighted mean value of the means of accepted sets of data; each set being obtained in a different 
laboratory  and/or  with  a  different  method  of  determination.  The  certified  value  and  its  uncertainty  are 
traceable to the International System of Units (SI). 
8)  The  uncertainty  of  the  certified  value  is  the  expanded  uncertainty  with  a  coverage  factor  k  =  2 
corresponding to a level of confidence of about 95 % estimated in accordance with ISO/IEC Guide 98-3, 
Guide to the Expression of Uncertainty in Measurement (GUM:1995), ISO, 2008. 

2 

 
  
 
 
 
Table of contents 

Summary .............................................................................................................................. 1 
Table of contents ................................................................................................................. 3 
Glossary ............................................................................................................................... 5 
Introduction .............................................................................................................. 9 
1 
Background ............................................................................................................... 9 
1.1 
Choice of the material ...............................................................................................12 
1.2 
Design of the project .................................................................................................12 
1.3 
Participants .............................................................................................................13 
2 
Project management and evaluation .........................................................................13 
2.1 
Processing ................................................................................................................13 
2.2 
Homogeneity study ...................................................................................................13 
2.3 
Stability study ...........................................................................................................13 
2.4 
Characterisation ........................................................................................................13 
2.5 
Material processing and process control .............................................................15 
3 
Origin of the starting material and available information ............................................15 
3.1 
Processing ................................................................................................................16 
3.2 
Homogeneity ...........................................................................................................17 
4 
4.1 
Between-unit homogeneity........................................................................................17 
4.2  Within-unit homogeneity and minimum sample intake...............................................18 
Stability....................................................................................................................20 
5 
Short-term stability study ..........................................................................................20 
5.1 
Long-term stability study ...........................................................................................21 
5.2 
Estimation of uncertainties ........................................................................................22 
5.3 
Characterisation .....................................................................................................24 
6 
Selection of participants ............................................................................................24 
6.1 
6.2 
Study setup ...............................................................................................................24 
6.3  Methods used ...........................................................................................................25 
6.3.1  Centrifugal liquid sedimentation ................................................................................25 
6.3.2  Dynamic light scattering ............................................................................................25 
6.3.3  Electron microscopy ..................................................................................................26 
6.3.4  Particle tracking analysis ...........................................................................................26 
6.3.5  Small-angle X-ray scattering .....................................................................................26 
6.4 
Evaluation of results .................................................................................................27 
6.4.1  Technical evaluation .................................................................................................27 
6.4.2  Statistical evaluation .................................................................................................29 
Value Assignment ...................................................................................................32 
7 
Certified values and their uncertainties .....................................................................32 
7.1 
Indicative values and their uncertainties....................................................................34 
7.2 

3 

 
Additional material information ..................................................................................35 
7.3 
7.3.1  Centrifugal liquid sedimentation (refractometry) ........................................................35 
7.3.2  Field flow fractionation ..............................................................................................35 
7.3.3  Electrochemical properties ........................................................................................36 
7.3.4  Sub-population of smaller particles ...........................................................................36 
Metrological traceability and commutability .........................................................37 
8 
8.1  Metrological traceability ............................................................................................37 
Commutability ...........................................................................................................40 
8.2 
Instructions for use ................................................................................................41 
9 
Safety information .....................................................................................................41 
9.1 
Storage conditions ....................................................................................................41 
9.2 
9.3 
Instructions for use and intended use .......................................................................41 
9.4  Minimum sample intake ............................................................................................42 
Use of the certified value ..........................................................................................42 
9.5 
Acknowledgements ................................................................................................43 
10 
References ..............................................................................................................44 
11 

Annexes: 

Annex A: Results of the homogeneity measurements 

Annex B: Results of the short-term stability measurements 

Annex C: Results of the long-term stability measurements 

Annex D: Summary of the methods used in the characterisation study 

Annex E: Results of the characterisation measurements 

4 

 
 
 
 
Glossary 

AF4 

AFM 

Asymmetrical flow field-flow fractionation 

Atomic force microscopy 

ANOVA 

Analysis of variance 

APD 

AUC 

b 

CCL 

CIPM 

CLS 

CRM 

D 

Dcal 

DLS 

ELS 

EM 
ERM® 

EU 

GUM 

IEC 

ILC 

ISO 

JRC 

k 

MALS 

MSbetween 

MSwithin  

n.a. 

n.c. 

n.d. 

NNLS 

p 

PMT 

PSA 

Avalanche photodiode detector 

Analytical ultracentrifugation 

Slope of regression line in stability study 

Consultative Committee for Length 

Comité  International  des  Poids  et  Mesures  (International  Committee  of 
Weights and Measures) 

Centrifugal liquid sedimentation 

Certified reference material 

Diameter of an equivalent sphere 

Assigned diameter of calibrant particles 

Dynamic light scattering 

Electrophoretic light scattering 

Electron microscopy 

Trademark of the European Reference Materials initiative 

European Union 

Guide to the Expression of Uncertainty in Measurement 

International Electrotechnical Commission 

Interlaboratory comparison 

International Organization for Standardization 

Joint Research Centre of the European Commission 

Coverage factor 

Multi-angle light scattering 

Mean of squares between-unit from an ANOVA 

Mean of squares within-unit from an ANOVA 

Not applicable (or not available) 

Not calculated 

Not detectable 

Non-negative least square 

Number of technically valid datasets 

Photomultiplier tube 

Particle size analysis 

5 

 
PSD 

PSL 

PTA 

PVC 

q 

QC 

R 

R 

rel 

Rg 

RG 

RI 

RM 

rpm 

RSD 

s 

SAXS 

sbb

sbetween 

SE 

SEM 

SI 

swithin 

swb 

t  

ti 

tsl 

ttt 

TEM 

TSEM 

U 

u 
u*

bb 

Particle size distribution 

Polystyrene latex 

Particle tracking analysis 

Polyvinyl chloride 

Scattering vector 

Quality control 

Correlation coefficient 

Radius of an equivalent sphere 

Relative value 

Radius of gyration 

Guinier radius 

Refractive index 

Reference material 

Revolutions per minute 

Relative standard deviation 

Standard deviation 

Small-angle X-ray scattering 

Between-unit standard deviation; an additional index "rel" is added when 
appropriate 

Standard deviation between groups as obtained from ANOVA 

Secondary electron 

Scanning electron microscopy 

International System of Units 

Standard deviation within groups as obtained from ANOVA 

Within-unit  standard  deviation;  an  additional  index  "rel"  is  added  when 
appropriate 

Mean of all ti 

Time elapsed at time point i 

Shelf life 

Transport time 

Transmission electron microscopy 

Transmission-mode scanning electron microscopy 

Expanded  uncertainty;  an  additional 
appropriate 

index  "rel" 

is  added  when 

Standard uncertainty; an additional index "rel" is added when appropriate 

Standard uncertainty related to a maximum between-unit inhomogeneity 
that could be hidden by method repeatability; an additional index "rel" is 
added when appropriate 

6 

 
 
ubb 

uc 

ucal 

uchar 

uCRM 

UCRM 

u∆ 

udeg 

ults 

usts 

ur  

x  
D meas 

n eff 
n MSwithin 
r cal 
r CRM 

Standard  uncertainty  related  to  a  possible  between-unit  inhomogeneity; 
an additional index "rel" is added when appropriate 

Combined uncertainty 

Standard uncertainty of a common calibrant 

Standard uncertainty of the material characterisation; an additional index 
"rel" is added when appropriate 

Combined standard uncertainty of the certified value; an additional index 
"rel" is added when appropriate 

Expanded  uncertainty  of  the  certified  value;  an  additional  index  "rel"  is 
added when appropriate 

Combined  standard  uncertainty  of  measurement  result  and  certified 
value 

Standard  uncertainty  corresponding  with  a  potential  degradation 
observed in the stability study 

Standard uncertainty of the long-term stability; an additional index "rel" is 
added when appropriate 

Standard  uncertainty  of the  short-term stability;  an  additional  index  "rel" 
is added when appropriate 

Standard uncertainty of the effective particle density; an additional index 
"rel" is added when appropriate 

Arithmetic mean 

Absolute  difference  between  mean  measured  value  and  the  certified 
value 

Effective degrees of freedom 

Degrees of freedom of MSwithin 

Effective particle density of a calibrant 

Effective particle density of the CRM 

7 

 
 
 
 
8 

 
 
 
1  Introduction 

1.1  Background 

According to the ISO definition, nanoparticles are particles with all three external dimensions 
between approximately 1 nm and 100 nm [5], that may exhibit unique properties due to their 
size. To implement the related European Commission's Recommendation (2011/696/EU) on 
the definition of a nanomaterial [6], and to better understand the properties of nanoparticles, 
reliable size and size distribution measurements are needed. In this respect, fit-for-purpose 
reference materials including quality control and calibration materials are necessary.  

A  variety  of  techniques  exists  to  analyse  the  size  and  size  distribution  of  nanoparticles.  As 
particles have rarely a perfectly spherical shape, different size parameters would need to be 
carefully  investigated  in  order  to  determine  the  effective  particle  size  [7].  Instead,  most 
measurement methods describe the size of the particles with an "equivalent" diameter. Due 
to differences in the measurement principles of the different methods, they do not all produce 
the  same  equivalent  particle  diameters for the  same  particle(s). Therefore  particle  size  is  a 
method-defined  measurand.  The  certified,  indicative  and  additional  material  information 
particle  size  values  of  ERM-FD101b  are  specified  in  this  report  as  equivalent  diameters 
corresponding to  the  methods  used,  and to  the  type  of  distribution  reported.  A  summary of 
the techniques used in this study is given below. 

1.  Centrifugal liquid sedimentation (CLS) methods [8] use a centrifugal force to settle 
particles from a suspension and thereby fractionate particles of different size and/or 
density. CLS instruments contain the suspension in a disc or a cuvette. 

(i) A disc centrifuge instrument is based on a hollow, optically clear spinning disc that is 
partly  filled  with  a  liquid  (e.g.,  a  sucrose  solution).  Under  the  influence  of  the  centrifugal 
force the liquid is held against the outside edge of the chamber forming a liquid ring. If this 
ring is created by a series of injections of liquid with decreasing density, the liquid at the 
outside edge of the ring is denser than the liquid near the inside edge. The latter density is 
chosen to be slightly higher than the density of the sample's dispersing medium. When the 
density  gradient  is  created  and  stabilised  a  small  volume  of  a  dilute  suspension  of 
particles is injected into the centre of the spinning disc. A laser light or X-ray beam passes 
through the liquid near the outside edge of the disc and particles passing the beam reduce 
the light intensity in proportion to their concentration. 

Disc centrifuges are operated in the line-start or in the homogeneous mode and are either 
equipped  with  (laser)  light  [9]  or  X-ray  absorption  [10]  optical  detection  systems.  Their 
rotational  speed  varies  from  500  revolutions  per  minute  (rpm)  to  24000 rpm.  The 
instruments  either  measure  the  attenuation  of  light  (turbidity)  or  that  of  X-rays.  The 
measured sedimentation time is converted to an equivalent particle size using Stokes' law 
of  sedimentation  and  the  effective  particle  density.  For  instruments  with  turbidity  optics, 
the  obtained  size  distribution  is  light  extinction-based,  and  this  can  be  converted  to  a 
particle mass-based distribution using Mie theory. The latter can be further converted to a 
number-based distribution by calculating the volume of the particles. For instruments with 
X-ray  absorption  optics  the  attenuation  of  the  X-ray  beam  is  directly  proportional  to  the 
mass of the detected particles and the size distribution is particle mass-based.1  

(ii)  In  the  cuvette  centrifuge,  particles  do  not  travel  through  a  pre-constructed  density 
gradient  but  sediment  out  of  their  native  dispersant  in  which  they  are  initially 

1  Note:  For  a  population  of  particles  with  uniform  density,  particle  mass-based  and  particle  volume-
based size distributions are identical. 

9 

 
 
                                                
types  of  cuvette  centrifuges  can  be 
two 
homogeneously  distributed.  Generally, 
distinguished  depending  on  their  maximum  rotational  speed.  The  first  group  of 
instruments have a rotational speed up to 4000 rpm and are equipped with turbidity optics 
[11].  The  second  group  of  cuvette  centrifuges,  which  are  known  as  analytical 
ultracentrifugation  (AUC)  instruments,  can  be  operated  at  rotational  speeds  up  to 
60000 rpm. Today's  commercially  available  AUC  instruments  are typically  equipped  with 
Rayleigh  interference  (refractometry)  optics.  Such  detection  system  determines  the 
distribution  of  sedimentation  coefficients  by  measuring  the  difference  in  refractive  index 
between  a  test  sample  and  a  reference  sample  [12].  The  difference  in  refractive  index 
directly  corresponds  to  the  mass  of  the  detected  particles.  Hence,  the  particle  size 
distribution  obtained  from  the  initial  sedimentation  coefficient  distribution,  using  Stokes' 
law, is particle mass-based. 

2.  Dynamic  light  scattering  (DLS),  also  referred to  as  photon  correlation  spectroscopy  or 
quasi-elastic light scattering, measures the fluctuation of light intensity that is scattered by 
a  quiescent  particle  suspension  upon 
irradiation  by  a  beam  of  coherent  and 
monochromatic  (laser)  light  [13].  The  intensity  of  the  scattered  light,  which  fluctuates  in 
time  due  to  the  Brownian  motion  of  the  particles,  is  registered  by  a  highly  sensitive 
photodetector  such  as  a  photomultiplier  tube  (PMT)  or  an  avalanche  photodiode  (APD). 
These detectors are commonly positioned at an angle of about 90° or at about 180° with 
respect to the incident light beam. DLS instruments are intrinsically absolute in nature and 
instrumental response or signal calibration or correction factors are thus not required. DLS 
instruments can be classified according to the principle of data acquisition and processing. 

(i) The majority of DLS instruments are operating in the time-domain. These instruments 
measure the scattered light intensity fluctuations and process this via a digital correlator to 
extract an intensity correlation function. 

For  highly  monomodal  dispersions  of  solid  spherical  particles,  the  correlation  function 
linearly  decays  with  a  rate  that  is  largely  proportional  to  the  translational  diffusion 
coefficient of the particles, except for some impact of the rotational diffusion component. 
The  decay  rate  can  be  determined  by  fitting  the  correlation  function  with  a  first-order  or 
exponential function. The (apparent) translational diffusion coefficient then yields, via the 
Stokes-Einstein  relationship,  the  (apparent)  hydrodynamic  particle  size.  In  reality,  also 
monomodal samples have a degree of polydispersity. As a result, the correlation function 
results from a distribution of decay constants and describes a multi-exponential behaviour 
that  can  only  be  fitted  approximatively  using  a  cumulants  generating  polynomial  power 
function.  This  is  generally  known  as  the  method  of  cumulants  [14,15,16].  The  first 
cumulant  of  the  power  function,  which  corresponds  to  the  initial  slope  of  the  correlation 
function,  provides  the  mean  translational  diffusion  coefficient.  The  Stokes-Einstein 
equation  then  relates  the  mean  translational  diffusion  coefficient  with  the  scattered  light 
intensity-weighted harmonic mean particle size (or the 'z-average' as it is called in certain 
commercial  DLS  software).  In  addition,  a  dimensionless  qualitative  estimation  of  the 
degree of polydispersity can be obtained from the ratio of the first and second cumulant. 
The cumulants method is the most robust, simple and widely used method for measuring 
the average size of nanoparticles in suspension.  

Contrary  to  the  cumulants  method,  distribution  calculation  algorithms  can  be  used  to 
compute full particle size distributions (PSDs) by deconvoluting the correlation function via 
algorithms  which  include  a  Laplace  transformation.  The  main  disadvantage  of  Laplace 
transformation is that the process is mathematically ill-posed: a given correlation function 
can  be  described  by  an  infinite  number  of  solutions.  The  existing  algorithms  try  to 
overcome  this  difficulty  by  using  criteria  to  limit  the  number  of  possible  solutions  and  to 
choose  the  most  reasonable  one.  One  such  algorithm,  called  CONTIN,  has  been 
developed  by  Provencher  [17,18].  CONTIN  is  a  generalised  inverse  Laplace  transform 
algorithm  that  seeks  the  simplest  (most  parsimonious)  solution  for  experimental  data 
based  on  prior  statistical  knowledge.  The  algorithm  additionally  contains  a  non-negative 

10 

 
least  square  (NNLS)  routine  that  is  often  effectively  used  as  stand-alone  PSD  algorithm 
[19]. Another algorithm, called Dynals, behaves similar to CONTIN.  

Since a single universally-accepted Laplace transformation algorithm does not exist, most 
manufacturers of DLS instruments have developed their own specific algorithms that are 
typically grafted on either the CONTIN or NNLS (or a combination of both) algorithm. Most 
of  these  algorithms  differ  from  each  other  in  the  grade  of  smoothing  of  the  correlation 
function. Because of the different approaches in deconvolution, smoothing and calculation 
of  mean  values  from  the  discrete  size  distributions,  and  due  to  sensitivity  to  noise  (from 
small  numbers  of  larger  particles),  the  results  of  the  distribution  calculation  algorithms 
tend  to  be  less  repeatable  and  reproducible  than  results  of  the  cumulants  method. 
Nevertheless,  the  advantage  of  producing  full  size  distributions  of  different  weightings 
(intensity-, volume- and number-based) has led to a very broad and common use of the 
distribution calculation algorithms. 

(ii)  The  second  group  of  DLS  instruments  are  equipped  with  spectrum  analysers  that 
obtain  the  frequency  spectra  by  Fourier  transforming  the  scattered  intensity  fluctuations 
into  a  power  spectrum.  The  power  spectrum  is  then  converted  into  a  PSD  using  a 
distribution calculation algorithm [20]. 

3.  Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) 
are  versatile  techniques  that  allow  analysing  the  morphology,  crystallographic  structure 
and  chemical  composition  of  nanomaterials.  An  electron  microscope  uses  an  electron 
source  to  generate  a  primary  electron  beam.  In  a  TEM  instrument,  a  parallel  electron 
beam is created and passes through the very thin specimen. The information contained in 
the electrons that pass through the specimen is used to create an image. The contrast in 
this image originates from the absorption and scattering of electrons in the specimen, due 
to the thickness and composition of the material (i.e. mass-thickness contrast), and from 
the crystal orientation (i.e. diffraction contrast). An SEM instrument scans the surface of a 
specimen  with  a  focused  beam  of  electrons.  An  image  is  created,  for  example,  by 
detecting  the  secondary  electrons  generated  from  the  interactions  of  the  electron  beam 
with the specimen surface at every point during the scan. 

SEM and TEM instruments have in common that they both produce 2D projections of 3D 
objects like nanoparticles. To image the nanoparticles, they are deposited onto a suitable 
substrate (e.g., carbon foils or metal grids, mica surfaces or silicon wafers). Based on the 
difference  in  contrast  between  the  nanoparticles  and  the  substrate,  well-dispersed 
nanoparticles  can  be  detected  in  an  image  analysis  process,  by  applying  grey-level 
threshold  limits,  and  then  be  quantified  e.g.  in  terms  of  particle  size  and  particle  size 
distribution. 

The  size  of  the  particles  in  the  electron  microscopy  (EM)  images  can  be  quantitatively 
defined using different size and shape parameters (e.g., minimum and maximum Feret's 
diameter,  Martin's  diameter,  projected  area-equivalent  circular  diameter)  [21,22,23,24]. 
Automated,  semi-automated  and  manual  image  analysis  can  be  performed  by  using 
designated software. 

4.  Particle  tracking  analysis  (PTA),  also  referred  to  as  nanoparticle  tracking  analysis, 
dynamic  ultramicroscopy,  or  orthogonal  tracking  microscopy,  combines  laser  light 
scattering microscopy with a digital video camera. The camera records the light scattered 
by the particles that are moving under Brownian motion. The PTA software detects, based 
on their scattering behaviour, individual particles suspended in a liquid and monitors their 
trajectories  in  the  suspension.  The  movement  of  the  particles,  expressed  as  a  mean 
square displacement, is related to their hydrodynamic diameter via a formula derived from 
the Stokes-Einstein equation. 

5.  In  small-angle  X-ray  scattering (SAXS),  a  narrow  beam  of X-rays  is  passed  through  a 
sample, e.g., a suspension of nanoparticles [25]. The X-rays are scattered at surfaces of 
increased  electron  density,  such  as  the  surface  of  the  particles,  thereby  creating  elastic 

11 

 
scattering waves (Rayleigh scattering). These scattering waves interfere with one another 
forming  a  scattering  pattern.  Particles  produce maximum  scattering  at  an  angle  which  is 
inversely related to the particle size. This angle, together with the individual unit vectors in 
the incident and scattered X-ray directions, is the basis for the scattering vector (q). The 
shape of the scattering curve contains information of particle size and particle shape.  

At small scattering vectors the scattered X-ray intensity only depends on the contrast (= 
difference  in  electron  density  of  the  particle  versus  dispersing  medium),  concentration, 
particle volume and radius of gyration (Rg). The latter is a size parameter that corresponds 
to the root mean square (quadratic mean) of the distance to the centre of mass weighted 
by the contrast of electron density and which can be determined by applying the Guinier 
law  [26].  This  initial  part  of  the  scattering  curve  can  be  approximated  by  a  Gaussian 
function,  hence  its  natural  logarithm  versus  q2  yields  a  straight  line.  The  value  of  the 
corresponding  Guinier  radius,  RG,  which  can  be  calculated  from  the  slope  of  the  fitted 
linear curve is a (volume)2-weighted mean of the size of the particles in the suspension. 
Following this Guinier approximation, only an overall mean particle size can be obtained.  

With the information in the SAXS scattering curve at larger q-values, it is also possible to 
obtain  a  full  PSD.  This  requires  assumption  of  a  certain  model  PSD  distribution,  the 
parameters of which must be fitted to make the experimental scattering curve match the 
theoretical  [27].  This  model  fitting  method  [28]  allows  computation  of  number-,  volume- 
and  (X-ray  scattering)  intensity-based  PSDs  under  the  assumption  of  homogeneous 
particle shape. 

1.2  Choice of the material 

Given  its  industrial  relevance,  and  the  ability  to  remain  colloidally  stable  for  several  years, 
silica nanoparticles were selected as target particles. 

ERM-FD101b  was  produced  from  a  commercially  available  sol  which  consisted  of  silica 
nanoparticles suspended in an aqueous solution of electrolytes. In order to allow the users of 
ERM-FD101b to use the CRM as-received (e.g., for DLS, SAXS and CLS), the original silica 
sols  were  strongly  diluted  with  purified  water  (18.2  MW
·cm  resistivity  at  25  °C).  This 
increases  the  versatility  with  respect  to  the  preparation  of  test  specimens  for  microscopy 
analysis  and  it  allows  using  values  of  physical  properties  (e.g.,  viscosity,  density,  refractive 
index) that are known for water and which are needed to calculate the equivalent diameters 
for several particle size analysis (PSA) methods. 

1.3  Design of the project 

The  stability  and  homogeneity  of  the  material  was  evaluated  through  PSA  using  DLS  and 
CLS. The combined knowledge on the material homogeneity and stability obtained from DLS 
and  CLS  measurements  is  sufficiently  detailed  to  also  use  the  resulting  homogeneity  and 
stability uncertainty contributions for the certification of the particle size values obtained with 
other  PSA  methods.  The  certified  and 
indicative  values  were  established  by  an 
interlaboratory comparison of different laboratories with different measurement methods and 
techniques. 

12 

 
 
 
 
2  Participants 

2.1  Project management and evaluation 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and 
Reference Materials, Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM) 

2.2  Processing 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and 
Reference Materials, Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM) 

2.3  Homogeneity study 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and 
Reference Materials, Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM; measurements 
under the scope of ISO/IEC 17025 accreditation BELAC No. 268-TEST) 

2.4  Stability study 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and 
Reference Materials, Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM; measurements 
under the scope of ISO/IEC 17025 accreditation BELAC No. 268-TEST) 

2.5  Characterisation 

The participants in the interlaboratory comparison study were (list alphabetical order): 

Agfa Gevaert NV, Agfa-Labs, Mortsel, BE 

Anton Paar GmbH, Graz, AT 

AQura GmbH, Marl, DE 
(accredited to ISO/IEC 17025, measurements under the scope of DAkkS No. D-PL-14093-01-00) 

Beijing Center for Physical and Chemical Analysis (BCPCA), Beijing, CN 
(accredited to ISO/IEC 17025, measurements under the scope of CNAS No. L0066) 

BAM Federal Institute for Materials Research and Testing, Berlin, DE 

Dannalab B. V., Enschede, NL 

Delft Solids Solutions B.V., Wateringen, NL 

Dr. Lerche KG, Berlin, DE 

European  Commission,  Joint  Research  Centre,  Directorate  F  –  Health,  Consumers  and 
Reference Materials, Geel, BE 
(accredited to ISO/IEC 17025, measurements under the scope of BELAC No. 268-TEST) 

Horiba Instruments Inc., Irvine, US 

Horiba Scientific, Longjumeau, FR 

Hungarian  Academy  of  Science,  Research  Centre  for  Natural  Sciences,  Biological 
Nanochemistry Research Group, Institute of Materials and Environmental Chemistry (RCNS 
HAS), Budapest, HU 

Leidos  Biomedical  Research  Inc,  Frederick  National  Laboratory  for  Cancer  Research, 
Nanotechnology Characterization Laboratory, Frederick, US 

13 

 
 
 
 
 
 
 
LGC Ltd., Teddington, UK 

LUM GmbH, Berlin, DE 

Malvern Instruments Inc., Westborough, US 

Malvern Instruments Ltd., Malvern, UK 

Malvern Instruments Ltd., Amesbury, UK 

MicroParticles GmbH, Berlin, DE 

MVA Scientific Consultants, Duluth, US 

Nanolytics, Gesellschaft für Kolloidanalytik mbH, Potsdam,DE 

National  Center  for  Nanoscience  and  Technology  (NCNST),  CAS  Key  Lab  for  Biomedical 
Effects of Nanomaterials and Nanosafety, Beijing, CN 

National 
Measurement, Beijing, CN 

Institute  of  Metrology  (NIM),  Division  of  Nano  Metrology  and  Materials 

National  Institute  of  Standards  and  Technology  (NIST),  Physical  Measurement  Laboratory, 
Semiconductor and Dimensional Metrology Division, Gaithersburg, US 

National Measurement Institute Australia (NMIA), Nanometrology, West Lindfield, AU 

National Physical Laboratory (NPL), Analytical Science Division, Teddington, UK 

Physikalisch-Technische  Bundesanstalt  (PTB),  Working  Group  7.11  "X-ray  Radiometry", 
Berlin, DE 

Physikalisch-Technische  Bundesanstalt 
Microscopy", Braunschweig, DE 

Sympatec GmbH, Clausthal-Zellerfeld, DE 

(PTB),  Working  Group  4.22 

"Quantitative 

Technical University (Bergakademie) Freiberg, Institute for Mechanical Process Engineering 
and Mineral Processing, Laboratory for Particle Characterisation, Freiberg, DE 

TNO, Delft, NL 

University of Konstanz, Physical Chemistry, Konstanz, DE 

University of Konstanz, Physics Department, Konstanz, DE  

University of Namur, Namur Nanosafety Centre, Namur, BE 

Veterinary  and  Agrochemical  Research  Centre  (CODA-CERVA),  Electron  Microscopy 
Service, Brussels, BE 

14 

 
 
 
 
3  Material processing and process control 

3.1  Origin of the starting material and available information 

The colloidal silica starting material Klebosol 30R50 was supplied by AZ Electronic Materials 
France SAS (Trosly-Breuil, FR). Material specifications for the starting material, as provided 
by the manufacturer, are listed in Table 1. 

In studies prior to the certification project, a number of material properties were assessed: 

- The nominal SiO2 (dry) mass fraction was confirmed by in-house measurements (309 g/kg). 

-  Preliminary  TEM  analyses  were  performed  by  an  independent  laboratory  (MVA  Scientific 
Consultants, Duluth, USA) qualified by the JRC. TEM grids were dipped in the as-received 
concentration of colloidal silica, and imaged with a Philips CM120 TEM at an acceleration 
voltage of 100 kV. A typical TEM micrograph is shown in Fig. 1. The particle aspect ratio, 
defined as the ratio of the major diameter (length) to the minor diameter (width) of a fitted 
ellipse, is close to 1.0, indicating a near-spherical morphology. 

The  TEM  micrograph  and  the  resulting  particle  size  distribution  (Fig.  1)  show  two  well-
separated particle populations: one at 80 nm and a second one of smaller particles around 
40 nm. The population of smaller particles accounts for about 30 % of the total number of 
particles.  The  certification  exercise  on  ERM-FD101b  was  focused  on  the  80  nm  particle 
population. 

Table 1: Information on Klebosol 30R50 starting material provided by the manufacturer 

Property 

Specifications / Observations 

Batch identification 

Appearance 

Nominal particle diameter 

Nominal SiO2 concentration 

Specific surface area 

Free alkalinity as Na2O 

pH (at 25 °C) 

Suspension density (at 20 °C) 

19019/L1 

Milky turbid 

80 nm 

300 g/kg 

40 m2/g to 60 m2/g 

≤ 0.2 g/kg 

8.5 to 9.5 

1.2 g/cm3 

15 

 
 
Fig 1 TEM micrograph and number-based area-equivalent PSD of Klebosol 30R50 

silica nanoparticles (MVA Scientific Consultants, Duluth, US) 

3.2  Processing 

The Klebosol 30R50 starting material was diluted to a target nominal SiO2 mass fraction of 
2.5 g/kg,  by  the  addition  of  purified  water  (resistivity  18.2 MW
·cm  at  25 °C)  produced  by  an 
Elix  35  water  purification  system  (Merck  Millipore,  Molsheim,  FR).  The  diluted  Klebosol 
30R50  was  prepared  by  addition  of  178  g  of  the  initial  starting  material  into  21822 g  of 
purified water. The batch was vigorously hand shaken and rolled several times, flushed with 
Ar, and left overnight. 

On the following day, before preparing the final batch, the suspension was transferred to a 
new clean bottle. In ensuring that potentially settled contamination was left behind, the lower 
end  of  the  transfer  tube  was  kept  about  5  cm  away  from  the  bottom  of  the  bottle.  During 
siphoning, special care was taken to minimise turbulence. Multiple samples were taken from 
both the originally prepared suspension, the fraction that was left behind in the original bottle 
and  from  the  final  batch.  Using  DLS,  no  significant  differences  were  found  between  the 
different  fractions.  Finally,  the  pH  of  the  final  batch  was  adjusted  to  pH  9  by  adding 
0.5 M NaOH. The solution was then flushed with Ar. 

Pre-scored 10 mL amber glass ampoules (Nederlandse Ampullenfabriek B.V., Nijmegen, NL) 
were chosen to provide a rugged and gas tight containment for the colloidal silica samples. 
Before filling, the ampoules were opened, rinsed with purified water and dried in an oven. 

The  ampoules  were  loaded  to  the  Rota  ampouling  machine  R  910  PA  (Rota,  Wehr,  DE). 
Every  ampoule  was  flushed  with  Ar  gas  immediately  before  filling  with  about  9  mL  of 
suspension.  The  suspension  in  the  supply  bottle  was  continuously  stirred  with  a  magnetic 
stirrer throughout the process of filling the ampoules. Immediately after filling, the ampoules 
were again flushed with Ar and closed. In total 1941 ampoules were produced. 

16 

 
 
 
 
 
4  Homogeneity 

A key requirement for any reference material (RM) produced as a batch of different units is 
the equivalence between the various units. In this respect, it is relevant whether the variation 
between units is significant compared to the uncertainty of the certified value. In contrast to 
that, it is not relevant if this variation between units is significant compared to the analytical 
variation.  Consequently,  ISO  Guide  34  [1]  requires  RM  producers  to  quantify  the  between-
unit variation. This aspect is covered in between-unit homogeneity studies. 

The within-unit inhomogeneity does not influence the uncertainty of the certified value when 
the minimum sample intake is respected, but determines the minimum size of an aliquot that 
is  representative  for  the  whole  unit.  Quantification  of  within-unit  heterogeneity  is  therefore 
necessary to determine the minimum sample intake. 

4.1  Between-unit homogeneity 

The between-unit homogeneity was evaluated to ensure that the certified values of the CRM 
are valid for all units of the material, within the stated uncertainty. 

The number of selected units corresponds to approximately the cubic root of the total number 
of the produced units. 15 units, selected using a random stratified sampling scheme covering 
the  whole  batch,  were  analysed  by  DLS  and  CLS  to  test  the  between-unit  homogeneity. 
From each of the 15 units, two independent sub-samples (aliquots) were taken and analysed 
by means of DLS (cumulants method) following the in-house validated method at a sample 
temperature of 25 °C. In addition to the DLS measurements, the 15 units were also analysed 
in  triplicate  by  means  of  line-start  CLS  (turbidimetry). The  temperature  of the  CLS  gradient 
was about 29 °C. 

The  DLS  and  CLS  measurements  were  performed  under  repeatability  conditions,  and  in  a 
randomised manner to be able to separate a potential analytical drift from a potential trend in 
the  filling  sequence.  Regression  analyses  were  performed  on  the  DLS  and  CLS  data  to 
evaluate potential trends in the analytical sequence as well as trends in the filling sequence. 
Analytical trends were visible and were linearly corrected in some of the CLS and DLS data 
sets. No trend on the filling was observed. 

Quantification  of  between-unit  inhomogeneity  was  accomplished  using  analysis  of  variance 
(ANOVA),  which  can  separate  the  between-unit  variation  (sbb)  from  the  within-unit  variation 
(swb).  The  latter  is  equivalent  to  the  method  repeatability  if  the  individual  samples  are 
representative for the whole unit. Evaluation by ANOVA requires unit means which follow at 
least a unimodal distribution and results for each unit that follow unimodal distributions with 
approximately  the  same  standard  deviations.  From  the  obtained  histograms  and  normal 
probability plots, it was concluded that all data followed a normal and unimodal distribution. 
Minor  deviations  from  unimodality  of  the  individual  values  do  not  significantly  affect  the 
estimate of between-unit standard deviations.  

Data  were  checked  and  scrutinised  for  single  and  double  outliers  by  applying  the  Grubbs’ 
test at a confidence level of 99 %. For both the DLS and CLS results, statistical outliers were 
neither detected for data grouped according to unit means, nor for data grouped according to 
individual results. 

One has to bear in mind that sbb and swb are estimates of the true standard deviations and 
are  therefore  subject  to  random  fluctuations.  Therefore,  the  mean  square  between  groups 
(MSbetween)  can  be  smaller  than  the  mean  squares  within  groups  (MSwithin),  resulting  in 
negative  arguments  under  the  square  root  used  for  the  estimation  of  the  between-unit 
variation,  whereas  the  true  variation  cannot  be  <  0.  In  this  case,  u*
bb,  the  maximum 
inhomogeneity that could be hidden by method repeatability, was calculated as described by 
Linsinger  et  al.  [29].  u*
bb  is  comparable  to  the  limit  of  detection  of  an  analytical  method, 

17 

 
yielding the maximum inhomogeneity that might be undetected by the given study setup. The 
larger value of sbb or u*
bb was used as uncertainty contribution for homogeneity, ubb. 
Method  repeatability  (swb,rel),  between–unit  standard  deviation  (sbb,rel)  and  u*
bb,rel  were 
calculated in accordance to ISO Guide 35 [2]. The results of the evaluation of the between-
unit variation are summarised in Table 2.  

s

wb,

rel

=

MS

within

y 

MS

between

MS

within

s

bb,

rel

=

u*
bb,

rel

=

n
y

4

y

MS
within
n

2

ν
MSwithin

Equation 1 

Equation 2 

Equation 3 

MSwithin 

mean of squares within a unit from an ANOVA 

MSbetween  mean of squares between-unit from an ANOVA 

y  

n 

ν

MSwithin

mean of all results of the homogeneity study 

mean number of replicates per unit 

degrees of freedom of MSwithin 

Table 2: Results of the homogeneity study for ERM-FD101b 

PSA method 
DLS 1) 

CLS 2) 

swb,rel 

[%] 

0.27 

0.65 

sbb,rel 

[%] 

0.10 

0.49 

u*

bb,rel 

[%] 

0.12 

0.19 

ubb,rel 

[%] 

0.12 

0.49 

1) Scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method) 
2) Light extinction-weighted modal Stokes particle diameter (turbidimetry) 

The  homogeneity  study  showed  no  outlying  unit  means  or  trends  in  the  filling  sequence. 
Therefore the between-unit standard deviation, sbb,rel, can be used as estimate of ubb. As u*
bb 
sets  the  limits  of  the  study  to  detect  inhomogeneity,  the  larger  value  of  sbb,rel  and  u*
bb,rel  is 
adopted as uncertainty contribution to account for potential inhomogeneity. 

4.2  Within-unit homogeneity and minimum sample intake 

The  within-unit  homogeneity  is  closely  correlated  to  the  minimum  sample  intake.  The 
minimum  sample  intake  is  the  minimum  amount  of  sample  that  is  representative  for  the 
whole unit and thus can be used in an analysis. Sample sizes equal to or above the minimum 
sample intake guarantee the certified value within its stated uncertainty.  

The minimum sample intake was determined from the results of the characterisation study, 
using  the  method  information  supplied  by  the  participants.  The  smallest  sample  intake  that 

18 

 
 
-
 
 
 
 
 
 
still  yielded  results  with  acceptable  accuracy  to  be  included  in  the  respective  studies  was 
taken  as  minimum  sample  intake.  Using  the  data  from  Annex  D  (Tables  D.1  to  D.5),  the 
following minimum sample intakes are derived: 

•  CLS (turbidimetry): 100 m L 
•  CLS (refractometry): 340 m L 
•  DLS: 100 m L 
•  EM: 2.5 m L of an as-received aliquot and analysis of at least 250 discrete particles 
•  PTA: 1 m L and at least 500 tracks 
•  SAXS: 20 m L 

19 

 
 
 
 
5  Stability 

Time  and  temperature  were  regarded  as  the  most  relevant  influences  on  stability  of  the 
material. The  influence of  visible  radiation  was  minimised  by  the  choice  of the  containment 
(amber glass ampoules) which eliminates most of the incoming light. 

Stability testing is necessary to establish conditions for storage (long-term stability) as well as 
conditions for dispatch to the customers (short-term stability). During transport, especially in 
summer time, temperatures up to 60 °C may be reached and stability under these conditions 
must be demonstrated if transport at ambient temperature will be applied. 

The  stability  studies  have  been  carried  out  using  an  isochronous  design  [30].  In  that 
approach,  samples  are  stored  for  a  certain  time  at  different  temperature  conditions. 
Systematically,  more  samples  are  moved  to  conditions  where  further  degradation  can  be 
assumed to be negligible (reference conditions), effectively "freezing" the degradation status 
of  the  material.  At  the  end  of  the  isochronous  storage,  the  samples  are  analysed 
simultaneously  under  repeatability  conditions.  Analysis  of  the  material  (after  various 
exposure  times  and  temperatures)  under  repeatability  conditions  greatly  improves  the 
sensitivity of the stability tests. 

5.1  Short-term stability study 

For  the  short-term stability  study,  samples  were stored  at 4  °C  and  60 °C for  0,  1,  2  and  4 
weeks (at each temperature). The reference temperature was set to 18 °C. For the storage 
temperatures 4 °C and 60 °C, 4 units were selected per storage time point. For the reference 
temperature of 18 °C, a total of 4 units were taken. All units were selected from the produced 
batch  using  a  random  stratified  sampling  scheme.  From  each  unit,  two  aliquots  were 
measured by DLS (cumulants method, sample temperature of 25 °C) and line-start CLS (disc 
centrifuge, temperature of the CLS gradient about 29 °C). Each DLS aliquot was measured 
three  times  in  a  consecutive  manner.  Measurements  were  equally  spread  over  two 
consecutive  days,  the  first  day  the  isochronous  study  at  4  °C,  the  second  day  the 
isochronous  study  at  60  °C.  All  measurements were  performed  in  a randomised  manner to 
be able to separate a potential analytical drift from a trend over storage time. 

Regression analyses were performed on the DLS and CLS data to evaluate potential trends 
in the analytical sequence. Analytical drift was visible and was linearly corrected in both CLS 
and  DLS  data  sets.  After  having  corrected  the  CLS  and  DLS  results  for  the  analytical  drift, 
data  were  pooled  according  to  the  storage  temperatures  4 °C  and  60 °C.  The  new  sets  of 
results were then evaluated individually for each temperature. The results were screened for 
outliers  using  the  single  and  double  Grubbs'  tests  on  99 %  confidence  levels.  No  outlying 
individual  results  were  found  (Table  3).  The  individual  DLS  and  CLS  measurement  results 
are shown in Fig.B1 and Fig.B2 of Annex B. 

Furthermore,  the  data  were  plotted  against  storage  time,  and  regression  lines  of  particle 
diameter versus time were calculated. The slopes of the regression lines were then tested for 
statistical significance (decrease/increase due to shipping conditions). For the DLS and CLS 
sets of results at 60 °C storage temperature, the slope of the regression lines were found to 
be significantly different from zero at a confidence level of 95 %. The values of both slopes 
(0.18 nm/week  for  DLS  and  −0.18 nm/week  for  CLS)  indicate  statistically  significant 
degradation. However, considering a shipping period of 1 week and the fact that the sample 
will not be exposed for more than 1 week at 60°C, and noting that the measured slopes are 
small  and  contradicting  each  other,  the  degradation  is  considered  technically  negligible. 
(During the production of a similar monodisperse, colloidal silica CRM, ERM-FD100, slopes 
of the regression lines were also found to be statistically significant at a confidence level of 

20 

 
 
95  %,  but  technically  negligible  [31].)  Nevertheless,  potential  degradation  over  one  week 
according to the measured slopes was included in the uncertainty value. 

Table 3: Results of the short-term stability tests 

Number of individual 
outlying results 

Significance of the trend on a 95 % 
confidence level 

PSA method 

4 °C 

60 °C 

DLS 1) 

CLS 2) 

None 

None 

None 

None 

4 °C 

No 

No 

60 °C 

Yes 

Yes 

1) Scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method) 
2) Light extinction-weighted modal Stokes particle diameter (turbidimetry) 

During the production of a similar colloidal silica CRM, ERM-FD304 [32], it was observed that 
freezing of the suspension significantly affected the particle size distribution measured after 
thawing. Therefore, ERM-FD101b must be protected against freezing. 

Supported by the experimental data and taking into account a maximum dispatch period of 
one  week,  it  can  be  concluded  that  the  material  can  be  safely  shipped  under  ambient 
conditions. Hence, cooled transportation is not required. 

5.2  Long-term stability study 

For  the  long-term  stability  study,  samples  were  stored  at  18 °C  for  0,  4,  8  and  12  months. 
The reference temperature was set to 4°C. A total of 4 units per storage time were selected 
using a random stratified sampling scheme. From each unit, two aliquots were measured by 
DLS  (cumulants  method,  sample  temperature  of  25 °C)  and  line-start  CLS  (disc  centrifuge, 
temperature of the CLS gradient about 29 °C). Each DLS aliquot was measured three times 
in  a  consecutive  manner.  Due  to  the  high  number  of  units  and  the  relatively  long  CLS 
measurement  time,  measurements  could  not  be  performed  under  strict  repeatability 
conditions. Instead, measurements were equally spread over two different days, with the first 
replicates  analysed  on  day  1  and  the  second  replicates  analysed  on  day  2.  Across  each 
measurement  sequence,  samples  were  randomised  with  respect  to  storage  time  and 
temperature. This approach allowed separating a potential analytical drift from a trend over 
storage time. 

The particle size results obtained by CLS revealed a clear systematic trend throughout each 
measurement  day:  the  modal  value  is  increasing  throughout  the  analysis  sequence.  A 
possible  reason  was  that  a  fraction  of  the  particles  from  the  previous  measurement  (e.g. 
particles from  the  40 nm  population)  had  not  yet  reached  the  detector  before  the  launch of 
the next measurement. No analytical drift was observed within the DLS sets of results. 

After  having  corrected  the  CLS  results  for  the  analytical  drift,  data  from  both  testing  days 
were  pooled  according  to  the  storage  temperature  and  time.  The  new  sets  of  results  were 
then  screened  for  outliers  using  the  single  and  double  Grubbs'  test.  No  outlying  individual 
results were found.  

The  individual  DLS  and  CLS  measurement  results  of  the  long-term  stability  measurements 
are  shown  in  Fig.C1  and  Fig.C2  of  Annex  C.  The  results  of  the statistical  evaluation  of  the 
long-term stability study are summarised in Table 4. 

21 

 
 
 
 
Furthermore,  the  data  were  plotted  against  storage  time  and  linear  regression  lines  of 
particle diameter versus time were calculated. The slope of the regression lines was tested 
for  statistical  significance  (decrease/increase  due  to  storage  conditions).  For  both  CLS  and 
DLS  sets  of  results,  the  slopes  of  the  regression  lines  were  not  significantly  different  from 
zero (on a 95 % confidence level) for a storage temperature of 18 °C. 

No  technically  unexplained  outliers  were  observed  and  none  of  the  trends  was  statistically 
significant  on  a  95  %  confidence  level  for  any  of  the  temperatures.  The  material  can 
therefore be stored at 18 °C. 

Table 4: Results of the long-term stability tests 

Number of individual 

Significance of the trend on a 

PSA method 

outlying results 

95 % confidence level 

DLS 1) 

CLS 2) 

None 

None 

No 

No 

1) Scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method) 
2) Light extinction-weighted modal Stokes particle diameter (turbidimetry) 

5.3  Estimation of uncertainties 

Due  to  the  intrinsic  variation  of  measurement  results,  no  study  can  rule  out  degradation  of 
materials completely, even in the absence of statistically significant trends. This means, even 
under ideal conditions, the outcome of a stability study can only be "degradation is 0 ± x % 
per  given  unit  of  time".  It  is  therefore  necessary  to  quantify  the  potential  degradation  that 
could be hidden by the method repeatability, i.e. to estimate the uncertainty of stability. The 
corresponding  uncertainties  of  stability  during  dispatch  and  storage  were  estimated  as 
described  in  [33]  for  each  measurand.  For  this  approach,  the  uncertainty  of  the  linear 
regression  line  with  a  slope  of  zero  is  calculated.  The  uncertainty  contributions  usts,rel  and 
ults,rel are calculated as the product of the chosen transport time/shelf life and the uncertainty 
of the regression lines as: 

u

sts

,

rel

=

u

lts

,

rel

=

RSD
(
t

i

∑

RSD
(
t

i

∑

t

tt

t

sl

)

2

t

)

2

t

Equation 4 

Equation 5 

RSD 

relative standard deviation of all results of the stability study 

ti 

t  

ttt 

tsl 

time elapsed at time point i 

mean of all ti 

chosen transport time (1 week at 60 ºC) 

chosen shelf life (24 months at 18 ºC) 

For  the  short-term  stability  studies,  an  uncertainty  component,  udeg,  corresponding  with  the 
observed  statistically  significant  degradation  trends,  needs  to  be  combined  with  the  above 
uncertainties. This contribution is calculated as: 

22 

 
 
(cid:215)
-
 
(cid:215)
-
 
 
=

u

deg

sltb
3

b 

slope of the regression line of the stability data 

Equation 6 

The following uncertainties were estimated: 

•  usts,  the  uncertainty  of  degradation  during  dispatch.  This  was  estimated  from  the 
60 °C  studies.  The  uncertainty  describes  the  possible  change  during  a  dispatch  at 
60 °C lasting for one week, which can be considered to be a realistic transport time. 
•  ults, the stability during storage. This uncertainty contribution was estimated from the 
18 °C  study. The  uncertainty  contribution  describes  the  possible  degradation  during 
48 months storage at 18 °C. 

The results of these evaluations are summarised in Table 5. 

Table 5: Uncertainties of stability during dispatch and storage. usts,rel was calculated for a 
temperature  of  60 °C  and  a  period  of  one  week;  ults,rel  was  calculated  for  a  storage 
temperature of 18 °C and a period of four years. 

PSA method 

DLS 1) 

CLS 2) 

usts,rel [%] 

ults,rel [%] 

0.12 

0.14 

0.98 

1.10 

1) Scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method) 
2) Light extinction-weighted modal Stokes particle diameter (turbidimetry) 

After  the  certification  campaign,  the  released  CRM  will  be  subjected  to  the  JRC's  regular 
stability monitoring programme to control and evidence its further stability. 

23 

 
(cid:215)
 
 
 
 
 
6  Characterisation 

The material characterisation is the process of determining the property values of a reference 
material. This process was based on a series of interlaboratory comparisons (ILCs) of expert 
laboratories. For each of the method-defined equivalent particle diameters an ILC was set up 
between  laboratories  that  applied  their  measurement  procedures,  but  with  respect  for  the 
standardised  elements  of  the  method-defined  measurand.  Crucial  in  this  characterisation 
approach  is  that  the  measurements  must  be  performed  under  intermediate  precision  (on 
different  days)  and  reproducibility  conditions  and  that  the  results  from  different  laboratories 
are independent. This approach aims at randomisation of laboratory bias, which reduces the 
combined uncertainty. 

6.1  Selection of participants 

36  laboratories  were  selected  based  on  criteria  that  comprised  both  technical  competence 
and quality management aspects. Each participant was required to operate a quality system 
and to deliver documented evidence of its laboratory proficiency in the field of size analysis 
of  nanoparticles.  Having  a  formal  accreditation  was  not  mandatory,  but  meeting  the 
requirements of ISO/IEC 17025 [3] was obligatory. Where measurements are covered by the 
scope  of  accreditation,  the  corresponding  accreditation  number  is  stated  in  the  list  of 
participants (Section 2). 

6.2  Study setup 

Each  laboratory  received  three  units  of  the  candidate  CRM  together  with  a  detailed 
measurement protocol. This protocol included a description of the measurement scheme as 
well as guidelines for sample handling and relevant measurement method parameters. The 
involved SAXS, EM, and CLS laboratories were requested to provide six independent results 
(two  replicates  per  unit).  The  participating  DLS  and  PTA  laboratories  were  requested  to 
provide nine independent results (three replicates/aliquots per unit) and each aliquot had to 
be  consecutively  measured  three  and  five  times,  respectively.  The  units  for  material 
characterisation were selected using a random stratified sampling scheme and covered the 
whole  batch.  At  each  laboratory,  the  sample  preparations  and  measurements  had  to  be 
spread over at least three different days to ensure intermediate precision conditions. 

Blinded CRMs were sent as quality control (QC) samples along with the candidate CRMs to 
allow  independent  assessment  of  method  trueness.  The  following  quality  control  samples 
were used: 

•  ERM-FD100 (colloidal silica, JRC) for SAXS and EM methods; 
•  ERM-FD304 (colloidal silica,JRC) for DLS and CLS (turbidimetry) methods; 
•  Nanosphere Size Standard 3080A (polystyrene latex, Thermo Scientific) for PTA; 
•  Nanosphere  Size  Standard  3100A  (polystyrene  latex,  Thermo  Scientific)  for  CLS 

(refractometry). 

The  results  of the  QC  samples  were  used  to  support  the  evaluation  of  the  characterisation 
results, i.e. to ensure reliability of the results obtained on the candidate CRM. 

Laboratories were also requested to give realistic estimations of the expanded uncertainties 
of the mean value of the replicate results. No approach for the estimation was prescribed, i.e. 
top-down and bottom-up or combinations of both were regarded as equally valid uncertainty 
estimation procedures. 

24 

 
6.3  Methods used 

6.3.1  Centrifugal liquid sedimentation 
ERM-FD101b  was  characterised  by  means  of  CLS  with  different  detection  systems  (i.e. 
turbidimetry and refractometry) and geometry (i.e. disc and cuvette type). Instruments were 
operated either in the line-start mode or in the so-called homogeneous mode. All laboratories 
analysed  ERM-FD101b  as-received  and  applied  the  general  guidelines  as  stipulated  in 
documentary standards ISO 13318-1 [8] and ISO 13318-2 [9]. These documentary standards 
include  both  the  line-start  and  homogeneous  CLS  methods.  Most  instruments  (in  particular 
the  disc-based  instruments)  do  not  permit  imposing  a  particular  sample  temperature. 
Therefore, the measurement protocol did not specify the measurement temperature. Instead, 
laboratories were requested to report the sample temperature, as measured during the test, 
or immediately after the end of the test. 

The  effective  particle  density  of  the test material  (ERM-FD101b)  is  needed  to  calculate  the 
measured  sedimentation  times  to  Stokes  diameters.  During  previous  studies,  the  effective 
density of the silica nanoparticles of the starting material Klebosol 30R50 was determined in 
situ  by  means  of  an  isopycnic  sedimentation  approach.  The  resulting  effective  particle 
density  was  found  to  be  2.03 g/cm3  ±  0.06 g/cm3  [34].  Based  on  these  isopycnic 
sedimentation  tests  on  similar  silica  sols  and  combining  this  with  other  "multiple-velocity" 
sedimentation approaches, the density of the ERM-FD101b silica nanoparticles is estimated 
to  be  2.0 g/cm3  with  an  expanded  uncertainty  of  0.1 g/cm3  (rectangular  probability 
distribution). This density value is considered SI-traceable because: 

- it is determined by combining results from multiple, independent measurement methods; 

-  the  uncertainty  of  the  results  of  the  individual  measurement  methods  mainly  depends  on 
the  gravimetric  preparation  of  suspensions,  which  is  done  using  SI-traceably  calibrated 
balances. (Other parameters in the measurement equations are the sedimentation time and, 
indirectly, the measurement temperature, via the viscosity values of the suspensions.) 

An overview of the instrument specifications and measurement conditions is given in Table 
D.1 of Annex D. The laboratory codes (e.g., L2) are random numbers and do not correspond 
to the order of laboratories listed in Section 2. The information in this annex is presented as 
reported by the ILC participants. 

6.3.2  Dynamic light scattering 
The characterisation of ERM-FD101b by DLS was performed in terms of the scattered light 
intensity-weighted mean size of the particle size distribution. 

Laboratories  were  asked  to  perform  sample  handling,  preparation  and  measurements 
according  to  a  protocol  that  followed  the  documentary  standards  ISO  13321  [15]  and  ISO 
22412  [16]  where  possible  and  that  provided  additional  guidelines  where  necessary. 
Measurements  on  the  monomodal  colloidal  silica  QC  sample  (ERM-FD304)  had  to  be 
performed  using  the  cumulants  method.  All  measurements  were  performed  on  samples  in 
the  as-received  state.  The  measurement  protocol  recommended  the  use  of  optical-quality 
glass  (e.g.,  quartz)  cuvettes,  but  disposable  plastic  cuvettes  were  acceptable  too. 
Furthermore,  the  protocol  required  measurements  to  be  performed  at  25  °C  ±  0.3 °C,  an 
equilibration time of 120 s, a viscosity (at 25 °C) of the dispersing medium of 0.8872 mPa.s 
and  a  refractive  index  (at  25 °C)  of  the  dispersing  medium  of  1.330.  From  each  ampoule, 
three  aliquots  had  to  be  taken  and  each  aliquot  had  to  be  measured  at  least  three  times 
under repeatability conditions.  

For the ERM-FD101b samples, the majority of the laboratories reported both harmonic mean 
values  according  to  the  cumulants  method,  as  well  as  mean  values  obtained  with  one  of 
several  different  (NNLS,  CONTIN)  distribution  calculation  algorithms.  The  use  of  PSD  data 
evaluation algorithms is not covered by the documentary standards ISO 13321 [15] and ISO 

25 

 
22412  [16]  valid  at  the time  of  the  characterisation  exercise. The  instrument  manufacturers 
follow different approaches to deconvolute the raw data, and to calculate the reported mean 
values  from  the  discrete  (binned)  size  distributions.  It  proved  difficult  to  group  the  datasets 
obtained  with  distribution  calculation  algorithms  according  to  the  kind  of  reported  average 
value  (e.g.,  arithmetic  mean,  harmonic  mean,  geometric  mean,  modal  (peak)  value)  [35]. 
However, it was noted that the main peak of the ERM-FD101b PSD is sufficiently narrow and 
symmetric to limit the theoretical differences between the peak averaging approaches used. 
Therefore, it was decided to group all results obtained with distribution calculation algorithms 
and  to  assign  one  particle  size  value,  representative  for  the  group  of  commonly  used  DLS 
distribution  calculation  algorithms.  This  has  consequences  for  the  estimation  of  the 
uncertainty (see 6.4.2). 

An  overview  of  the  instrument  specifications  and  measurement  conditions  is  given  in 
Table D.2 of Annex D. The laboratory code consists of a number assigned to each laboratory 
(e.g.,  L2)  and  abbreviation  of  the  distribution  calculation  algorithm  used  (e.g.,  NNLS, 
CONTIN).  

6.3.3  Electron microscopy 
SEMs and TEMs were used to characterise ERM-FD101b in terms of area-equivalent circle 
diameters.  It  was  asked  in  the  technical  specifications  to  count  and  measure  only  particles 
with  an  equivalent  diameter  larger  than  60  nm.  For  the  obtained  number-based  PSDs,  the 
modal and median values were determined. To minimise the risk of contamination during the 
preparation  of  EM  test  specimens,  participants  were  recommended  to  prepare  the  test 
specimens  in  a  clean  room  or  in  a  low  contamination  environment.  The  laboratories  were 
asked to analyse the acquired micrographs according to documentary standard ISO 13322-1 
[21].  For  each  prepared  test  specimen,  at  least  1000  discrete  (i.e.  non-touching)  particles 
had  to  be  measured.  During  the  course  of  the  study  this  requirement  was  relaxed:  it  was 
allowed to also count touching particles, if they were clearly non-overlapping.  

An overview of the instrument specifications and measurement conditions is given in Table 
D.3  of  Annex  D.  The  laboratory  code  consists  of  a  random  number  assigned  to  each 
laboratory (e.g., L2) and abbreviation of the type of EM instrument used (e.g., SEM, TEM).  

6.3.4  Particle tracking analysis 
The  intensity  of  the  light  scattered  by  the  particles  in  undiluted  ERM-FD101b  in  a  PTA 
instrument is too high and as a result individual particles cannot easily be resolved. As a rule 
of  thumb,  PTA  test  samples  should  contain  between  107  and  109  particles/mL.  Therefore, 
laboratories were asked to dilute the as-received material using purified water (resistivity of 
18.2 MW
.cm at 25°C and additionally filtered through a membrane with nominal pore size of 
0.1 m m) depending on the optical specifications of their instruments. To allow determination 
of  the  particle  concentration,  sample  loading  (and  dilution)  had  to  be  performed  using 
accurately calibrated micropipettes and/or syringes. Additional measurement conditions that 
were fixed in the test protocol were: measurement temperature (25 °C ± 0.5 °C), equilibration 
time between measurements (30 s), viscosity (0.8872 mPa.s at 25 °C) and refractive index of 
the  dispersion  liquid  (1.330  at  25 °C). The mode,  mean  and  median  values  of  the  number-
based PSDs had to be reported, as well as the particle concentration. 

An  overview  of  the  instrument  specifications  and  measurement  conditions  is  given  in 
Table D.4 of Annex D.  

6.3.5  Small-angle X-ray scattering 
ERM-FD101b  was  also  characterised  using  SAXS.  Measurements  were  performed  on  the 
as-received  material.  The  experimental  scattering  curves  were  corrected  for  background 
noise. The  data  were  analysed  both  using the Guinier  approximation  and  the  'model fitting' 
approach. 

26 

 
In the Guinier approximation, the initial parts (at low q-ranges) of the scattering curves were 
approached  with  linear  regression fits from  which  the  (volume)2-weighted  Guinier  radii  (RG) 
were  deduced.  These  results  were  then  converted  into  the  mean  equivalent  spherical 
diameter (D) following Equation 7. 

D =

2

5
R
G3

Equation 7 

In addition, PSDs were determined by applying the 'model fitting' approach to a broader part 
of the scattering curve [28].  

The laboratories were asked to perform the measurements at a temperature in the range of 
20 °C to 30 °C, preferably at 25 °C.  

An overview of the instrument specifications, measurement conditions and chosen models is 
given in Table D.5 of Annex D.  

6.4  Evaluation of results 

The  characterisation  campaign  resulted  in  a  total  of  55  independent  datasets.  Table  6 
summarises the measurands reported for each of the techniques and methods.  

Table 6: Measurands and number of independent datasets per technique 

Technique (method) 

Measurands 

Sets 

CLS (turbidimetry) 

CLS (refractometry) 

DLS (cumulants method) 

DLS (distribution calculation 
algorithms) 

EM  

PTA 

Light extinction-weighted modal Stokes diameter 
Mass-based modal Stokes diameter 

Scattered light intensity-weighted harmonic mean 
hydrodynamic diameter  
Scattered light intensity-weighted mean hydrodynamic 
diameter  

8 

2 

20 

14 

Number-based area-equivalent diameter (modal, mean) 

14 

Number-based hydrodynamic diameter (modal, 
arithmetic mean, median) 

SAXS (Guinier approximation) 

((Volume)2)-weighted mean diameter  

SAXS (model fitting) 

Number-, volume-, intensity-weighted modal diameter 

7 

5 

5 

All  individual  results  of  the  participants,  grouped  per  technique/method/measurand  are 
displayed in tabular and graphical form in Annex E. 

6.4.1  Technical evaluation 
The  obtained  data  were  first  checked  for  compliance  with  the  requested  measurement 
protocol  and  for  their  validity  based  on  technical  criteria.  The  following  criteria  were 
considered during the evaluation: 

•  appropriate validation of the measurement procedure; 
•  compliance  with  the  provided  measurement  protocol:  sample  preparations  and 

measurements performed on three days; 

27 

 
 
 
 
•  method  performance,  i.e.  agreement  of  the  measurement  results  with  the  assigned 
value of the QC sample following the procedure described in ERM Application Note 1 
[36]. 

Table 7 shows the datasets rejected as not technically valid based on the above criteria. 

Table  7:  Datasets  that  showed  non-compliances  with  the  analysis  protocol  and  technical 
specifications, and action taken 

Description of problem 

Action taken 

Lab-
code 
L9a 

L9b 

PSA 
method 
EM 

EM 

EM 

Results for QC sample ERM-FD100 did not agree with 
the certified value within the reported uncertainty 
Results for QC sample ERM-FD100 did not agree with 
the certified value within the reported uncertainty 

L29  Modal values for QCM not reported 

DLS 

L1 

DLS 

L12 

DLS 

L29 

Results for QC sample ERM-FD304 did not agree with 
the certified value within the reported uncertainty 
Results for QC sample ERM-FD304 did not agree with 
the certified value within the reported uncertainty 
Protocol not followed 

DLS 

L32 

Results for QC sample ERM-FD304 did not agree with 
the certified value within the reported uncertainty 

Data not used for 
evaluation 
Data not used for 
evaluation 
Data not used for 
evaluation 
Data not used for 
evaluation 
Data not used for 
evaluation 
Data not used for 
evaluation 
Data not used for 
evaluation 

Dynamic  light  scattering:  19  laboratories  were  involved  in  the  DLS  ILC  study.  One 
laboratory submitted 2 data sets. Three laboratories failed on the measurements of the QCM 
and  one  laboratory  did  not  follow  the  measurement  protocol.  In  total  16  data  sets  were 
accepted  for  the  cumulants  method,  and  11  data  sets  were  accepted  for  the  distribution 
calculation algorithms. 

The results of the technical evaluation of the DLS datasets are summarised in Annex E1. 

Centrifugal liquid  sedimentation:  10  laboratories  were  involved  in  the  CLS  ILC  study. Of 
the  10  laboratories,  4  participated  with  homogeneous  CLS  (ultracentrifugation  or  cuvette) 
methods. This group is further split into laboratories that used AUC with refractometry (2) and 
laboratories  that  performed  measurements  using  benchtop  ultracentrifuges  equipped  with 
turbidimetry  (2).  The  other  six  laboratories  applied  line-start  CLS  (disc  centrifugation) 
methods  with  turbidimetry.  Because  of  the  different  measurands,  datasets  originating  from 
CLS  instruments  with  turbidimetric  detection  systems  and  CLS  instruments  equipped  with 
refractometric detection (L18 and L28), were evaluated separately. 

The results of the technical evaluation of the CLS datasets are summarised in Annex E2. 

Electron  microscopy:  Ten  laboratories  participated  in  the  ILC  study  with  SEM  and  TEM 
methods. A total of 14 datasets were received. Three participants performed measurements 
using both SEM and TEM, and one participant used an SEM both in scanning mode and in 
transmission  (TSEM)  mode.  An  unpaired  two-tailed  Students'  t-test  showed  no  significant 
difference (significance level 0.05) between the mean values of the two instrument groups.  

The results of the technical evaluation of the EM datasets are summarised in Annex E3.  

Particle  tracking  analysis:  Seven  laboratories  participated  in  the  ILC  study  with  PTA. 
Laboratories  also  reported  the  absolute  particle  number  concentration.  However,  the 
concentration  results  varied  too  much  between  laboratories.  Therefore,  the  concentration 
data  were  considered  insufficiently  reliable  for  certification  purposes.  For  PTA,  the  as-

28 

 
 
 
received  material  was  too  concentrated  and  all  laboratories  therefore  applied  high  dilution 
factors (Annex Table D.4). Dilution does affect the ionic strength of the suspension and can 
thus  also  have  an  effect  on  the  colloidal  stability,  i.e.  increased  interaction  between 
nanoparticles  can  lead  trigger  the  formation  of  agglomerates.  While  no  excessive 
agglomeration  has  been  reported  by  the  laboratories,  the  presence  of  a  limited  number  of 
small  agglomerates  explains  the  slightly,  but  significantly,  higher  value  for  the  arithmetic 
mean particle size compared to the median and modal particle size measurands. 

The results of the technical evaluation of the PTA datasets are summarised in Annex E4. 

Small-angle  X-ray  scattering:  Five  laboratories  participated  in  the  SAXS  ILC  study.  Test 
samples were analysed as-received and the experimental scattering curves were evaluated 
following both the Guinier approximation and the model fitting approach. An overview of the 
q-ranges used for the Guinier approximation and of the imposed particle morphology models 
is given in Annex Table D.5-2 and Table D.5-3. The model fitting data were not requested by 
the JRC, but spontaneously reported by the participants, using one or several of the number-
based,  volume-based  and  intensity-based  types  of  PSD.  To  have  a  complete  set  of  five 
PSDs  of  the  same  distribution  type,  several  of  the  reported  average  values  were 
mathematically  transformed  at  the  JRC  from  one  to  the  missing  PSD.  This  was  possible 
using the straightforward relation between the modal (peak) value of the intensity-, volume- 
and number-based versions of the near-Gaussian shaped peak in the PSD. 

The results of the technical evaluation of the SAXS datasets are summarised Annex E5. 

6.4.2  Statistical evaluation 
The DLS, CLS (turbidimetry), EM, SAXS and PTA datasets that were accepted on the basis 
of  technical  criteria,  and  used  for  certification,  were  statistically  evaluated.  This  evaluation 
included testing for the normality of dataset means using kurtosis/skewness tests as well as 
normal  probability  plots,  and  testing  for  outlying  means  and  standard  deviations  (both  at 
99 %  confidence  level)  using  the  Grubbs'  and  Cochran  tests,  respectively.  Standard 
deviations  within  (swithin)  and  between  (sbetween)  laboratories  were  calculated  using  one-way 
ANOVA. The results of these evaluations are shown in Table 8.  

The CLS (refractometry) data sets were not evaluated for their normality and outlying means 
and  standard  deviations  because  only  two  datasets  were  available.  The  main  observations 
are:  

CLS: Statistical evaluation of the ERM-FD101b datasets flagged the variances of laboratory 
L5  and  L14  as  outliers  for  the  modal  Stokes  particle  diameter  by  CLS  (turbidimetry).  In 
essence,  outliers  of  variance  show  that  repeatability  varies  between  laboratories.  The 
heterogeneity  of  variances  prevents  pooling  of  all  individual  results,  so  the  evaluation  is 
based on the mean of laboratory means instead. In conclusion, outlying variances are not a 
reason for exclusion of data. 

DLS:  The  mean  value  of  the  mean  particle  diameter  by  DLS  (cumulants  method)  of 
laboratory L6 was detected as an outlier by the Grubbs' test. This mean value is lower than 
the  values  reported  by  the  other  laboratories.  However  the  difference  between  the  mean 
value of laboratory L6 and the others is covered by the measurement uncertainties. As there 
is no clear technical evidence that the results of laboratory L6 significantly deviate from the 
other results, the data is retained.  

SAXS: The variances of laboratories L25 and L35 were determined as outliers for the mean 
particle  diameter  by  SAXS  (Guinier  approximation).  For  the  model  fitting  approach,  all 
laboratories  have  an  outlying  variance  compared  to  the  very  low  variance  of  L22 
(synchrotron  radiation).  As  mentioned  before,  outlying  variances  are  not  a  reason  for 
exclusion of data.  

DLS and PTA: For DLS and PTA methods, the statistical evaluations by Cochran test were 
out  of  range,  pointing  to  the  difference  of  standard  deviations  between  the  laboratories. 

29 

 
However,  as  explained  before,  outlying  variances  are  not  a  sufficient  reason  for  data 
exclusion. 

Table  8  Statistical  evaluation  of  the  technically  accepted  datasets  for  ERM-FD101b  (p  = 
number of technically valid datasets). 

p 

Outliers 

Means 

8 
16 

None 
L6 

Varian-
ces 
L5,L14  
L5, L24 

PSA method 
CLS 1) 
DLS 2) 
(cumulants) 

DLS 3) 
(distribution calculation 
algorithms) 
EM 4) 
(modal) 
EM 5) 
(median) 
PTA 6) 
(modal) 
PTA 7) 
(median) 
PTA 8) 
(arithmetic mean) 
SAXS 9) 
(Guinier) 
SAXS 10) 
(model fit, number) 
SAXS 11) 
(model fit, volume) 
SAXS 12) 
(model fit, intensity) 

11 

None 

L3b 

11 

None 

None 

11 

None 

None 

7 

7 

7 

5 

5 

5 

5 

None 

None 

None 

None 

None 

L20 

None 

L25, L35 

None 

None 

None 

L21, L25, 
L26, L35 
L21, L25, 
L26, L35 
L25, L35 

Normally 
distributed 

Yes 
Yes (acc. 
skewness test) 
/ No (acc. 
kurtosis test), 
due to 1 
outlier 
Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Statistical parameters 

Mean 
[nm] 
86.7 
89.5 

s 
[nm] 
3.6 
1.9 

sbetween 
[nm] 
3.6 
1.9 

swithin 
[nm] 
0.6 
0.7 

93.1 

2.1 

2.1 

0.8 

83.7 

2.4 

2.3 

1.3 

83.5 

2.2 

2.1 

1.2 

81.8 

3.8 

3.7 

1.2 

82.2 

4.0 

4.0 

1.2 

86.5 

4.3 

4.3 

1.2 

86.7 

3.6 

3.5 

1.6 

80.9 

0.5 

0.5 

0.7 

Yes 

81.7 

0.6 

0.5 

0.6 

Yes 

82.5 

0.8 

0.8 

0.3 

1) light extinction-weighted modal Stokes diameter (turbidimetry);  
2) scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method); 
3) scattered light intensity-weighted mean hydrodynamic particle diameter (distribution calculation algorithms);  
4) number-weighted modal area-equivalent particle diameter;  
5) number-weighted median area-equivalent particle diameter;  
6) number-weighted modal hydrodynamic particle diameter;  
7) same as 6) but median;  
8) same as 6) but arithmetic mean;  
9) ((volume)2)-weighted mean particle diameter (Guinier approximation);  
10) number-weighted modal particle  
diameter (model fitting approach);  
11) same as 10) but volume-weighted; 12) same as 10) but scattered-X-ray intensity-weighted. 

The  uncertainty  related  to  the  characterisation  (uchar)  was  then  estimated  as  the  standard 
error of the means, i.e. s/(cid:214) p with s and p taken from Table 8. One exception was made for 
the  values  obtained  with  DLS  distribution  calculation  algorithms.  As  explained  in  section 
6.3.2,  it  is  not  straightforward  to  link  the  particle  size  results  obtained  with  different 
distribution calculation algorithms to a particular type of mean particle size. Therefore, rather 
than  calculating  uchar  as  the  standard  error  of  the  11  measured  values,  it  was  decided  to 
consider the range of reported values as a rectangular distribution (range = 6.0 nm), with the 

30 

 
 
 
midpoint of the interval being the expected value and the corresponding standard uncertainty 
equal to (range)/(2·(cid:214) 3). This approach provides the most neutral assessment of the reported 
data and assigns the same weight to each instrument and software represented in the set of 
accepted data. 

An overview of the estimated uncertainties for characterisation is shown in Table 9. 

Table 9: Uncertainty of characterisation for ERM-FD101b  

PSA method 
CLS 1) 
DLS 2) 
(cumulants) 
DLS 3) 
(distribution calculation algorithms) 
EM 4) 
(modal) 
EM 5) 
(median) 
PTA 6) 
(modal) 
PTA 7) 
(median) 
PTA 8) 
(arithmetic mean) 
SAXS 9) 
(Guinier) 
SAXS 10) 
(model fit, number) 
SAXS 11) 
(model fit, volume) 
SAXS 12) 
(model fit, intensity) 

p 

8 

16 

11 

11 

11 

7 

7 

7 

5 

5 

5 

5 

Mean [nm] 

s [nm] 

uchar [nm] 

86.7 

89.5 

3.6 

1.9 

92.5 13) 

6.0 13) 

83.7 

83.5 

81.8 

82.2 

86.5 

86.7 

80.9 

81.7 

82.5 

2.4 

2.2 

3.8 

4.0 

4.3 

3.6 

0.5 

0.6 

0.8 

1.3 

0.5 

1.7 

0.8 

0.7 

1.4 

1.7 

1.6 

1.6 

0.2 

0.3 

0.4 

1) light extinction-weighted modal Stokes diameter (with turbidimetry);  
2) scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method);  
3) scattered light intensity-weighted mean hydrodynamic particle diameter (distribution calculation algorithms);  
4) number-weighted modal area-equivalent particle diameter;  
5) as in 4) but median;  
6) number-weighted modal hydrodynamic equivalent particle diameter;  
7) as in 6) but median;  
8) as in 6) but arithmetic mean;  
9) ((volume)2)-weighted mean particle diameter (Guinier approximation);  
10) number-weighted modal particle diameter (model fitting approach);  
11) as in 10) but volume-weighted; 12) As in 10) but scattered-X-ray intensity-weighted;  
12) not 'mean' and 's' but 'midpoint of interval' and 'range' (maximum value – minimum value). 

31 

 
 
 
 
 
7  Value Assignment 

Certified, indicative and information values were assigned for ERM-FD101b. 

Certified  values  are  values  that  fulfil  the  highest  standards  of  accuracy.  Procedures  at  the 
JRC, Directorate F require generally pooling of not less than six datasets to assign certified 
values.  (An  exception  was  made  for  the  highly  reproducible  SAXS  data  analysed  with  the 
model fitting approach (uchar < 0.5 %). The low number of accepted data sets (5) is accounted 
for  by  applying  the  Welch-Satterthwaite  equation  when  expanding  the  corresponding 
combined measurement uncertainty.) Full uncertainty budgets in accordance with the 'Guide 
to the Expression of Uncertainty in Measurement' [4] were established. 

Indicative  values  are  values  where  either  the uncertainty  is  deemed  too large  or  where  too 
few  independent  datasets  were  available  to  allow  certification.  Uncertainties  are  evaluated 
according to the same rules as for certified values. 

Additional material information refers to values that were obtained in the course of the study. 
For example, results reported from only one or two laboratories or in cases where individual 
measurement uncertainty is high, would fall under this category. 

7.1  Certified values and their uncertainties 

The  unweighted  mean  of  the  means  of  the  accepted  CLS  (turbidimetry),  DLS,  PTA,  SAXS 
(model  fitting  approach)  and  EM  datasets,  as  shown  in  Table  8  were  assigned  as  certified 
values for the different measurands.  

The  assigned  uncertainties  consist  of  uncertainties  related  to  characterisation,  uchar 
(Section 6),  potential  between-unit  inhomogeneity,  ubb  (Section  4),  potential  degradation 
during transport (usts) and long-term storage, ults (Section 5). 

For  the  CLS  method,  an  additional  uncertainty  stems from  the  density  of  the  ERM-FD101b 
particles.  Furthermore,  as  the  majority  (6  out  of 8)  CLS  datasets  were  obtained  with  a  disc 
centrifuge  requiring  calibration,  and  since,  with  the  exception  of  one,  all  CLS  datasets 
obtained with calibration used PVC calibrants that were supplied by CPS Instruments, Inc., 
an uncertainty contribution corresponding with the accuracy of the assigned size and density 
values of these calibrants is also added to the overall uncertainty budget: 

- The uncertainty related to the effective particle density (ur ,CRM) was included, following the 
approach  in  a  previous  certification  exercise  [37].  As  described  in  section  6.3.1,  the  ERM-
FD101b  particle  density  is  r  = 2.0  g/cm3  ±  0.1  g/cm3.  Assuming  a  rectangular  probability 
distribution  (n p  =  ¥
),  ur   is  0.058 g/cm3.  To  calculate  from  this  value  the  corresponding 
uncertainty of the Stokes diameter requires calculation of the partial derivative of the Stokes' 
law  to  the  particle  density.  For  a  typical  set  of  sedimentation  times,  Stokes  diameters  and 
densities of PVC calibrants and ERM-FD101b, the conversion factor is 33 nm/(g/cm3). 
-  The  density  of  the  calibrant  particles  r cal = 1.385  g/cm3  has  an  expanded  uncertainty  of 
3.5 %  [38].  The  corresponding  standard  uncertainty  is  0.048 g/cm3.  To  calculate  from  this 
value the corresponding uncertainty of the Stokes diameter, ur ,cal, requires calculation of the 
partial  derivative  of  the  Stokes'  law  to  the  calibrant  particle  density.  For  a  typical  set  of 
sedimentation  times,  Stokes  diameters  and  densities  of  PVC  calibrants  and  ERM-FD101b, 
the conversion factor is 85 nm/(g/cm3). 

-  The  uncertainty  of  the  Stokes  diameters  assigned  to  the  calibrants  is  5 %  [38].  For  an 
average  calibrant  size  of  250 nm,  the  corresponding  standard  uncertainty  is  6.3 nm.  To 
calculate from this value the corresponding uncertainty of the Stokes diameter, ud,cal, requires 
calculation  of  the  partial  derivative  of  the  Stokes'  law  to  the  calibrant  particle  size.  For  a 
typical  set  of  sedimentation  times,  Stokes  diameters  and  densities  of  PVC  calibrants  and 
ERM-FD101b, the conversion factor is 0.25 nm/nm.  

32 

 
The different contributions were combined to estimate the expanded, relative uncertainty of 
the certified value (UCRM) with a coverage factor k as: 

U

CRM,

rel

(cid:215)=
k

u

2
char,

rel

+

u

2
bb,

rel

+

u

2
sts,

l
re

+

u

2
lts,

l
re

+

u

2
ρ,

CRM,

rel

+

u

2
r

,

cal,

rel

+

u

2
cal,D,

rel

Equation 8 

•  uchar was estimated as described in Section 6 
•  ubb was estimated as described in Section 4 
•  usts was estimated as described in section 5 
•  ults was estimated as described in Section 5 
•  ur ,CRM was estimated as described in Section 7.1 (only for CLS) 
•  ur ,cal was estimated as described in Section 7.1 (only for CLS) 
•  uD,cal was estimated as described in Section 7.1 (only for CLS) 

Because  of  the  sufficient  numbers  of  the  degrees  of  freedom  of  the  different  uncertainty 
contributions, a coverage factor k = 2 was applied, to obtain the expanded uncertainties. 

The certified values and their uncertainties are summarised in Table 10. 

Table 10: Certified values and their uncertainties12) for ERM-FD101b  

UCRM,
rel 
[%] 

UCRM  
[nm] 

8.4 

2.5 

4.3 

2.7 

2.6 

4.0 

4.5 

4.3 

2.1 

2.1 

2.2 

8 

2.3 

4 

2.2 

2.2 

4 

4 

4 

1.7 

1.8 

1.8 

PSA method 

Certifie
d value 
[nm] 

uchar 
[%] 

ubb 
[%] 

usts 
[%] 

ults 
[%] 

ur

,CRM 
[%] 

ur
,cal 
[%] 

uD,cal 
[%] 

1.80 

n.a. 

n.a 

87 

82 

82 

87 

93 

n.a 

n.a 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

0.12 

0.98 

0.98 

2.38 

2.20 

0.14 

0.85 

1.74 

0.12 

0.12 

0.12 

0.12 

0.12 

0.98 

0.98 

0.12 

0.98 

1.88 

0.12 

0.12 

0.12 

0.12 

0.12 

1.10 

0.80 

0.54 

83.5 

83.7 

1.86 

1.46 

89.5 

0.49 

2.01 

0.98 

CLS 1) 
DLS 2) 
(cumulants) 
DLS 3) 
(distribution calculation 
algorithms) 
EM 4) 
(modal) 
EM 5) 
(median) 
PTA 6) 
(modal) 
PTA 7) 
(median) 
PTA 8) 
(arithmetic mean) 
SAXS 9) 
(model fit, number) 
SAXS 10) 
(model fit, volume) 
SAXS 11) 
(model fit, intensity) 
1) light extinction-weighted modal Stokes diameter (turbidimetry);  
2) scattered light intensity-weighted harmonic mean hydrodynamic particle diameter (cumulants method);  
3) scattered light intensity-weighted arithmetic mean hydrodynamic particle diameter;  
4) number-weighted modal area-equivalent particle diameter;  
5) number-weighted median area-equivalent particle diameter; 
6) number-weighted modal hydrodynamic particle diameter;  
7) same as 6) but median;  
8) same as 6) but arithmetic mean; 
9) number-weighted modal particle diameter (model fitting approach); 
10) as in 9) but volume-weighted; 11) as in 9) but scattered-X-ray intensity-weighted;  
11) expanded (k = 2) and rounded uncertainty 

80.9 

0.30 

81.7 

0.32 

82.5 

0.43 

0.12 

0.12 

0.98 

0.98 

0.12 

0.98 

0.12 

0.98 

0.12 

0.12 

0.12 

0.12 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

33 

 
 
r
r
r
r
r
r
the  given  study,  homogeneity  and  stability  experiments  were  conducted  under 
In 
repeatability conditions by means of DLS and CLS (turbidimetry) methods. While CLS has a 
superior resolution for detecting particles of different size and density, DLS is more sensitive 
in  detecting  agglomerates.  For  that  reason,  the  combined  knowledge  on  the  material 
homogeneity  and  stability  obtained  from  DLS  measurements  is  considered  sufficiently 
detailed to also use the resulting homogeneity and stability uncertainty contributions (i.e. ubb, 
usts and ults) as realistic contributions in the calculations for the uncertainties of the SAXS, EM 
and PTA measurands. 

7.2  Indicative values and their uncertainties 
Indicative values were assigned to the ((volume)2)-weighted mean particle diameter obtained 
by  SAXS  measurements  (Guinier  approximation).  Technically  valid  datasets  were  received 
from five different laboratories. The size data were obtained in a range of scattering vectors q 
varying between 0.025 nm-1 and 0.070 nm-1. For the size of the ERM-FD101b particles, this 
q-range  is  near  the  edge  of  validity  of  the  Guinier  approximation.  Therefore  the  resulting 
value was not certified but is provided as an indicative value instead.  

The uncertainty budget (Table 11) was set up in a similar way as for the certified values. The 
individual standard uncertainties were taken from the DLS homogeneity and stability studies. 
In contrast to the certified values, where a fixed coverage factor of k = 2 was used to expand 
the uncertainty, the combined uncertainty of the indicative value was multiplied with a t-factor 
extracted from the t-distribution table following calculation of the effective degrees of freedom 
according to the Welch-Satterthwaite equation [4].  

n

eff

=

n eff  
uc  

ui  

i  

4
)(
yu
c
4
N
yu
)(
∑
i
n

=

1

i

i

(Eq. 8) 

effective number of degrees of freedom 

combined uncertainty 

individual standard measurement uncertainties 

degrees of freedom corresponding to ui 

This  approach  is  required  if  the  standard  uncertainties  are  estimated  from  evaluations  with 
an  insufficient  number  of  degrees  of  freedom  to  obtain  the  required  95 %  confidence  level 
when  using  a  k = 2  coverage  factor.  For  the  (volume)2-weighted  mean  particle  diameter 
(SAXS, Guinier approximation) the main uncertainty contribution is uchar,rel. Since at the same 
time this  uncertainty  contribution  has  the  smallest  number  of  degrees  of freedom, n eff could 
be estimated by n char = 4. Hence the t-factor is 2.78 (for a 95% confidence level). 

Table 11: Indicative value and uncertainty for ERM-FD101b  

PSA 

Indicative value 

uchar,rel 

ubb,rel 

usts,rel 

ults,rel 

method 

SAXS1) 

[nm] 

87 

[%] 

[%] 

[%] 

[%] 

1.86 

0.12 

0.12 

0.98 

5.9 

UCRM,rel 
[%] 

UCRM 

[nm] 

6 2) 

1) ((Volume)2)-weighted mean particle diameter (Guinier approximation) valid in a temperature range of [25 ± 5] °C 
2) with coverage factor t = 2.78 (for a n eff = 4, calculated from Equation 9) 

34 

 
 
 
 
n
 
 
 
7.3  Additional material information 

A number of particle properties were also assessed but only at one or two laboratories. The 
evaluated results are listed as additional material information values. These values must be 
regarded as informative only on the general properties of the material and cannot be, in any 
case, used as certified or indicative value. 

7.3.1  Centrifugal liquid sedimentation (refractometry) 
For  the  mass-weighted  modal  Stokes  diameter  obtained  by  CLS  (refractometry)  two  well-
matching sets of data were received. The average value of both laboratories is provided as 
an information value. 

Table 12: Additional material information value obtained by CLS (refractometry) 

Particle sizing method 
CLS 1) 

Information value [nm] 

84 

1)  Mass-weighted  modal  Stokes  particle  diameter  (refractometry)  at  25  °C  ±  1  °C,  using  an  effective  particle 
density of 2.0 g/cm3 

7.3.2  Field flow fractionation 
The  JRC,  Directorate  F  analysed  ERM-FD101b  using  an  asymmetrical  flow  field-flow 
fractionation  (AF4)  instrument  which  was  coupled  to  a  multi-angle  light  scattering  (MALS) 
detector.  Instrumental  details  are  listed  in  Table  D.6  of  Annex  D.  This  set-up  was  used  to 
measure particle size in two different ways.  

1) At any given elution time, the particles entering the MALS induce a scattering intensity that 
depends on the scattering angle. The MALS detectors, placed at 21 different angles, capture 
the angular dependence of the scattering intensity. This dependency was used to calculate 
radii of gyration, which were transformed into hydrodynamic radii or diameters. A fractogram 
representative for ERM-FD101b is given in Fig. 2. 

0

0.14

]

V

[
e
s
n
o
p
s
e
r

°
2
9
S
L
L
A
M

0.12

0.10

0.08

0.06

0.04

0.02

0.00

 Retention time [min]

10

20

30

40

300

270

240

210

180

150

120

90

60

30

0

]

m
n
[

i

i

r
e
t
e
m
a
d
c
m
a
n
y
d
o
r
d
y
H

0

10

20

30

40

Retention time [min]

Fig 2 Light scattering intensity fractogram (solid line) obtained using AF4-MALS (92º signal). 
The  dotted  curve  indicates  the  hydrodynamic  diameter  of  the  particles  eluting  after  the 
indicated retention time. 

35 

 
 
 
 
 
 
 
 
2)  Calibration  curves  (retention  time  versus  particle  size)  were  established  using  different 
polystyrene latex (PSL) size standards with narrow size distribution and using the maxima of 
the analytical peaks as given by the 92º angle signal of the MALS detector. Both a linear as 
well as a double-logarithmic calibration curve were established, the latter providing a better 
correlation coefficient (R2 = 0.995) than the former (R2 = 0.989). The corresponding results 
(averages of 12 replicates) are given in Table 13. 

Table 13: Additional material information values for ERM-FD101b obtained by AF4 

Particle sizing method 
AF4-MALS (92°) 1) 
AF4-MALS (92°) 2) 

Information value [nm] 

86 

83 

1)  Scattered light intensity-weighted  modal  hydrodynamic  diameter  at   35  °C  ±  1  °C, calculated  via  a  calibration 
curve using a linear scale 
2) Same as 1) but calculated via calibration curve using a double logarithmic scale 

7.3.3  Electrochemical properties 
JRC,  Directorate  F  also  reported  pH,  zeta  potential  and  electrolytic  conductivity  results 
(Table 14); instrumental details are listed in Table D.7 of Annex D.  

Table 14: Additional material information values 

Measurement method 
Zeta potential 1) 
pH 2) 
Electrolytic conductivity 3) 

Information value 

-49 mV 

8.7 

0.03 mS/cm 

1) As obtained by electrophoretic light scattering (ELS) at 25 °C ± 1 °C 
2) As determined by a potentiometric method at 22 °C ± 1 °C 
3) Determined at 25 °C ± 1 °C using a folded capillary cell (Malvern Zetasizer Nano ZS instrument)  

7.3.4  Sub-population of smaller particles 
As  mentioned  in  section  3.1  (Origin  of  the  starting  material),  a  sub-population  of  smaller 
particles can be seen in the EM micrographs (Fig. 1). Due to the small size of this particles 
and their lower number, not all the techniques used during the ILC could detect or quantify 
them. However, some laboratories did report the presence of this population: 
•  Lab 18 performed Analytical Ultracentrifugation (AUC). Assuming that the 40 nm particles 

have the same particle density, Lab 18 estimated their mass fraction at 5 %. 

•  Lab 16 showed that this sub-population is revealed in the volume-weighted PSD obtained 

with line-start CLS (turbidimetry), but not in the intensity-weighted PSD. 

•  The  technical  specifications  for  the  EM  tests  asked  to  count  and  measure  only  particles 
above  60  nm.  Nevertheless,  L13  reported  that  the  sub-population  of  small  particles 
accounts  for  around  40  %  of  the  total  number  of  particles.  Also  Lab  20  performed  EM 
tests and reported the presence of the sub-population with a mode centred around 40 nm. 
The  total  number  of  particles  in  that  mode  was  30 %  of  that  observed  for  the  main 
population centred at 80 nm. In the PSD, both particle populations are baseline separated. 
•  Lab 25 reported SAXS data showing a sub-population at 30 nm (volume-weighted PSD). 
Also Lab 26 mentioned a sub-population near 40 nm, but their contribution to the total 
scattering intensity was too small to enable a reliable determination of their mean size. 

36 

 
 
 
 
 
8  Metrological traceability and commutability 

8.1  Metrological traceability 

Identity 

The term 'particle size' is not very specific, as there are multiple size parameters for a single 
particle,  especially  if  it  is  not  of  regular  shape.  Most  PSA  methods  produce  an  equivalent 
diameter (or radius). The actual realisation of the term "equivalent" strongly depends on the 
nature of the measurement principle of each method.  

ERM-FD101b  has  been  characterised  using  multiple  PSA  methods  and  the  different 
assigned property values are intrinsically linked to their corresponding operationally-defined 
measurands.  The  certified  values  can  be  regarded  as  reliable  estimates  of  the  true 
equivalent particle sizes, and are underpinned by the agreement of the laboratories' results 
with  the  assigned  values  for  the  CRMs  (ERM-FD100  and  ERM-FD304)  that  were  used  as 
QC samples. 

All  certified  size  values  of  ERM-FD101b,  except  the  values  obtained  with  electron 
microscopy, are valid only in a specified temperature range: 

-  For  the  DLS,  SAXS  and  PTA  methods,  the  certified  values  are  valid  in  the  range 
[25 ± 5] °C. JRC performed DLS tests during which sample temperature was systematically 
varied in this range. The observed trend was statistically significant (0.1 nm/°C) but, due to 
the  restriction  of  the  allowed  testing  temperature,  it  is  technically  insignificant  compared  to 
the  uncertainty  of  the  apparent  hydrodynamic  diameter.  For  the  SAXS  method,  the  size 
values reflect the size of the core silica particles, and are less dependent on changes of the 
surface  layer  of  the  silica  particles.  This  justifies  extrapolating  the  conclusions  on 
temperature effects from the DLS experiments to the SAXS data. 

- For the CLS method, the certified value is valid in the range [25 °C, 36°C].  

All  accepted  CLS  characterisation  data  were  obtained  in  the  above  specified  temperature 
range.  Within  the  specified  temperature  ranges,  there  is  no  significant  trend  between  the 
reported size values and the reported measurement temperatures.  

The  combination  of  restricting  the  validity  of  the  certified  value  to  a  limited  temperature 
range, and the absence of a technically significant trend within the specified range, justifies 
the  absence  of  a  specific  temperature  uncertainty  contribution  in  the  uncertainty  budget  of 
the certified values. 

Quantity value 

Metrological  traceability  of  the  assigned  particle  size  values  to  a  higher  order  accepted 
reference,  ultimately  to  a  practical  realisation  of  the  definition  of  a  unit  of  the  International 
System  of  Units  (SI)  such  as  the  metre,  requires  traceability  of  all  relevant  input  factors 
through  an  unbroken  network  of  calibrations.  Properly  characterised  and  fit-for-purpose 
calibrants  are  essential  links  in  order  to  establish  the  traceability  network.  Considering  the 
method-defined  nature  of  the  different  measurands,  the  traceability  network  and  final 
references have been specific for each method. A summary of the traceability network for the 
different  methods  used  in  this  study  is  given  below.  Detailed  information  regarding  the 
calibrants used by the different laboratories in the study is listed in the tables of Annex D. 

Centrifugal liquid sedimentation (turbidimetry):  

The measurand of the CLS method, being the light extinction-weighted modal Stokes particle 
diameter, is defined by the procedures described in documentary standards ISO 13318-1 [8] 
and 13318-2 [9].  

37 

 
 
Whereas the cuvette methods  do  not require  calibration for  particle  size, the  sedimentation 
time scales of the disc centrifuges were all calibrated using particle size standards. With the 
exception  of  one  laboratory,  all  laboratories  requiring  calibration  used  PVC  calibrant  beads 
that were supplied by CPS Instruments, Inc. The certificates of these calibrants suggest that 
the assigned values are SI-traceable but there is no documentation available to substantiate 
this  claim.  Therefore,  a  series  of  these  PVC  standards  of  different  particle  size  has  been 
checked  in-house  against  each  other  as  well  as  against  colloidal  silica  and  polystyrene 
materials. The  obtained results [38]  indicate  consistency,  in  terms  of  particle  size,  amongst 
the  different  PVC  standards.  In  addition,  a  few  of  the  CPS  calibrants  were  subject  of  a 
collaborative  study  involving  different  laboratories  [38].  The  study  showed  that  the  modal 
particle  diameter,  as  well  as  the  effective  particle  density,  which  are  assigned  to  the  PVC 
calibrants  are  accurate  within  5  %  and  3.5  %  respectively  (95  %  confidence  level).  The 
conclusion is that, when using the PVC calibrants with traceable particle density and size, the 
particle size results from well-executed disc-CLS measurements are traceable to the SI unit 
of length. The uncertainties about the PVC calibrants size and density, as well as that of the 
SI-traceable particle density value of the ERM-FD101b particles, have been included in the 
uncertainty  of  the  certified  value  obtained  by  CLS  (turbidimetry),  which  therefore  is  also 
traceable to the SI. 

Dynamic light scattering:  

The scattered light intensity-weighted mean hydrodynamic particle diameters, as determined 
by the cumulants method and by the distribution calculation algorithms, are method-defined. 
Since  the  DLS  method  is  intrinsically  absolute  in  nature,  there  is  no  need  for  instrument 
response  calibration  or  for  introducing  corrective  terms.  Traceability  of  the  measured 
diameter values depends on the traceability of the values corresponding with the parameters 
occurring in the Stokes-Einstein equation: 
•  Temperature: the sample temperatures have been measured by sensors which had either 
been  accurately  calibrated  by  their  manufacturer  or  which  had  been  verified  following 
alpha testing using Pt100 sensors. 

•  Detector  angle:  the  angles  at  which  the  detectors  were  fixed  had  been  geometrically 
determined as they depend on the mechanical design of the DLS systems. The accuracy 
of the angle is assured by respecting the applied mechanical tolerances. 

•  Measured  decay  rate:  the  traceability  of  the  measured  decay  rates  depends  on  the 
accurately  known  constant  resonant  frequency  of  quartz  crystal  oscillators  that  are 
integrated  in  programmable  logic  devices  such  as  a  field-programmable  gate  array 
(FPGA). 

•  Refractive index and viscosity of the sample/particle: refractive index and viscosity values 
were  obtained  from  tables  in  the  literature  (e.g.,  CRC  Handbook)  reporting  traceably 
measured values. 

•  Laser  wavelength:  traceability  of  the  wavelength  value  to  the  SI  was  assured  by  using 
helium-neon lasers with a nominal wavelength of 633 nm. In the ILC study, 15 out of the 
20  DLS  instruments  were  equipped  with  such  a  laser.  Unstabilised  He-Ne  lasers  of 
633 nm  are  used  in  most  laser  interferometers  and  many  instruments  used  for  length 
measurements.  These  instruments,  including  DLS  instruments,  are  very  often  used  at 
uncertainty  levels  that  are  large  compared  to  the  possible  variation  of  the  He-Ne  laser 
vacuum  wavelength.  Based  on  these  considerations,  the  International  Committee  of 
Weights and Measures (CIPM) recognised the need for providing documentary evidence 
regarding the value of the vacuum wavelength and its uncertainty that can be expected in 
the  absence  of  calibration.  During  its  96th  meeting,  the  CIPM  adopted  a  wavelength  of 
632.9908 nm  with  a  relative  standard  uncertainty  of  1.5  x  10-6  [39].  Following  thorough 
evaluation  of  the  Consultative  Committee  for  Length  (CCL)  of  the  CIPM,  the  CCL 

38 

 
 
recommended  including  unstabilised  red  He-Ne  lasers,  operating  on  the  633  (3s2→2p4) 
neon  transitions,  in  the  new  list  of  standard  frequencies,  "Recommended  values  of 
standard  frequencies  for  applications  including  the  practical  realization  of  the  metre  and 
secondary representations of the second". This list replaces the Mise en Pratique for the 
definition of the metre. 

Because of the calibration or traceable values of these input parameters, the certified value 
and uncertainty of the hydrodynamic diameters obtained with DLS are traceable to the SI. 

Electron microscopy:  

The  measurands  investigated  by  SEM  and  TEM  were  the  number-weighted  modal  and 
median  area-equivalent  particle  diameters.  The  magnifications  of  the  instruments  were 
calibrated using different types of standards which were all suitable for calibration of lateral 
dimensional measurement systems. Some laboratories used in-situ calibrants in the form of 
PS particle CRMs. Several laboratories used pitch size standards that were calibrated on a 
metrological atomic force microscope, thereby linking the assigned pitch dimensions to the SI 
unit  metre  through  the  calibrated  laser  light  wavelength  of  the  interferometer.  Other 
laboratories  relied  on  a  calibration  via  the  interplanar  distance  of  a  Si  crystal.  Hence,  the 
certified value and uncertainty are traceable to the SI through the SI traceable values of the 
calibrants used by the different laboratories. 

Particle tracking analysis:  

The measurands investigated by PTA are the number-weighted-modal, arithmetic mean and 
median  hydrodynamic  particle  diameters.  Traceability  of  the  measured  diameter  values 
depends on the traceability of the values corresponding with the parameters occurring in the 
modified Stokes-Einstein equation: 
•  Temperature: temperature has a direct impact on the measured hydrodynamic diameters, 
as  it  is  a  parameter  in  the  Stokes-Einstein  equation  itself,  but  also  because  it  has  an 
influence on viscosity. Sample temperature therefore is measured by sensors (thermistor) 
and  controlled  by  an  integrated  temperature  control  (a  Peltier  controlled  with  a  PID 
feedback  loop).  Temperature  sensors  are  usually  calibrated  by  their  manufacturer. 
Possible  inaccuracies  or  instabilities  of  the  temperature  sensor  calibrations  are  not 
expected  to  contribute  to  the  uncertainty  of  the  certified  values  of  the  hydrodynamic 
diameters measured with PTA beyond the values reported in Section 7. 

•  Viscosity of the sample/particle: viscosity values were obtained from tables in the literature 

(e.g., CRC Handbook) reporting traceably measured values. 

•  PTA  instruments  measure  the  length  of  particles'  trajectories  and  as  a  consequence  a 
calibration  of  the  measured  distances  is  required.  The  corresponding  calibration  of  the 
video  image  pixel  size  has  been  performed  by  the  instrument  manufacturer,  first  via 
measurements on 10 micrometre pitch graticules and later, more indirectly, via PSA tests 
on certified polystyrene latex particle size standards (ThermoScientific, 3200 series). The 
certified  values  of  the  latter  size  standards  are  SI-traceable,  as  indicated  on  their 
certificate and as documented in [40]. 

•  Time:  the  traceability  on  time  measurement  depends  on  the  accurately  known  constant 
resonant frequency of quartz crystal oscillators that are integrated in programmable logic 
devices.  The  accuracy  of  time  measurements  with  such  devices  is  sufficient  to  exclude 
significant effects on the uncertainty of the hydrodynamic diameters measured with PTA. 
•  In  order  to  detect  deviations  from  a  proper  calibration  status,  most  of  the  participating 
laboratories  verify  their  instruments  with  a  variety  of  quality  control  materials  and 
calibrants (see Table D.4). 

39 

 
 
 
Because of the calibration or traceable values of these input parameters, the certified value 
and uncertainty of the hydrodynamic diameters obtained with PTA are traceable to the SI. 

Small-angle X-ray scattering:  

The  measurands  investigated  by  SAXS  are  the  scattered  X-ray  intensity-,  number-  and 
volume-weighted modal particle diameters. Traceability of the measured diameter values to 
the SI unit of length directly depends on the traceability of the values corresponding with the 
parameters  occurring  in  the  equation  linking  scattering  vector  (q)  and  measured  scattering 
angle  (q ):  q = 4p /l   sinq   .  Several  set-ups  were  used  in  the  laboratories  participating  in  the 
interlaboratory characterisation study, and different calibration approaches were followed. 
•  Half of the scattering angle (q ): either directly measured using (i) a calibrated goniometer, 
or  (ii) geometrically  calculated from  the  calibrated  detector  pixel  size  and  the  sample-to-
detector  distance.  For  the  second  approach, the absolute  sample-to-detector  distance  is 
determined  through  standard  distance  measurements.  The  detector  pixel  size  is  derived 
from the scattering angle(s) of a suitable reference material (e.g., silver behenate).  

•  X-ray  wavelength:  traceability  of  the  x-ray  wavelength  value  to  the  SI  unit  metre  was 
assured  via  back-reflection  from  appropriate  monochromators  or  single  crystals  (e.g. 
silicon)  with  known  lattice  constant,  or  through  the  nominal  wavelength  of  the  Cu-Ka
characteristic X-radiation. 

The variety of calibration approaches used in the interlaboratory study ensures absence of a 
systematic bias between laboratories and therefore justifies the decision not to add a specific 
uncertainty component into the overall uncertainty budget. 

8.2  Commutability 

According  to  ISO  Guide  34  [1],  the  assessment  of  commutability  for  a  CRM  requires  a 
comparison  and  establishment  of  a  numerical  correlation  between  the  property  value 
assigned  to  the  CRM  and  to  routine  test  samples  using  both  a  "higher-order"  reference 
measurement procedure and one or more "lower-order" routine measurement procedures. If 
the ratio between the results of the compared measurement procedures is the same as the 
ratio of the results for routine test samples, then the CRM is said to be commutable. 

ERM-FD101b  has  been  characterised  by  multiple  techniques  and  methods  which  all  target 
different  method-defined  measurands  and  the  size  measurement  results  for  near-spherical 
particles  are  uncorrelated.  Therefore,  commutability  cannot  be  assessed.  It  can  be 
mentioned  however  that  the  ERM-FD101b  colloidal  silica  is  representative  for  the  routine 
tests samples encountered in daily laboratory practice. 

40 

 
 
 
 
 
9  Instructions for use 

9.1  Safety information 

This  material  should  be  handled  with  care.  Nanoparticles  can  have  an  impact  on 
environment and human health. Any spilling of the suspension should be handled according 
to the usual laboratory safety precautions. 

For further details refer to the safety data sheet. 

9.2  Storage conditions 

The materials shall be stored at 18 °C ± 5 °C. Ampoules must not be allowed to freeze, as 
this will irreversibly compromise the integrity of the material. 

Please  note  that  the  European  Commission  cannot  be  held  responsible  for  changes  that 
happen  during  storage  of  the  material  at  the  customer's  premises,  especially  of  opened 
ampoules. 

9.3  Instructions for use and intended use 

The  intended  use  of  ERM-FD101b  is  to  check  the  performance  of  instruments  and/or 
methods that characterise the size distribution of particles in the range around 100 nm that 
are either suspended in a liquid medium or deposited onto a suitable substrate. The certified 
values are regarded as reliable estimates of the true values and ERM-FD101b can therefore 
be used for calibration purposes.  

Before  opening,  the  ampoule  should  be  gently  inverted  several  times  to  ensure  the 
homogeneity  of  the  suspension  and  to  re-suspend  any  settled  particles.  Remove  any 
suspension that remains in the upper part (conical tip) of the ampoule by gently flicking the 
conical part with the forefinger while tilting the ampoule. The ampoule is pre-scored and can 
be opened by applying moderate pressure with one's thumb to snap off the conical part. The 
contents  of  an  ampoule  should  be  used  the  same  day  as  opened  and  should  be  gently 
homogenised before every measurement, without introducing air bubbles. 

DLS  method:  Aliquots  of  ERM-FD101b  shall  be  measured  as-received,  i.e.  without  dilution. 
The  measurement  temperature  shall  be  within  the  range  [25 ± 5] °C.  The  viscosity  and 
refractive  index  of  the  dispersing  medium  (water)  at  25 °C  are  0.8872  mPa·s  and  1.330, 
respectively.  The  value  of  the  viscosity  must  be  adjusted  when  tests  are  not  performed  at 
25 °C. 

CLS (turbidity) method: Aliquots of ERM-FD101b shall be measured as-received, i.e. without 
dilution.  The  effective  density  of  the  silica  particles  is  2.0  g/cm3.  The  temperature  of  the 
sample (for cuvette methods) or of the density gradient (for disc methods) shall be within the 
range [25 °C, 36 °C]. 

Electron microscopy method: The material should be transferred to a suitable grid/substrate; 
after drying, at least 250 discrete (non-overlapping) particles of the large particle population 
should  be  counted  and  measured.  If  necessary,  ERM-FD101b  can  be  diluted  with  purified 
water before transferring the particles to the grid/substrate. 

Particle tracking analysis: ERM-FD101b should be diluted with purified water (filtered through 
a  membrane  with  nominal  pore  size  of  0.1  µm)  to  a  particle  concentration  suitable  for  the 
user's instrument. Neither the as-received, nor the diluted silica suspension shall be treated 
by  filtration,  centrifugation  or  sonication.  The  measurement  temperature  shall  be  within  the 
range [25 ± 5] °C. 

41 

 
Small-angle  X-ray  scattering:  ERM-FD101b  should  be  either  analysed  as-received,  i.e. 
without dilution, or diluted in 2-fold in purified water. The measurement temperature shall be 
within the range [25 ± 5] °C. 

9.4  Minimum sample intake 

The minimum amount of sample to be used is for: 

•  CLS (turbidimetry): 100 m L; 
•  CLS (refractometry): 340 m L; 
•  DLS: 100 m L; 
•  EM: 2.5 m L of an as-received aliquot and analysis of at least 250 discrete particles;  
•  PTA: 1 m L. One shall analyse at least 500 tracks per measurement; 
•  SAXS: 20 m L. 

9.5  Use of the certified value 

The  main  purpose  of  this  material  is  to  assess  the  performance  of  instruments  and/or 
methods  that  are  used  for  measuring  the  size  of  nanoparticles.  As  any  reference  material, 
ERM-FD101b can also be used for control charts or validation studies. 

Use as a calibrant 

The  certified  values  that  have  been  assigned  to  the  equivalent  diameters  are  regarded  as 
reliable estimates of the true values and ERM-FD101b can therefore be used for calibration 
purposes  for  EM  and  PTA  methods.  ERM-FD101b  can  also  be  used  for  the  calibration  of 
CLS 
the 
sedimentation  of  the  smaller  particles  of  the  40 nm  population,  which  otherwise  would 
interfere with the signal of the first sample measured after calibration. 

is  allowed  after  calibration 

instruments,  provided 

that  sufficient 

time 

for 

Comparing an analytical result with the certified value 

A result is unbiased if the combined standard uncertainty of measurement and certified value 
covers the difference between the certified value and the measurement result (see also ERM 
Application Note 1, www.erm-crm.org [36]. 

For  assessing  the  method  performance,  the  measured  values  of  the  CRMs  are  compared 
with the certified values. The procedure is described here in brief: 

•  Calculate  the  absolute  difference  between  mean  measured  value  and  the  certified 

value (D meas). 

•  Combine  measurement uncertainty  (umeas)  with  the  uncertainty  of  the  certified  value 

(uCRM): 

=D

u

u

2
meas

+

u

2
CRM

•  Calculate the expanded uncertainty (UD ) from the combined uncertainty (uD

,) using an 
appropriate coverage factor, corresponding to a level of confidence of approximately 
95 % 
If  D meas  £   UD   no  significant  difference  between  the  measurement  result  and  the 
certified value, at a confidence level of about 95 % exists. 

• 

Use in quality control charts 

ERM-FD101b can be used for quality control charts. Different CRM-units will give the same 
result as inhomogeneity was included in the uncertainties of the certified values.  

42 

 
 
 
 
10 Acknowledgements 

The  authors  would  like  to  acknowledge  the  support  received  from  Jean  Charoud-Got  and 
John  Seghers from  the JRC,  Directorate  F  related  to  the  processing  of this  CRM  and  from 
Maria Contreras Lopez concerning the set-up of the required isochronous studies. 

Furthermore,  the  authors  would  like  to  thank  Katrien  Busschots  and  Tsvetelina  Gerganova 
(JRC, Directorate F) for the reviewing of the certification report, as well as the experts of the 
Certification  Advisory  Panel  "  Physicochemical-physical  properties",  Mark  Gee  (NPL, 
Teddington  ,  UK),  Jan  Mast  (CODA-CERVA,  Brussels,  BE)  and  Ludwig  Niewöhner 
(Bundeskriminalamt, Wiesbaden, DE) for their constructive comments. 

43 

 
 
 
11 References 

1  

2  

3  

4  

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6 

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9 

ISO  Guide  34,  General  requirements  for  the  competence  of  reference  materials 
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ISO/IEC  17025:2005,  General  requirements  for  the  competence  of  testing  and 
calibration 
for  Standardization,  Geneva, 
Switzerland, 2005 

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10 

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11  T. Detloff, T. Sobisch, D. Lerche, Particle size distribution by space or time dependent 
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12  H.  Cölfen,  W.  Wohlleben,  Analytical  ultracentrifugation  of  latexes,  In:  L.M.  Gugliotta, 
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Signpost, Kerala, India, 2010, pp 183-222 

13  B.J. Berne, R. Pecora, Dynamic light scattering with applications to chemistry, biology, 

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16 

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ISO  22412,  Particle  size  analysis  –  Dynamic  light  scattering  (DLS),  International 
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ISO  13322-1,  Particle  size  analysis  –  Image  analysis  methods  –  Part  1:  Static  image 
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22  P.-J.  De  Temmerman,  E.  Van  Doren,  E.  Verleysen,  Y.  Van  der  Stede,  M.A.D. 
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23  P.-J.  De  Temmerman,  J.  Lammertyn,  B.  De  Ketelaere,  V.  Kestens,  G.  Roebben,  E. 
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transmission  electron  microscopy:  an 
interlaboratory comparison case study, Metrologia 50 (2013) 663-678 

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27  O.  Glatter,  A  new  method  for  the  evaluation  of  small-angle  scattering  data,  J.  Appl. 

Crystallogr. 10 (1977) 415-421 

28 

ISO  17867,  Particle  size  analysis  –  Small-angle  X-ray  scattering,  International 
Organization for Standardization, Geneva, Switzerland, 2015 

29  T.P.J.  Linsinger,  J.  Pauwels,  A.M.H.  van  der  Veen,  H.  Schimmel,  A.  Lamberty, 

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by isochronous measurements, Fres. J. Anal. Chem. 360 (1998) 359-361 

31  A. Braun, K. Franks, V. Kestens, G. Roebben, A. Lamberty, T. Linsinger, Certification 
of equivalent spherical diameters of silica nanoparticles in  water – Certified reference 
material  ERM-FD100,  EUR  Report  24620  –  European  Union,  Luxembourg  -  2011  – 
ISBN 978-92-79-18676-9 

32  K. Franks, A. Braun, V. Kestens, G. Roebben, A. Lamberty, T. Linsinger, Certification 
of  the  equivalent  spherical  diameters  of  silica  nanoparticles  in  aqueous  solution  – 
Certified  reference  material  ERM-FD304,  EUR  Report  25018  –  European  Union, 
Luxembourg - 2012 – ISBN 978-92-79-21866-8 

33  T.P.J.  Linsinger,  J.  Pauwels,  A.  Lamberty,  H.  Schimmel,  A.M.H.  van  der  Veen,  L. 
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Chem. 370 (2001) 183-188 

34  H.  Woehlecke,  T.  Detloff,  K.  Franks,  V.  Kestens,  G.  Roebben,  D.  Lerche,  In-situ 
determination of the effective particle density of suspended colloidal silica particles by 
means  of  analytical  centrifugation,  International  Congress  on  Particle  Technology 
(PARTEC), Nuremberg, Germany, 23-25 April 2013 

45 

 
 
 
35 

ISO 9276-2, Representation of results of particle size analysis – part 2: Calculation of 
average  particle  sizes/diameters  and  moments  from  particle  size  distributions, 
International Organization for Standardization, Geneva, Switzerland, 2014 

36  T. Linsinger, ERM Application note 1, www.erm-crm.org, last accessed 25/1/2016 

37  V. Kestens and G. Roebben, Certification of equivalent diameters of a mixture of silica 
nanoparticles  in  aqueous  solution  :  ERM-FD102,  EUR  26656  –  European  Union, 
Luxembourg - 2014 – ISBN 978-92-79-38396-0 

38  V.  Kestens,  V.A.  Coleman,  P.-J.  De  Temmerman,  C.  Minelli,  H.  Woehlecke,  G. 
Roebben, Improved metrological traceability of particle size values measured with line-
start incremental centrifugal liquid sedimentation, accepted for publication in Langmuir 
(2017), DOI: 10.1021/acs.langmuir.7b01714 

39  CIPM, Report of the 96th meeting (2007) – BIPM, Paris, FR  

40  S.  D.  Duke,  E.  B.  Layendecker,  Internal  standard  method  for  size  calibration  of  sub-
micrometer  spherical  particles  by  electron  microscope,  Technical  Note  TN-010.3, 
Thermo Fisher Scientific, Fremont, CA, USA, 2009 

46 

 
 
 
Annexes 

Annex A: Results of the homogeneity measurements 

Fig. A1 and Fig. A2 show the averages of the replicates measured per ampoule and their 95 % 
confidence  intervals  (error  bar).  These  confidence  intervals  are  based  on  the  expanded 
measurements  uncertainties  (k = 2)  of  the  DLS  (cumulants  method)  and  line-start  CLS  (disc 
centrifuge)  methods  operated  in  repeatability  conditions,  as  obtained  during  in  house  method 
validation  studies.  Absolute  values  do  not  necessarily  agree  with  the  certified  values  due  to 
potential laboratory bias, but this is irrelevant for the evaluation of homogeneity. 

)

m
n
(

100

i

i

r
e
t
e
m
a
d
n
a
e
m
c
n
o
m
r
a
h
d
e
h
g
e
w
-
y
t
i
s
n
e
n

t

t

i

i

t

h
g

i
l

d
e
r
e

t
t

a
c
s

95

90

85

80

75

70

0

500

1000

1500

2000

Unit number

Fig. A1 Homogeneity data (average results of two replicates) of ERM-FD101b; DLS (cumulants 
method)  scattered  light  intensity-weighted  harmonic  mean  particle  diameter;  error  bars 
correspond  to  the  expanded  measurement  uncertainties  (k = 2)  for  use  of  the  method  in 
repeatability conditions. 

47 

 
 
 
 
 
 
 
 
110

105

100

95

90

85

80

75

70

)

m
n
(

i

r
e
t
e
m
a
d
s
e
k
o
t
S

l

i

a
d
o
m
d
e
t
h
g
e
w
-
n
o
i
t
c
n
i
t
x
e

t
h
g
L

i

0

500

1000

1500

2000

Unit number

Fig. A2 Homogeneity data (average results of three replicates) of ERM-FD101b; line-start CLS 
(disc centrifuge) light extinction-weighted modal Stokes particle diameter; error bars correspond 
to  the  expanded  measurement  uncertainties  (k  = 2)  for  use  of  the  method  in  repeatability 
conditions. 

48 

 
 
 
 
 
 
 
 
 
Annex B: Results of the short-term stability measurements 

Graphs depicted in Fig. B1 and Fig. B2 show the short-term stability data as obtained with DLS 
(cumulants method)  and  with  line-start  CLS (disc  centrifuge),  respectively.  Absolute  values  do 
not  necessarily  agree  with  the  certified  values  due  to  potential  laboratory  bias,  but  this  is 
irrelevant for the evaluation of the stability. 

Fig.  B1  Short-term  stability  data  (results  of  individual  replicates)  of  ERM-FD101b;  DLS 
(cumulants  method)  results  (scattered 
intensity-weighted  harmonic  mean  particle 
diameter), when stored several weeks at 4 °C (a) and 60 °C (b). Results at time point 0 weeks 
correspond to units that were stored at the reference temperature of 18 °C. 

light 

)

m
n
(

r
e

92

t

e
m
a
d

i

s
e
k
o
S

t

l

a
d
o
m
d
e

t

i

h
g
e
w

-
n
o

i
t
c
n

i
t
x
e

t

h
g
L

i

90

88

a)

)

m
n
(

92

i

t

r
e
e
m
a
d
s
e
k
o
S

t

l

a
d
o
m
d
e

t

i

h
g
e
w
-
n
o

0

1

2

3

4

Time (week)

i
t
c
n

i
t
x
e

t

h
g
L

i

90

88

0

1

2

3

4

Time (week)

b)

Fig. B2 Short-term stability data (results of individual replicates) of ERM-FD101b; line-start CLS 
(disc centrifuge) light extinction-weighted modal Stokes' particle diameter, when stored several 
weeks  at  4 °C  (a)  and  60 °C  (b).  Results  at  time  point  0  weeks  correspond  to  units  that  were 
stored at the reference temperature of 18 °C. 

49 

 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex C: Results of the long-term stability measurements 

Fig.  C1  and  C2  show  the  long  term  stability  data  obtained  with  DLS  (cumulants  method)  and 
line-start CLS (disc centrifuge), respectively. Absolute values do not necessarily agree with the 
certified  values  due  to  potential  laboratory  bias,  but  this  is  irrelevant  for  the  evaluation  of 
stability. 

Fig.  C1  Long-term  stability  data  (results  of  individual  replicates)  of  ERM-FD101b;  DLS 
(cumulants  method)  results  (scattered 
intensity-weighted  harmonic  mean  particle 
diameter), when stored several months at 18 °C. Results at time point 0 months correspond to 
units that were stored at the reference temperature of 4 °C. 

light 

50 

 
 
 
 
)

m
n
(

r
e
t
e
m
a
d

i

'

s
e
k
o
t
S

l

a
d
o
m
d
e
t
h
g
e
w
-
n
o
i
t
c
n

i

i
t
x
e

t

h
g
L

i

92.0

91.5

91.0

90.5

90.0

89.5

89.0

88.5

88.0

87.5

87.0

0

2

4

6

8

10

12

Time (Month)

Fig. C2 Long-term stability data (results of individual replicates) of ERM-FD101b; line-start CLS 
(disc  centrifuge) light  extinction-weighted modal  Stokes'  particle  diameter  results,  when  stored 
several months at 18 °C. Results at time point 0 months correspond to units that were stored at 
the reference temperature of 4 °C. 

51 

 
 
 
 
 
 
 
 
 
Annex D: Summary of methods used in the characterisation study 

Table D.1 Centrifugal liquid sedimentation: relevant instrumental and method details (as reported by the participants) 

Lab 
code 

Instrument 
make 

Type  Mode of 
operation 

Rota-
tional 
speed 
[rpm] 

Type of 
optics 

Laser 
wave-
length 
[nm] 

Density gradient 

Type / 
Temperature 

Concentra-
tion 
interval 
[g/kg] 

Sample 
volume 
m L] 
[m

Type 

Calibrant 

Particle 
density 
[g/cm3] 

Assigned 
modal 
value 
[nm] 

1.385 

377 

1.83 

490 

1.385 

239 

PVC particles 
(CPS 
Instruments 
Inc.) 

Silica 
microspheres 
(Duke 
Scientific) 

PVC particles 
(CPS 
Instruments 
Inc.) 

Disc 

Line-start 

20000 

Turbidity 

470 

Sucrose 

0 to 80 

200 

28 °C 

Disc 

Line-start 

24000 

Turbidity 

470 

Sucrose 

80 to 240 

500 

30 °C 

Disc 

Line-start 

20000 

Turbidity 

405 

Sucrose 

40 to 120 

100 

L2 

L5 

L9 

Disc 
Centrifuge 
DC24000 
(CPS 
Instruments 
Inc.) 

Disc 
Centrifuge 
DC24000 
(CPS 
Instruments 
Inc.) 

Disc 
Centrifuge 
DC24000 
(CPS 
Instruments 
Inc.) 

29.5 °C 

n.a. 

25 °C 

L14 

LUMiSizer 
651 

Cu-
vette 

Homoge-
neous 

4000 

Turbidity 

470 

(LUM 
GmbH) 

n.a. 

n.a 

n.a. 

n.a. 

n.a. 

L16  Disc 

Disc 

Line-start 

22000 

Turbidity 

405 

Sucrose 

80 to 240 

300 

Centrifuge 
DC24000 
(CPS 
Instruments 
Inc.) 

(not reported) 

52 

1.385 

226 

PVC particles 
(CPS 
Instruments 
Inc.) 

 
 
m
m
L17 

LUMiSizer 
651 

Cu-
vette 

Homoge-
neous 

4000 

Turbidity 

470 

(LUM 
GmbH) 

L18  Optima XL-I 

(Beckman-
Coulter) 

Cu-
vette 

Homoge-
neous 

3000 

Refractive 
index 

675 

n.a. 

25 °C 

n.a. 

25 °C 

n.a. 

n.a 

n.a. 

n.a. 

n.a. 

n.a. 

n.a 

Counter 
balance cells 

n.a. 

n.a. 

L20  Disc 

Disc 

Line-start 

24000 

Turbidity 

405 

Sucrose 

80 to 240 

100 

Centrifuge 
DC24000 
(CPS 
Instruments 
Inc.) 

36 °C 

L23  Disc 

Disc 

Line-start 

20000 

Turbidity 

405 

Sucrose 

20 to 80 

300 

1.385 

263 

1.385 

239 

PVC particles 
(CPS 
Instruments 
Inc.) 

PVC particles 
(CPS 
Instruments 
Inc.) 

n.a. 

340 

Counter 
balance cells 

n.a 

n.a 

Centrifuge 
DC20000 
(CPS 
Instruments 
Inc.) 

L28  Optima  XL-I/ 
XL A 

Cu-
vette 

Homoge-
neous 

3000 

Refractive 
index  

(Beckman-
Coulter) 
n.a. = not applicable 

29 °C 

n.a 

25 °C 

53 

 
 
 
 
Table D.2 Dynamic light scattering: relevant instrumental and method details (as reported by the participants) 

Lab 
code 

Instrument type / 
make 

Method 

Source 

Laser 

Wave-
length 
[nm] 

Detector 

Sample cell 

Analysis type 

Power 
[mW] 

Type ‡ 

Angle  
[°°°°] 

Type 

Cuvette 
dimension 
length 

[mm] 

Aliquot 
volume 
[mL] 

(in addition to 
the 
Cumulants 
method)  

L1 

L2 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

4 

4 

APD 

173 

APD 

173 

L3a 

Malvern Zetasizer 
Nano ZS90 

Correlation 
function analysis 

He-Ne 

633 

n.a. 

APD 

90 

L3 b 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

n.a. 

APD 

173 

WhL
4 

Malvern Zetasizer 
Nano ZSP 

Correlation 
function analysis 

He-Ne 

633 

10 

APD 

173 

L5 

L6 

L7 

L8 

L9 

L12 

L13 

Coulter N4 PLUS 
Submicron 
Particle Sizer 
Coulter N4 PLUS 
Submicron 
Particle Sizer 
Horiba 
NanoPartica SZ-
100Z 

Correlation 
function analysis 

Correlation 
function analysis 

Correlation 
function analysis 

He-Ne 

633 

4 

PMT 

90 

He-Ne 

633 

10 

PMT 

90 

Diode 

532 

10 

PMT 

90 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

4 

4 

4 

4 

APD 

173 

APD 

173 

APD 

173 

APD 

173 

54 

Quartz glass 
microcuvette 
Disposable 
polystyrene 
cuvette 
Standard 
quartz glass 
cuvette 
Standard 
quartz glass 
cuvette 
Disposable 
Polystyrene  
cuvette 
Disposable 
Polystyrene  
cuvette 
Disposable 
acrylic 
cuvette 
Disposable 
plastic 
cuvette 
Disposable 
PMMA 
cuvette 
Disposable 
polystyrene 
cuvette 
Disposable 
polystyrene 
cuvette 
Disposable 
polycyclic 
olefin 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

0.125 

NNLS 

1 

1 

1 

1 

1 

3 

1 

NNLS 
GP 

n. a. 

n. a. 

NNLS 
MNM 

CONTIN 
(SDP analysis) 

CONTIN 
(SDP analysis) 

n. a. 

1.5 

NNLS 

0.5 

NNLS 
GP 

3 

NNLS 

0.1 

NNLS MNM + 
Q-corrrection 

 
 
L19 

L20 

L23 

L24 

L27 

L29 

L32 

L36 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

He-Ne 

633 

4 

4 

4 

APD 

173 

APD 

173 

APD 

173 

HORIBA SZ-100 
Nano Partica 

Correlation 
function analysis 

NANOPHOX 
PARTICLE 
ANALYSER, 
NX0059 

3D-cross-
correlation 
function analysis 

Malvern Zetasizer 
Nano ZS 

Correlation 
function analysis 

ALV-6010/160 

NANOPHOX 
PCCS 

3D-cross-
correlation 
function analysis 
3D-cross 
correlation 
function analysis 

Diode 

532 

10 

PMT 

90 

He-Ne 

633 

15 

APD 

90 

He-Ne 

633 

4 

APD 

173 

YAG 

532 

500 

Photo-
diode 

90 

Diode 

658 

30 

APD 

90 

Disposable 
polystyrene 
cuvette 
Disposable 
polystyrene 
cuvette 
quartz 
cuvette 
Disposable 
plastic 
cuvette 

Disposable 
acrylic 
cuvette 

Standard 
quartz glass 
cuvette 
Disposable 
polystyrene 
Cuvette 
Disposable 
acrylic 
cuvette 

10 

10 

10 

10 

1 

1 

1 

1.5 

NNLS 
MNM 

NNLS 
MNM 

NNLS 
GP 
Inverse 
Histogram 
method 

10 

0.3 

n.a. 

10 

10 

10 

1 

2 

1 

NNLS 

n.a. 

NNLS 

n.a.  =  not  available  or  reported  by  participant,  ‡  APD:  avalanche  photodiode  detector;  PMT:  photomultiplier  tube;  GP:  General 
Purpose, regulariser 0.01; MNM : Multi Narrow Mode, regulariser 0.001 

55 

 
 
 
 
 
Table D.3 Electron microscopy: relevant instrumental and method details (as reported by the participants) 

Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

Bright-field 
Condenser 
aperture n°4 

CCD 
camera 
type 

Total 
sam-
pled 
area 

m m2] 
[m

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

Image analysis software 
- Image analysis strategy 

Bin 
size 
[nm] 

Olympus 
SIS 
Mega-
view II 
CCD 

Pixels 
1k x 1k 

0.935 

78 

0.84 

6307 

iTEM (Olympus Soft 
Imaging Solutions GmbH) 

2 

Manual optimisation of 
image brightness 
/contrast, each particle 
was manually measured 
as a polygon clicking on 
the perimeter of the 
particles 

2.5 m L of the as-
received 
suspension 
brought onto a 
Carbon-coated 
grid (Cu 200 
mesh) and 
vacuum dried 

Lab 
code 

Instru-
ment 
type / 
Make 

Accele-
ration 
voltage - 
SEM 
working 
distance 

L2 

TEM 

80 kV 

Philips 
CM200 

L9a 

SEM 

10 kV 

Zeiss 
Supra 
40 Field 
Emissio
n Gun 
SEM 

Between 
3 nm 
and 3.6 
mm 

Optical diffraction 
cross-grating (No. 
607) with 2160 
lines/mm and 
463 nm line spacing 
(Ted Pella, Inc.) 

Reference 
polystyrene particles 
with certified mean 
diameters of 112 nm 
(S130-1), 305 nm 
(S130-5) and 1036 
nm (S130-7) (Plano 
GmbH) 

Traceability 
statement not 
available 

Grid with 2160 
lines/mm or a pitch 
spacing of 463 nm  
in both X and Y 
direction (Agar 
Scientific) 

Si specimen, 
RM8820 with line 
spacing of 201 nm 
(NIST) 

In-lens 
secondary 
electron 

20 µm 

10x to 500x 
diluted with 
ultrapure water 

20 m L of the 
diluted solution 
brought on a 1 
5Alcian blue 
modified 200 
mesh carbon 
coated copper 
grid. 

Grid dried with 
compressed air 

n.a. 

1250 

100 

1.8 

18362 

Zeiss SmartStich software. 

1 

ImageJ v1.48 was used to 
crop the images to remove 
areas where overlap with 
others scans had occurred 
and remove the top part of 
the image where minor 
drift had occurred. 

Software Microsoft Excel 
2010 and OriginPro 9.1 for 
data analysis 

56 

 
 
m
m
 
 
 
 
Lab 
code 

Instru-
ment 
type / 
Make 

Accele-
ration 
voltage - 
SEM 
working 
distance 

L9b 

SEM 

10 kV 

Zeiss 
Supra 
40 Field 
Emis-
sion 
Gun 
SEM 

Between 
3 nm 
and 3.6 

L10 

TEM 

120 kV 

Tecnai 
G2 
Spirit 
TEM  
(FEI, 
Eindho-
ven, 
The 
Nether-
lands) 

Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

CCD 
camera 
type 

Total 
sam-
pled 
area 

m m2] 
[m

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

Image analysis software 
- Image analysis strategy 

Bin 
size 
[nm] 

Grid with 2160 
lines/mm or a pitch 
spacing of 463 nm  
in both X and Y 
direction (Agar) 

Si specimen, 
RM8820 with lines 
by 201 nm ( NIST) 

Optical diffraction 
cross-grating (S106) 
with 2160 lines/mm 
and 463 nm line 
spacing (Agar 
Scientific) 

Traceability 
statement not 
available 

10 to 500x diluted 
with ultrapure 
water 

Scanning 
TEM diode 
detector 

20 m L of the 
diluted QCM\CRM 
solution on a 
Alcian blue 
modified 200 
mesh carbon 
coated copper 
grid. 

Grid dried with 
compressed air 

50 m L taken from 
the as-received 
material and 10x 
diluted with 
ultrapure water 

Bright-field 
with 
objective 
aperture of 
150 m m 

Alcian blue pre-
treated 
Pioloform® 
carbon-coated 
grid (Cu 400 
mesh) placed on 
a drop (15 m L) of 
diluted 
suspension, 
10 min 
incubation, 
washed and air 
dried 

n.a. 

1250 

100 

1.8 

18362 

Zeiss SmartStich software. 

1  

Excel 2010 and OriginPro 
9.1 for data analysis 

ImageJ v1.48 was used to 
crop the images to remove 
areas where overlap with 
others scans had occurred 
and remove the top part of 
the image where minor 
drift had occurred. 

93 

13 

0.82 

13015 

iTEM (Olympus Soft 
Imaging Solutions GmbH) 
and Sigmaplot® 

1 

Manual grey-scale 
thresholding, and 
automated particle size 
analysis, lognormal fit 
using Fityk software 

Particles were detected 
based on their sphericity 
(>0.4) and convexity 
(>0.8) 

Eagle 
CCD 
Bottom-
mount 

Pixels 
4k x 4k 

57 

 
 
m
m
 
 
 
Lab 
code 

Instru-
ment 
type / 
Make 

Accele-
ration 
voltage - 
SEM 
working 
distance 

L13 

SEM 

5 kV 

FEI 
Helios 
Dual-
Beam 

4 mm 

L15a 

TEM 

120 kV 

Carl 
Zeiss 
Libra 
120 

NanoLatticeTM 
standard with 
nominal 100 nm 
pitch (VLSI 
Standards, Inc.) 

Pitch size standard 
calibrated on 
metrological AFM, 
pitch value of 
99.9 nm ± 1.5 nm 
(k = 2) traceable to 
SI Unit, metre 

Magnification 
verified using 100 
nm PSL spheres 
(NIST SRM 1963a) 

Optical diffraction 
cross-grating (S106) 
with 2160 lines/mm 
and 463 nm line 
spacing) (Plano 
GmbH) 

Traceability 
statement not 
available 

Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

CCD 
camera 
type 

Total 
sam-
pled 
area 

m m2] 
[m

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

Image analysis software 
- Image analysis strategy 

Bin 
size 
[nm] 

65 

1.9 

8690 

ImageJ v1.47 

<0.1 

Manual setting of contrast 
and brightness 

Binarisation by baseline 
thresholding and 
automated particle size 
analysis and lognormal fit, 
particles touching each 
other or cut by image 
border were excluded  

500 

20 

0.7 

6198 

iTEM (Olympus Soft 
Imaging Solutions GmbH) 

0.1 

Manual grey-scale 
thresholding, touching 
particles were manually 
excluded, particle sizes 
were automatically 
measured. No image filter 
was used 

Single-crystal 
silicon chips  

Standard SE 
detector  

n.a 

7 to 
315 

coated with 
poly-L-lysine. 
The substrates 
are incubated 
with 50 μL 
sample 

solution without 
dilution for 3 
seconds, and 
then washed 
with Dl water 
and dried with 
air.  

TRS 
slow 
scan 
CCD 

Pixels 
2k x 2k 

Dilution of the 

Sample 10 to 20x 
diluted with 
ultrapure water 

Bright-field 
with 
objective 
aperture of 
30 m m 

2 m L of the 
diluted 
suspension 
brought onto a 
carbon-coated 
grid (Cu 400 
mesh) and air 
dried in a clean 
room 

58 

 
 
m
m
 
 
 
Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

Lab 
code 

Instru-
ment 
type / 
Make 

Accele-
ration 
voltage - 
SEM 
working 
distance 

L15b 

SEM 

20 kV 

Carl 
Zeiss 
Neon-
40-EsB 

5 mm 

Calibration sample 
with Series No. IMS-
HR 083641-01380 
from Carl Zeiss 
Microscopy GmbH. 
Deviation: specified 
< 3 %, measured 
1 %. 

L20 

TEM 

100 kV 

NIST RM 8013 

JEOL 
2100 

Dilution of the 

Sample 10 to 20x 
diluted with 
ultrapure water 

2 m L of the 
diluted 
suspension 
brought onto a 
carbon-coated 
grid (Cu 400 
mesh) that was 
placed on a 
carbon pad and 
air dried in a 
clean room 

1:800 dilution 
with 0.1 m m 
filtered ultrapure 
water 
(18.2 MW

.cm) 

6  m L of the 
diluted 
suspension drop 
cast onto a 
carbon coated 
grid (Cu 400 
mesh), > 6 hr 
drying in 
cytotoxic cabinet 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

In-lens SE, 
aperture of 
30 m m 

CCD 
camera 
type 

Total 
sam-
pled 
area 

m m2] 
[m

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

Image analysis software 
- Image analysis strategy 

Bin 
size 
[nm] 

n.a. 

500 

100 

1.2 

6336 

iTEM and ImageJ 

0.1 

Manual grey-scale 
thresholding, touching 
particles were manually 
excluded, particle sizes 
were automatically 
measured. Image filters 
were not used 

168 to 
341 

10 

1 to 
2 

Bright-field 
with 
objective 
aperture of 
40 m m 

Gatan 
Ultra-
scan 
1000 
CCD 

Pixels 
2k x 2k 

8945 

ImageJ version 1.45s 

1.0 

Semi-automated particle 
detection, manual setting 
of contrast and brightness, 
7x7 filter, particles 
touching or cut by field of 
view excluded from 
analysis 

59 

 
 
m
m
 
 
 
Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

Dilution 50x with 
ultrapure water 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

Bright-field 
with 
objective 
aperture of 
500 m m 

Lab 
code 

Instru-
ment 
type / 
Make 

Accele-
ration 
voltage - 
SEM 
working 
distance 

L27a 

TEM 

200 kV 

LIBRA 
200 FE 
(Zeiss) 

L27b 

SEM 

5 kV 

4.5 mm 

Carl 
Zeiss 
Ultra 55 
FE 

Mag*I*Cal single-
crystal silicon 
reference standard 
(Technoorg-Linda 
Ltd.) 

Traceable to SI Unit, 
metre, through 
interplanar lattice 
spacing of a silicon 
crystal 

400 nm pitch 
spacing standard 
(Chinese Academy 
of Sciences) 

Pitch size standard 
SI traceably 
calibrated on 
metrological AFM 

CCD 
camera 
type 

Total 
sam-
pled 
area 

m m2] 
[m

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

Image analysis software 
- Image analysis strategy 

Bin 
size 
[nm] 

20 

0.6 

6389 

Telesco-
pic 
SSCCD 
Camera 
(Gatan 
894.20p.
2) 

1 

1 

The Buehler Omnimet 
image system was used 
for image processing and 
diameter measurement. 
The particles were 
separated manually with 
thresholds and finally the 
diameters are derived 
from the area of the 
particles. 

Image J software for 
image processing and 
diameter measurements. 
The particles were 
separated automatically 
with threshold methods 
and the diameters are 
derived from the areas of 
the particles. 

The modal diameter were 
estimated by fitting the 
histogram with a normal 
distribution 

Dilution 100x 
with ultrapure 
water 

30 m m 
aperture 
size 

n.a. 

30 

3.7 

6720 

60 

 
 
m
m
 
 
 
 
 
 
Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

CCD 
camera 
type 

Total 
sam-
pled 
area 

m m2] 
[m

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

Image analysis software 
- Image analysis strategy 

Bin 
size 
[nm] 

Lab 
code 

Instru-
ment 
type / 
Make 

Accele-
ration 
voltage - 
SEM 
working 
distance 

L33 a 

SEM 

20 kV 

JEOL 
JSM-
6500F 

Lens 
detector 

n.a 

11.8 to 
18.2 

Agar Scientific Ltd. 
S1930 Silicon Test 

Specimen Certified 
Specimen No. A877 

Sample was 
used as 
received. 

The TEM grids 
(copper, 200 
mesh, carbon 
only film) was 
dipped in silica 
solution 

100 

0.9 

6154 

NIH ImageJ software. 

Touching particles were 
measured only if their 
complete circumference 
was clearly visible. 
Microsoft Excel was used 
to determine the area 
equivalent circular 
diameter, median and 
modal particle diameters, 
and generate particle size 
distributions. Brightness, 
contrast and grayscale 
were not adjusted for 
particle sizing. 

Brightness,contrast and 
grayscale were not 
adjusted for CRM particle 
sizing 

100 

0.5 

6177 

NIH ImageJ software. 

Touching particles were 
measured only if their 
complete circumference 
was clearly visible. 
Microsoft Excel was used 
to determine the area 
equivalent circular 
diameter, median and 
modal particle diameters, 
and generate particle size 
distributions. Brightness, 
contrast and grayscale 
were only adjusted for 
particle sizing of QCM. 

L33 b 

TEM 

100 kV 

Philips 
CM120 

Lens 
detector 

Mag-i-Cal calibration 
standard 

Traceable to SI Unit, 
metre, through 
interplanar lattice 
spacing of a silicon 
crystal 

The TEM grids 
(copper, 200 
mesh, carbon 
only film) was 
dipped in the as 
received silica 
solution 

12 to 
17.1 

Scienti-
fic 
Instru-
ments & 
Applica-
tions 
(SIA) 2k 
by 2k 
CCD 
camera 

61 

 
 
m
m
 
 
Accele-
ration 
voltage - 
SEM 
working 
distance 

30 kV 

5mm 

Lab 
code 

Instru-
ment 
type / 
Make 

L34 

SEM 

Zeiss 
Leo 
Supra 
35 VP 

Magnification 
calibration - 
Metrological 
traceability 

Specimen 
preparation 

Calibration with 
calibrated artefact 
containing a 2D 
grating with a pitch 
of 144 nm and 
700 nm 

Traceable 
calibration of the 
grating by laser 
diffractometry 

Sample was 
used as 
receveid. 

1  to 2 µL on 
200 mesh TEM 
grids covered 
with carbon film 
( type S160 from 
Plano GmbH) 

L29 

TEM 

200 kV 

Tecnai 
G2 20 
S-
TWIN, 
FEI 

Calibration with gold 
standard sample 
with grid (300 
meshes). 

Calibration at low 
magnification with 
grId and Au lattice at 
high magnification 
grid 

1000x dilution 
with ultrapure 
water 

3 µL of diluted 
sample copper 
grid with carbon 
film, 200 
meshes 

SEM 
electron 
detector 
type - TEM 
imaging 
mode 

Five solid 
state 
electron 
detectors, 

Four 
detectors 
used as dark 
field 
detectors 
and the fifth 
one use for 
bright-field 
imaging 

(transmis-
sion mode) 

Bright field 

CCD 
camera 
type 

n.a 

Total 
sam-
pled 
area 

m m2] 
[m

329 to 
3848 

Image 
magnifi-
cation 
[1000x] 

Pi-
xel 
size 
[nm] 

Total 
# of 
parti-
cles 
coun-
ted 

100 

4.5 

9058 

Bin 
size 
[nm] 

1 
and 

2  

Image analysis software 
- Image analysis strategy 

Image Analysis in several 
steps : 

Global thresholding 
Iterative determination of 
threshold (and size) 
Interpolation of the image 
and final size determination 
Particle selection (only 
particle in the size range 60-
100 nm with circularity 
above 0.8 were analysed) 
statistics 

200 KV, 
CCD 
camera, 

1.295 
per 
sam-
ple 

13.8 

1.1 

8066 

Image J 

1 

treatment of particles cut 
by the measurement 
frame (ISO 13322-1:2004) 

62 

 
 
m
m
 
 
 
Table D.4 Particle tracking analysis: relevant instrumental and method details (as reported by the participants) 

Lab 
code 

Instrument 
type / make 

Aliquot preparation 

Laser 

Source  Wave-
length 
[nm] 

Power 
[mW] 

Came-
ra type 

Aliquot 
volume 
[mL] 

Measu-
rement 
duration  

[s] 

Came
-ra 
shut-
ter 

Analy
-sis 
soft-
ware 

Bin 
width 
[nm] 

Calibra-
tion or 
PQ 

L9 

NanoSight 
LM10 HSB 

1 µL diluted in ultrapure water 
1:5000 (final dilution) 

Diode 

640 

40 

CCD 

0.25 

60 

n.a 

L10 

NanoSight 
LM10 HSBF 

L11 

NanoSight 
NS500 

L20 

NanoSight 
LM14 

Aliquots were prepared in a 
class II cabinet. Test portions 
were taken from the ampoule 
with a clean pipette using a new 
tip for each aliquot. 50 m L of the 
as-received material was diluted 
in 450 µL MilliQ water ( filtered 
through 0.1 µm filter), 20 000x 
dilution 

Test portions taken from the 
ampoules were 6000x diluted in 
pre-filtrated ultrapure water that 
was passed through a filter with 
nominal pore sizes of 0.1 m m. 

The sample was diluted (1000x 
to 2000x) by ultrapure water. 
Sample preparation was done 
in a cytotoxic cabinet fitted with 
a high-efficiency particulate air 
(HEPA) filter. 

L27 

NanoSight 

LM20 

Original sample dilute 2500 to 
5000x in ultrapure water 

Sample prepare in clean room 
(class 6) 

NTA 
2.3 

n.a 

By 
manufac-
turer 

NTA 
2.3 

n.a 

QC with 
NIST RM 
8012-
8013 

Diode 

405 

60 

sCMOS 

0.25 

60 

1000 

Diode 

405 

< 60 

EMCC
D 

0.25 

60 

n.a. 

NTA 
3.0 

n.a. 

Diode 

532 

50 

sCMOS 

0.5 

60 

5 

laser 

638 

40 

0.25 

60 

n.a 

NTA 
2.3 

n.a. 

NTA 
2.3 

n.a 

QC with 
NIST RM 
8012 

Calibra-
tion with 
Thermo-
Scientific 
PSL 
(100 nm) 

Calibra-
tion with 
Thermo-
Scientific 
PSL 
3010 
(100 nm) 

63 

 
 
L30 

NanoSight 
NS500 

10 µL sample is diluted 100 x 
with ultrapure water (filtered on 
0.1 µm filter). 20 µL of this first 
dilution is diluted in 1980 µL 
ultrapure water to reach a 10 
000 X final dilution. 

laser 

532 

EMCC
D 

0.25 

60 

33 

NTA 
2.3 

n.a 

L31 

NanoSight 
LM10 HSB 

Dilution 10000x with ultrapure 
water 

Diode 

405 

<40 

sCMOS 

0.5 

60 

n.a 

NTA 
3.0 

n.a 

QC with 
Thermo-
Scientific 
PSL 
3100A, 
3200A 

QC with 
Thermo-
Scientific 
PSL 
3050A, 
3100A, 
3200A 

Table D.5-1 Small-angle X-ray scattering (SAXS): relevant instrumental details (as reported by the participants) 

Lab 
code 

L21 

Base instrument 

X-ray generator / 
source 

X-ray beam / optics 

Detector 

Aliquot 
preparation 
Temperature 

/ 

Sample 
holder / 
container 

X'Pert 
(PANalytical), 
Expert 
camera 

PRO 

SAXS 

X-ray tube, Cu-Ka

Line 
with 
focus, 
elliptical  X-ray  mirror  / 
monochromator 

PIXcel linear, 
solid state 
detector 
(PANalytical) 

1 part sample / 2 
parts  ultrapure 
water / 24 °C 

Disposable 
Mark tubes 
(quartz glass) 

Half of the 
scattering 
angle range [°°°°] 

-0.10 to 4 

Aliquot 
volume  

m L] 
[m

30 

L22 

BESSY 
synchrotron 

L25 

SAXSpace  (Anton 
Paar) 

L26  CREDO 

L35 

SAXSess 
Paar) 

(Anton 

Synchroton 
radiation (photon 
energy 6000 eV) 

X-ray tube IDE 
3003, Cu-Ka,  40 kV 
/ 50 mA (General 
Electric) 

Microfocus X-ray 
tube, GeniX3D Cu 
ULD, Cu-Ka 
(Xenocs) 

X-ray tube, Cu-Ka,
2.2 kW 
(PANalytical) 

4-crystal 
monochromator, 
pinhole 
(0.5 x 0.5 mm2) 

collimation 

Line collimation 

Pilatus 1M 
(Dectris) 

received 

As 
25 °C 

/ 

Borosilicate 

0.037 to 1.6 

20 

capillary 

Mythen 1k, strip 
size 50 µm 

received 

As 
25 °C 

/ 

Quartz 
capillary 

0.03 to 2.84 

25 

Parabolic  X-ray  mirror 
/ 
monochromator, 
pinhole collimation 

Pilatus-300k 
(Dectris) 

received 

As 
25 °C 

/ 

Capillary 

0.031 to 1.31 

20 

Line collimation 

1D diode array  As 

received 

/ 

23 °C 

Flow-through 
capillary  

0.025 to 1.40 

20 

64 

 
 
 
m
m
 
 
 
Table D.5-2 SAXS - Guinier approximation: relevant instrumental and method details (as reported by the participants) 

Lab 
code 

Measured q-
range 

Largest measurable size 
according to ISO 17867* 

q-range used for linear 
fit of the QCM data 

q-range used for linear 
fit of FD101b data 

Calculation of RG 

[1/nm] 

L21 

[0.045, 2.8] 

L22 

[0.021, 0.87] 

L25 

[0.021, 2] 

L26 

[0.044, 1.862] 

L35 

[0.03652, 2] 

[nm] 

140 

300 

300 

143 

172 

[1/nm] 

[1/nm] 

[0.065, 0.135] 

[0.045, 0.061] 

[0.05, 0.15] 

[0.025, 0.041] 

[0.042, 0.07] 

[0.042, 0.07] 

[0.048, 0.080] 

[0.044, 0.1] 

Fit part of Gaussian to I(q) (via linear fit in 
ln(I(q)) – q2 plot) 

Fit part of Gaussian to I(q); iteratively over 
different q-ranges, until qG,max = 1.3/RG 

Fit part of Gaussian to I(q) (via linear fit in 
ln(I(q)) – q2 plot) 

Fit part of Gaussian to I(q) (via linear fit in 
ln(I(q)) – q2 plot) 

[0.036, 0.16] 

[0.036, 0.070] 

Fit of part of Gaussian to I(q) 

Table D.5-3 SAXS - model fitting approach: relevant instrumental and method details (as reported by the participants) 

Lab 
code 

Name used by lab to 
describe method (reference) 

Assumptions 
about particles 

Assumptions about 
size distribution 

Weighting 

Averaging / 
calculation of 
mean/reported value  

Transformed to 
other 
weighting(s)? 

in acc. with ISO 17867, 9.3, 
Model Fitting (using EasySAXS 
v 2.0 software, PANalytical) 

Homogeneous 
spheres 

in acc. with ISO 17867, 9.3, 
Model Fitting 

solid spheres 

Monomodal, Gaussian 
shaped, 100 size 
classes 

Bimodal  (main mode 
Gaussian, 2nd mode 
lognormal) 

in acc. with ISO 17867, 9.3, 
Model Fitting 

Homogeneous 
spheres 

Multimodal (20 cubic 
splines used to fit data) 

in acc. with ISO 17867, 9.3, 
Model Fitting 

homogeneous 
spheres 

Bimodal, Gaussian 

L21 

L22 

L25 

L26 

L35 

Volume 

Mode (R50) of fitted 
Gaussian peak 

Analytically, to 
intensity 

Number  

Mode of Gaussian peak 

Analytically, to 
volume and intensity 

Separate fits for 
volume and 
intensity 

Separate fits for 
Number, volume 
and intensity 

Mode of peak in size 
distribution curve 

Mode of Gaussian peak 

- 

- 

- 

Model fit 

spheres 

Monomodal, Gaussian 

Number 

Mode of Gaussian peak 

65 

 
 
 
 
 
Table D.6 Asymmetrical flow field-flow fractionation: relevant instrumental and method details (as reported by the participant) 

Lab code 

Instrument 
make 

type 

/ 

Aliquot preparation 

Measurement parameters 

Calibration 

JRC -
Directorate F 

AF2000 MF 
(Postnova Analytics 
GmbH, DE) 

Test samples were diluted 
to 
using 
1 mg/mL 
ultrapure water 

0.2  min  with  a  constant  cross  flow  of  1.0  mL/min, 
40  min  with  linearly  decaying  cross  flow  (1.0 
mL/min to 0 mL/min), 5 min without cross flow 

standards 
latex 
Polystyrene 
Thermofisher (3030A, 3040A, 3050A, 
3080, 3100A, 3125A, 3150A) 

Table D.7  Zeta potential, pH and electrolytic conductivity: relevant instrumental and method details (as reported by the participant) 

Lab code 

Physical 
property 

Instrument  type  /  make 
/ specifications 

Aliquot preparation 

Instrument calibration 

Sample holder 

Aliquot 
volume 
[mL] 

Malvern Zetasizer Nano 
ZS 

Light source: He-Ne, 
Power 4 mW, 
Wavelength 633 nm, 
Detector: APD, 13° 
angle, Model: 
Smoluchowski 

744 pH Meter (Metrohm 
AG) 

Solitrode electrode with 
Pt1000 temperature 
sensor (Metrohm AG) 

Malvern Zetasizer Nano 
ZS 

JRC -
Directorate F 

Zeta 
potential 

JRC -
Directorate F 

pH 

JRC -
Directorate F 

Electrolytic 
conductivit
y 

Test samples were analysed as-
received. Before loading of the 
sample, the measurement cell 
was pre-rinsed with ethanol of 
analytical grade and abundantly 
rinsed with ultrapure water. 

n.a. 

Polycarbonate folded 
capillary cell with gold-
plated beryllium/copper 
electrodes 

0.75 

2-point calibration using 
buffer solutions (Metrohm 
AG) with nominal pH values 
of 9.00 ± 0.02 (art. no. 
6.2305.030) and 4.00 ± 
0.02 (art. no. 6.2305.010) 

Measurement in 
ampule before sample 
uptake 

n.a 

Polycarbonate folded 
capillary cell with gold-
plated beryllium/copper 
electrodes 

0.75 

Test samples were analysed as-
received. 

Test samples were analysed as-
received 

66 

 
 
 
 
 
 
 
 
 
 
 
 
Annex E: Results of the characterisation measurements 

Annex E1: Results of the characterisation measurements – DLS 

Table  E1.1  –  Scattered  light  intensity-weighted  harmonic  mean  particle  diameter  obtained  by 
DLS (cumulants method). 

Lab -
code 

Replicate results [nm] 

1 

2 

3 

4 

5 

6 

7 

8 

9 

Mean 
[nm] 

s 1) 
[nm] 

U 2) 
[nm] 

L2 

90.7 

90.6 

90.6  90.6 

90.1 

90.3  90.9  90.7 

90.7 

90.6 

L3 a 

87.1 

86.9 

87.2  87.3 

87.6 

86.8  87.1  87.2 

86.8 

87.1 

L3 b 

90.0 

89.5 

89.5  89.9 

90.0 

90.0  89.3  89.0 

89.3 

89.6 

L4 

L5 

L6 

L7 

L8 

L9 

L13 

L19 

L20 

L23 

L24 

L27 

L36 

90.4 

90.2 

90.1  90.5 

90.2 

90.3  90.4  90.3 

90.4 

90.3 

95.3 

90.8 

90.2  91.4 

90.8 

90.6  91.5  90.7 

92.1 

91.5 

83.8 

83.6 

83.6  83.7 

83.6 

83.5  83.7  83.6 

83.6 

83.6 

90.6 

88.5 

89.0  88.5 

89.2 

88.5  90.1  89.7 

88.8 

89.2 

90.4 

90.4 

90.9  91.8 

92.5 

91.9  91.1  91.1 

90.3 

91.2 

91.1 

90.6 

91.4  89.9 

91.0 

91.2  91.2  91.3 

91.3 

91.0 

90.5 

90.3 

90.3  89.9 

90.4 

90.2  90.1  89.7 

89.8 

90.1 

90.3 

90.3 

90.9  90.6 

90.5 

91.0  91.2  91.0 

91.2 

90.8 

90.3 

90.6 

90.4  90.6 

90.6 

90.4  90.5  90.6 

91.2 

90.6 

90.1 

89.6 

89.5  90.0 

90.2 

90.3  89.8  88.8 

90.1 

89.8 

88.6 

89.2 

89.7  90.5 

90.0 

90.2  86.8  89.9 

90.2 

89.5 

89.9 

88.3 

89.9  89.4 

89.9 

88.6  89.2  88.3 

89.2 

89.2 

88.5 

89.1 

88.2  87.7 

89.9 

88.5  89.0  87.4 

87.4 

88.4 

Results not used for certification 

L1 

94.5 

93.5 

93.2  93.2 

94.2 

94.4  93.7  92.7 

92.0 

93.5 

L12 

L29 

L32 

90.4 

90.0 

90.3  90.2 

91.0 

90.4  89.7  89.6 

89.7 

90.1 

89.3 

90.1 

89.3  90.1 

89.41 

90.1  89.8  89.1 

89.3 

89.6 

86.9 

87.1 

87.1  87.9 

88.3 

87.9  87.5  87.8 

87.6 

87.6 

0.2 

0.3 

0.4 

0.1 

1.5 

0.1 

0.8 

0.8 

0.5 

0.3 

0.4 

0.3 

0.5 

1.2 

0.7 

0.8 

0.8 

0.4 

0.4 

0.4 

1.6 

3.0 

3.0 

3.1 

8.2 

6.7 

17.8 

6.9 

4.0 

3.4 

1.5 

1.6 

1.8 

17.9 

4.5 

2.7 

0.4 

2.5 

3.6 

1.8 

1) Standard deviation of the mean aliquot results 
2) Expanded uncertainty (k = 2) as reported by the participants 

67 

 
 
 
 
 
 
 
 
Table  E1.2  –  Scattered  light  intensity-weighted  mean  particle  diameter  obtained  by  DLS 
(distribution calculation methods) 

Replicate results [nm] 

1 

2 

3 

4 

5 

6 

7 

8 

9 

Mean 
[nm] 

s 1) 
[nm] 

U 2) 
[nm] 

Lab -
code 

L2 

L4 

L5 

L8 

L9 

94.9 

94.5  94.9  94.4  94.4  94.1 

95.1 

94.8 

94.7 

94.6 

0.3 

91.9 

91.8  91.9  91.9  92.1  92.0 

92.0 

92.2 

92.1 

92.0 

0.1 

93.2*  91.2  90.8  92.1  89.2  90.7 

93.9 

90.4*  92.9 

91.6 

1.5 

94.5 

94.5  95.2  96.3  96.9  96.6 

95.6 

95.5 

94.4 

95.5 

0.9 

94.5 

94.7  94.7  94.4  94.8  95.1 

94.8 

95.2 

95.5 

94.9 

0.3 

L13 

90.4 

90.1  90.2  89.9  90.3  90.1 

90.0 

89.6 

89.6 

90.0 

0.3 

L19 

93.8 

93.6  94.7  94.1  94.2  94.9 

94.9 

94.9 

94.7 

94.4 

0.5 

L20 

91.7 

91.9  91.6  92.0  91.6  91.6 

92.3 

92.5 

94.2 

92.2 

0.8 

L23 

93.5 

93.6  93.5  94.3  94.3  94.0 

93.8 

92.6 

94.0 

93.7 

0.5 

1.7 

3.1 

8.2 

7.3 

4.2 

3.4 

1.5 

1.7 

1.9 

L24 

94.9 

94.5  94.9  94.4  94.4  94.1 

95.1 

94.8 

94.7 

94.6 

0.3 

19.0 

L36 

89.6 

89.6  88.3  89.0  91.3  89.8 

90.3 

89.5 

88.4 

89.5 

0.9 

2.7 

Results not used for certification 

L1 

L12 

L29 

97.8 

97.8  97.4  97.0  97.8 

98.8  97.2 

96.7 

96.2  97.4 

94.0 

94.2  94.8  94.2  94.8 

94.3  94.8 

94.6 

95.6  94.6 

93.2 

93.7  93.7  93.5  93.3 

93.3  93.2 

93.8 

93.6  93.5 

0.3 

0.3 

0.2 

0.4 

1.9 

3.7 

1) Standard deviation of the mean aliquot results 
2) Expanded uncertainty (k = 2) as reported by the participants 

* For these aliquots, only two replicates were used for the calculations of the mean. One 
replicate was removed after evaluation of the data. 

68 

 
 
 
 
)

m
n
(

i

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e
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e
m
a
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e
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e
t
t
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110

105

100

95

90

85

80

75

70

)
°
3
7
1
(

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L

)
°
0
9
(

a
3
L

)
°
3
7
1
(

b
3
L

)
°
3
7
1
(

4
L

)
°
0
9
(

5
L

)
°
0
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(

6
L

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°
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(

7
L

)
°
3
7
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(

8
L

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°
3
7
1
(

9
L

)
°
3
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(

3
1
L

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°
3
7
1
(

0
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L

)
°
3
7
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(

3
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L

)
°
0
9
(

7
2
L

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°
3
7
1
(

9
1
L

)
°
0
9
(

4
2
L

)
°
0
9
(

6
3
L

Fig. E1.1  Laboratory  mean  values  (used for  certification)  of the DLS (cumulants method)  light 
intensity-weighted harmonic mean particle diameters as obtained by 15 laboratories; error bars 
indicate  the  expanded  (k = 2)  measurement  uncertainties  as  reported  by  the  participants.  The 
two horizontal lines reflect the certified range. 

115

110

105

100

95

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L
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4 N
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TIN 
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(m

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m

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a
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n

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ersio
L
N
6 N
o
m
4 In
ar
(h

v

n)
a
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(

3
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1
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2
L

Fig.  E1.2  Laboratory  mean  values  (used  for  certification)  of  the  DLS  (distribution  calculation 
methods) light intensity-weighted mean particle diameters as obtained by 11 laboratories; error 
bars indicate the expanded (k = 2) measurement uncertainties as reported by  the participants. 
The two horizontal lines reflect the certified range. 

69 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex E2: Results of the characterisation measurements – CLS 

Table  E2.1  Light  extinction-weighted  modal  Stokes  particle  diameter  results  obtained  by  CLS 
(turbidimetry) 

Lab- 
code 

L2 

L5 

L9a) 

L14 

1 

2 

87.1 

87.3 

81.5 

78.9 

87.7 

87.5 

85.7 

84.3 

L16b) 

92.3 

92.0 

L17 

L20 

L23 

83.3 

83.3 

87.9 

87.5 

89.5 

89.9 

Aliquot results [nm] 

3 

86.8 

83.0 

87.8 

84.6 

92.9 

83.1 

88.0 

89.5 

4 

87.3 

80.9 

87.9 

84.1 

91.9 

83.0 

87.6 

89.8 

5 

86.6 

82.8 

87.7 

84.4 

93.1 

83.1 

87.9 

89.2 

6 

87.1 

80.5 

87.8 

83.9 

92.3 

83.2 

87.7 

89.4 

Mean 
[nm] 

s 
[nm] 

U 
[nm] 

87.0 

81.3 

87.7 

84.5 

92.4 

83.2 

87.8 

89.6 

0.2 

1.5 

0.1 

0.6 

0.4 

0.1 

0.2 

0.3 

9.1 

8.9 

10.5 

8.5 

3.7 

8.3 

6.5 

9.0 

a) Laboratory L9 measured 4 aliquots per sample. Two replicates per sample (second and third 
measurements) were used for the calculations. 
b) Laboratory L16 measured 6 aliquots per sample. Two replicates per sample were used for the 
calculations. 

Table  E2.2  Mass-weighted  modal  Stokes  particle  diameter  results  obtained  by  CLS 
(refractometry). 

Lab- 
code 

L18 

L28 

Aliquot results [nm] 

1 

83.9 

84.7 

2 

83.1 

84.3 

3 

84.4 

84.7 

4 

82.5 

84.3 

5 

83.4 

85.3 

6 

83.0 

84.7 

Mean 
[nm] 

s 
[nm] 

U 
[nm] 

83.4 

84.7 

0.6 

0.3 

1.4 

3.1 

70 

 
 
 
 
 
)

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L

i

100

90

80

70

L 2

L 5

L 16

L 20

L 23

L 14

L 17

L 9

Fig  E2.1  Laboratory  mean  values  of  the  light  extinction-weighted  modal  Stokes  particle 
diameter  obtained  by  8  laboratories  using  CLS  (turbidimetry);  the  error  bars  indicate  the 
expanded (k = 2) measurement uncertainties as reported by the participants, the two horizontal 
lines reflect the certified range. 

100

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L18

L28

Fig  E2.2  Laboratory  mean  values  of  the  mass-weighted  modal  Stokes  particle  diameter 
obtained  by  2  laboratories  using  CLS  (refractometry);  the  error  bars  indicate  the  expanded 
(k = 2) measurement uncertainties as reported by  the participants (L28) or calculated from the 
QCM results (L18). 

71 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex E3: Results of the characterisation measurements – EM 

Table  E3.1  Number-weighted  modal  area-equivalent  particle  diameter  results  obtained  using 
EM 

Lab - 
code 

PSA 
Method 

Replicate [nm] 

1 

2 

3 

4 

5 

6 

L2 

L10 

L13 

TEM 

85.1 

87.0 

88.9 

87.0 

88.8 

88.9 

TEM 

85.5 

85.6 

85.8 

85.8 

85.8 

85.5 

SEM 

82.7 

82.4 

81.9 

82.4 

82.9 

82.7 

L15 a 

TEM 

82.7 

82.8 

83.8 

83.0 

84.4 

83.5 

L15 b 

SEM 

87.4 

87.6 

84.2 

86.6 

88.0 

88.3 

L20 

TEM 

81.0 

81.0 

82.0 

82.0 

84.0 

84.0 

L27 a 

TEM 

83.0 

82.2 

82.5 

80.9 

82.6 

82.0 

L27 b 

SEM 

83.0 

80.7 

81.6 

83.1 

79.5 

81.8 

L33 a 

SEM 

81.1 

79.4 

84.9 

81.5 

78.5 

78.8 

L33 b 

TEM 

81.4 

81.9 

83.8 

81.7 

82.2 

80.6 

L34 

TSEM 

85.9 

85.1 

84.7 

85.0 

86.0 

86.5 

Results not used for certification 

L9 a 

L9 b 

L29 

TSEM 

91.5 

90.7 

88.5 

88.1 

88.5 

90.4 

SEM 

85.5 

88.5 

88.1 

85.1 

87.5 

83.5 

TEM 

n. r. 

n. r. 

n. r. 

n. r. 

n. r. 

n. r. 

Mean 
[nm] 

s 
[nm] 

U 
[nm] 

87.6 

85.7 

82.5 

83.3 

87.0 

82.3 

82.2 

81.6 

80.7 

81.9 

85.5 

89.6 

85.9 

n. r. 

1.5 

0.2 

0.4 

0.7 

1.5 

1.4 

0.7 

1.4 

2.4 

1.1 

0.7 

1.4 

1.7 

n. r. 

5.3 

3.9 

3.1 

5.0 

5.2 

11.5 

5.4 

3.6 

3.6 

2.8 

5.1 

5.0 

10.6 

n. r. 

n.r.: no results reported 

72 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table E3.2   Number-weighted  median  area-equivalent  particle  diameter  results  obtained 
using EM 

Lab - 
code 

PSA 
Method 

Replicate [nm] 

1 

2 

3 

4 

5 

6 

Mean 
[nm] 

s 
[nm] 

U 
[nm] 

L2 

L 10 

L 13 

TEM 

TEM 

85.3 

86.2 

87.7 

86.0 

87.2 

87.3 

84.9 

85.2 

85.3 

85.7 

85.0 

85.0 

SEM 

82.8 

82.5 

82.0 

82.5 

83.1 

82.8 

L15 a 

TEM 

83.0 

81.6 

84.1 

82.8 

83.1 

84.3 

L15 b 

SEM 

87.9 

87.9 

83.7 

86.9 

88.0 

88.3 

L 27 a 

TEM 

81.3 

82.0 

83.0 

81.2 

82.1 

81.7 

L 27 b 

SEM 

83.1 

80.7 

82.3 

83.6 

80.2 

82.2 

L 20 

TEM 

80.0 

80.0 

81.0 

81.0 

84.0 

83.0 

L 33 a 

SEM 

81.2 

79.7 

82.5 

83.5 

78.6 

81.0 

L 33 b 

TEM 

80.0 

82.2 

83.2 

84.0 

80.5 

81.7 

L34 

TSEM 

85.1 

84.5 

83.9 

84.4 

85.3 

85.9 

Results not used for certification 

L9 

L9 

L29 

TSEM 

89.3 

89.0 

87.0 

85.7 

86.3 

89.0 

SEM 

TEM 

84.5 

84.8 

86.2 

85.0 

85.2 

83.5 

81 

78 

87 

80 

76 

76 

86.6 

85.2 

82.8 

83.2 

87.1 

81.9 

82.0 

81.5 

81.1 

81.9 

84.9 

87.7 

84.9 

79.7 

0.9 

0.3 

0.4 

1.0 

1.7 

0.7 

1.3 

1.6 

1.8 

1.5 

0.8 

1.6 

0.9 

4.1 

5.2 

3.9 

3.0 

5.0 

5.3 

5.4 

3.6 

8.2 

3.6 

3.0 

3.8 

4.9 

10.6 

15.9 

73 

 
 
 
 
 
 
100

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L2 -TEM

L10 -TEM

L 13 SEM

L15 a -TEM

L15 b-SEM

L 27 a-TEM

L 27 b-SEM

L 20-TEM

L 33 a-SEM

L 33 b-TEM

L34 -TSEM

Fig  E3.1  Laboratory  mean  values  of  the  number  weighted  modal  area-equivalent  diameter  as 
obtained by 9 laboratories using EM; the error bars indicate the expanded (k = 2) measurement 
uncertainties as reported by the participants, the two horizontal lines reflect the certified range. 

)

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90

80

70

L2 -TEM

L 10 -TEM

L 13 SEM

L15 a -TEM

L15 b-SEM

L 27 a-TEM

L 27 b-SEM

L 20-TEM

L 33 a-SEM

L 33 b-TEM

L34 -TSEM

Fig E3.2 Laboratory mean values of the number weighted median area-equivalent diameter as 
obtained by 9 laboratories using EM ; the error bars indicate the expanded (k = 2) measurement 
uncertainties as reported by the participants, the two horizontal lines reflect the certified range. 

74 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex E4: Results of the characterisation measurements – PTA 

Table E4.1 Number-weighted modal hydrodynamic particle obtained by PTA  

Lab-
code 

Aliquot mean results [nm] 

1 

2 

3 

4 

5 

6 

7 

8 

9 

mean 
[nm] 

s 
[nm] 

U  
[nm] 

L9 

73.8  76.8  78.0  76.8  77.6  77.0  77.4  77.0  76.0  76.7 

L10 

L11 

L20 

L27 

L30 

L31 

82.5  81.8  83.3  84.2  83.5  79.5  81.5  81.3  83.2  82.3 

80.4  80.6  80.5  81.5  81.1  80.6  81.3  79.8  80.0  80.6 

84.4  82.0  85.2  84.0  83.2  84.6  82.8  83.4  84.0  83.7 

76.8  77.2  76.8  77.6  77.6  78.6  77.4  77.8  77.4  77.5 

87.6  85.0  85.4  83.6  86.8  84.2  85.4  83.6  84.2  85.1 

87.1  85.7  85.9  89.1  89.1  87.0  85.6  85.3  85.7  86.7 

1.2 

1.4 

0.6 

1.0 

0.5 

1.4 

1.5 

8.3 

9.2 

1.2 

7.0 

4.3 

1.7 

6.1 

Table E4.2 Number weighted arithmetic mean hydrodynamic diameter obtained by PTA  

Aliquot mean results [nm] 

1 

2 

3 

4 

5 

6 

7 

8 

9 

mean 
[nm] 

s 
[nm] 

U  
[nm] 

84.3  83.6  85.9  85.9  84.4  86.3  83.6  84.1  83.7  84.6 

1.1 

87.2  86.7  88.4  90.6  88.4  86.1  88.3  87.1  87.6  87.8 

82.7  82.1  82.7  82.9  82.9  82.4  82.2  80.2  80.0  82.0 

1.3 

1.1 

9.0 

9.8 

2.4 

93.0  92.1  95.2  94.8  92.6  94.5  96.0  94.3  96.4  94.3 

1.5 

17.4 

83.0  84.2  83.8  84.8  84.6  84.4  83.2  84.8  84.6  84.2 

90.8  89.8  89.0  89.6  91.0  88.0  90.2  87.8  88.4  89.4 

L31   84.3  82.1  82.7  84.7  85.5  84.6  82.3  81.0  81.7  83.2 

0.7 

1.2 

1.6 

4.4 

1.4 

5.8 

Lab-
code 

L9 

L10 

L11 

L20 

L27 

L30 

Table E4.3 Number weighted median hydrodynamic diameter obtained by PTA  

Lab-
code 

Aliquot mean results [nm] 

1 

2 

3 

4 

5 

6 

7 

8 

9 

mean 
[nm] 

s 
[nm] 

U  
[nm] 

L9 

78.3  79.2  80.8  81.0  80.2  80.8  79.8  80.0  79.2  79.9 

0.9 

L10  83.8  83.2  84.3  86.0  84.7  81.8  83.2  83.0  84.0  83.8 

1.2 

L11  80.5  80.7  80.9  80.5  80.3  80.9  81.1  78.3  79.9  80.3 

0.8 

L20  87.3  86.4  88.8  88.2  86.7  88.3  89.0  88.3  88.4  87.9 

0.9 

L27  80.6  80.6  79.6  80.4  81.0  81.0  80.6  80.4  82.0  80.7 

0.6 

L30  87.8  86.8  86.2  86.2  88.0  85.4  87.2  85.2  85.4  86.4 

1.1 

L31  78.0  76.2  76.5  71.8  79.4  78.0  76.1  75.5  76.1  76.4 

2.1 

8.3 

9.4 

1.8 

9.1 

4.0 

1.2 

5.3 

75 

 
 
 
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90

80

70

L9

L10

L11

L20

L27

L30

L31 

Fig. E4.1  Laboratory  mean  values  of  the  number-weighted modal  hydrodynamic  diameters  as 
obtained  by  7  laboratories  using  PTA;  error  bars  indicate  the  expanded  (k = 2)  measurement 
uncertainties as reported by the participants, the two horizontal lines reflect the certified range. 

)

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100

90

80

70

L9

L10

L11

L20

L27

L30

L31 

Fig.  E4.2  Laboratory  mean  values  of  the  number-weighted  arithmetic  mean  hydrodynamic 
diameters  as  obtained  by  7  laboratories  using  PTA;  error  bars  indicate  the  expanded  (k = 2) 
measurement  uncertainties  as  reported  by  the  participants,  the  two  horizontal  lines  reflect  the 
certified range. 

76 

 
 
 
 
 
 
 
 
 
 
 
 
)

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110

100

90

80

70

L9

L10

L11

L20

L27

L30

L31 

Fig. E4.3 Laboratory mean values of the number-weighted median hydrodynamic diameters as 
obtained  by  7  laboratories  using  PTA;  error  bars  indicate  the  expanded  (k = 2)  measurement 
uncertainties as reported by the participants, the two horizontal lines reflect the certified range. 

77 

 
 
 
 
 
 
 
 
Annex E5: Results of the characterisation measurements – SAXS 
Table E5.1 (Volume)2-weighted mean particle diameter (Guinier's approximation)  

Lab 
code 

L21 

L22 

L25 

L26 

L35 

1 

86.8 

84.4 

86.6 

90.8 

81.4 

Replicate [nm] 

3 

85.9 

84.8 

87.9 

90.7 

83.8 

4 

86.3 

84.7 

88.5 

90.9 

82.4 

2 

86.5 

84.4 

89.0 

90.8 

81.4 

5 

86.5 

84.5 

89.2 

90.9 

81.6 

6 

86.9 

85.1 

96.4 

90.8 

81.3 

Mean  
[nm] 

s 
[nm] 

U 
[nm] 

86.5 

84.7 

89.6 

90.8 

82.0 

0.4 

0.3 

3.5 

0.1 

1.0 

4.2 

n.a. 

3.6 

7.4 

4.9 

Table E5.2 Number-weighted modal particle diameter (model fitting)  

Lab 
code 

L21 

L22 

L25 

L26 

L35 

Replicate [nm] 

1 

2 

3 

4 

5 

6 

81.87 

81.60 

81.32 

81.78 

81.54 

81.74 

81.09 

81.09 

81.12 

81.12 

81.10 

81.10 

80 

80.7 

80.4 

80.7 

79.4 

80.9 

79.4 

80.9 

82.9 

80.8 

79.2 

80.9 

80.52 

80.44 

81.28 

80.46 

80.20 

80.20 

Mean  
[nm] 

s 
[nm] 

U 
[nm] 

81.6 

81.1 

80.2 

80.8 

80.5 

0.20 

0.01 

1.39 

0.10 

0.40 

4.2 

1.3 

3.0 

3.4 

2.2 

Table E5.3 Volume-weighted modal particle diameter (model fitting)  

Lab 
code 

L21 

L22 

L25 

L26 

L35 

Replicate [nm] 

1 

2 

3 

4 

5 

6 

82.68 

82.41 

82.13 

82.59 

82.35 

82.55 

81.66 

81.66 

81.69 

81.68 

81.67 

81.67 

83.3 

81.4 

82.1 

81.4 

81.3 

81.34 

83.4 

80.48 

81.6 

81.6 

81.6 

81.6 

80.94 

80.86 

81.70 

80.88 

80.62 

80.62 

Mean  
[nm] 

s 
[nm] 

U 
[nm] 

82.5 

81.7 

82.0 

81.5 

80.9 

0.20 

0.01 

1.17 

0.10 

0.40 

4.2 

1.4 

3.0 

3.4 

2.2 

Table E5.4 Scattered X-ray intensity-weighted modal particle diameter (model fitting) 

Lab 
code 

L21 

L22 

L25 

L26 

L35 

Replicate [nm] 

1 

2 

3 

4 

5 

6 

83.22 

83.06 

83.05 

83.14 

83.07 

83.24 

82.22 

82.22 

82.24 

82.24 

82.23 

82.23 

84 

83 

82.8 

83.46 

83.6 

83.26 

82.2 

82.1 

82.3 

82.2 

82.3 

82.3 

81.36 

81.28 

82.12 

81.30 

81.06 

81.06 

Mean  
[nm] 

s 
[nm] 

U 
[nm] 

83.1 

82.2 

83.4 

82.2 

81.4 

0.08 

0.01 

0.43 

0.08 

0.39 

4.2 

1.5 

3.0 

3.4 

2.2 

78 

 
 
 
 
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L21

L22

L25

L26

L35

Fig.  E5.1  Laboratory  mean  values  of  the  (volume)2-weighted-mean  particle  diameters  as 
obtained  by  5  laboratories  using  SAXS  (Guinier  approximation).  The  error  bars  indicate  the 
expanded (k = 2) measurement uncertainties as reported by the participants, the two horizontal 
lines reflect the indicative range. 

100

95

90

85

80

75

70

)

m
n
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b
m
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L21

L22

L25

L26

L35

Fig.  E5.2  Laboratory  mean  values  of  the  number-weighted-modal  particle  diameters  as 
obtained  by  5  laboratories  using  SAXS  (model  fitting  approach).  The  error  bars  indicate  the 
expanded (k = 2) measurement uncertainties as reported by the participants, the two horizontal 
lines reflect the certified range. 

79 

 
 
 
 
 
 
 
 
 
 
 
100

95

90

85

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75

70

)

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n
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L21

L22

L25

L26

L35

Fig. E5.3 Laboratory mean values of the volume-weighted-modal particle diameters as obtained 
by  5  laboratories  using  SAXS  (model  fitting  approach).  The  error  bars  indicate  the  expanded 
(k = 2)  measurement  uncertainties  as  reported  by  the  participants,  the  two  horizontal  lines 
reflect the certified range. 

)

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100

95

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80

75

70

L21

L22

L25

L26

L35

Fig.  E5.4  Laboratory  mean  values  of  the  scattered  X-ray  intensity-weighted-modal  particle 
diameters  as  obtained  by  5  laboratories  using  SAXS  (model  fitting  approach).  The  error  bars 
indicate  the  expanded  (k = 2)  measurement  uncertainties  as  reported  by  the  participants,  the 
two horizontal lines reflect the certified range. 

80 

 
 
 
 
 
 
 
European Commission 

EUR 28362 EN – Joint Research Centre – Directorate F – Health, Consumers and Reference Materials 
Title: 

CERTIFICATION REPORT The certification of equivalent diameters of silica nanoparticles in aqueous 
solution: ERM®-FD101b 

Author(s): Y. Ramaye, V. Kestens, A. Braun, T. Linsinger, A. Held, G. Roebben 
Luxembourg: Publications Office of the European Union 
2017 – 80 pp. – 21.0 x 29.7 cm 
EUR – Scientific and Technical Research series
ISSN 1831-9424 
ISBN 978-92-79-64637-9 
doi:10.2787/212519 

As the Commission's in-house science service, the Joint Research Centre's mission is to provide EU policies 
with independent, evidence-based scientific and technical support throughout the whole policy cycle. 

Working in close cooperation with policy Directorates-General, the JRC addresses key societal challenges 
while stimulating innovation through developing new methods, tools and standards, and sharing its know-
how with the Member States, the scientific community and international partners. 

Key policy areas include: environment and climate change; energy and transport; agriculture and food 
security; health and consumer protection; information society and digital agenda; safety and security, 
including nuclear; all supported through a cross-cutting and multi-disciplinary approach. 

L
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doi:10.2787/212519

ISBN 978-92-79-64637-9