Developing Reference Methods for Nanomaterials 1 Date Event title www.nanovalid.eu Title of Event: NanoValid Summer School, Tallinn/ Estonia Date: June 16 th , 2014 Dispersion and characterisation of nanoparticles Partner representative name: Annegret Potthoff, Tobias Meißner Partner organisation name: Fraunhofer-Gesellschaft (Germany)
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Developing Reference Methods for Nanomaterials … · Developing Reference Methods for Nanomaterials 3 Slide title Topics Physico-chemical characterisation for toxicologic assessment
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Developing Reference Methods for Nanomaterials
1
Date
Event title
www.nanovalid.eu
Title of Event: NanoValid Summer School, Tallinn/ Estonia
Applications and examples Powder characterization Dispersion of nanoparticles prior to toxicological tests Proteins – natural dispersant aids How to analyze a nanostructured powder?
Conclusions
Developing Reference Methods for Nanomaterials
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Physico-chemical characterisation for toxicological assessment
ISO/TR 13014:2012
Physical description
Chemical composition
Extrinsic properties
Time
Batch
Which parameters?
How often?
When?
From what?
Nanomaterial
Developing Reference Methods for Nanomaterials
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What does it mean – nanomaterial?
Definition according to ISO/TS 80004-4:2011
Nanostructuredmaterial
Nanostructuredpowder
Agglomerates, aggregates,
nanoparticles
Nanocomposite Solid nanofoam
Nanoporousmaterial
Fluid nanodispersion
Material with an nanoscaled (1 nm – 100 nm) internal or external structure
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COMMISSION RECOMMENDATION on the definition of nanomaterial
2011/696/EU, recommendation of 18 October 2011
`Nanomaterial` means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm – 100 nm.
“Nanomaterials” by EU definition are among “nanostructured materials” according to ISO norm.
EU commission recommends, that about 50 % of particles – calculatedfrom a number weighted distribution – have to comply with this condition, while there is no similar definition in ISO norm.
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Materials considered in this presentation
Nanostructuredmaterial
Nanostructuredpowder
Agglomerates, aggregates,nanoobjects
Nanocomposite Solid nanofoam
Nanoporousmaterial
Fluid nanodispersion
Nanostructuredmaterial
Nanostructuredpowder
Agglomerates, aggregates,
nanoparticles
Nanocomposite Solid nanofoam
Nanoporousmaterial
Fluid nanodispersion
Independent on amount of nanoscaled material.
Developing Reference Methods for Nanomaterials
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Which pc parameters are the most relevant for toxicological assessment
ISO/TR 13014:2012
Physical descriptionParticle size/ particle size distributionState of agglomeration and aggregationParticle shapeSpecific surface area
Chemical compositionCompositionPurity/ impuritiesSurface chemistry
Colloid chemical stable suspensions might be stable against sedimentation,but they do not necessarily have to!
Value of zeta potential Repulsive forces between particles Electrostatic stability Agglomeration of particles
Value of zeta potential 0 Repulsive forces between particles Electrostatic stability Agglomeration of particles
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Slide title
Analysis of zeta potential
Measurement principle ConcentrationElectrophoretic light scattering * Up to 5 vol%Streaming potential Up to 5 vol%Electroosmosis Below 0,5 wt%Sedimentation potential Below 0,5 wt%Electro sonic amplitude * 1 to 40 vol%
ISO norms are available for
- sampling and sample splitting and- *-marked measurement principles.
Acceleration of particles in an electric field FE – Accelerating force Stokes friction acts against particle movement FR – Retarting force
v / E = q / 6 r When FE = FR constant particle movement
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Slide title
Zeta potential – calculation from electrophoresis
r < 0.1 r > 100
Large Particle with a thin Double Layer Small Particle with thick Double Layer
v / E = µe = 2 or f[r] / 3 HENRY Equation
µe = 2 r / 3 µe = r) / SMOLUCHOWSKI Equation
HUECKEL Equation
v / E = q / 6 r
Correction for surface conductivity Correction function for changes of dielectricity and viscosity Correction for particle shape Corrections for high ion and particle concentrations
Developing Reference Methods for Nanomaterials
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Slide title
Analysis of particle sizes
Sample preparation finalized successfully
Developing Reference Methods for Nanomaterials
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Slide title
Analysis of particle size distributions of dispersed nanomaterials in liquids
Diffusion velocity – lowScattered light intensity – high
Correlogram
Correlation function
Developing Reference Methods for Nanomaterials
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Slide title
Dynamic light scattering (DLS) – result
Depending on sample preparation hydrodynamic diameter xDLS maydescribe diameter of primary particles, aggregates or agglomerates
Calculation of intensity-weighted particle size distribution,conversion into volume-weighted particle size distribution possible
DTkx B
3 Calculation of particle size from diffusion coefficient: xDLS
Polydispersity index PI (between 0 and 1)
Developing Reference Methods for Nanomaterials
Intensity-weighted distribution Volume-weighted distribution
Exam
ple
1Ex
ampl
e 2
Dynamic light scattering (DLS) – Comparison of different approachesPresence of nanoparticles and serum proteins in cell culture medium Analysis using mean particle size and PI (cumulant method) difficult
Usage of complex algorithm calculating size distributions
Developing Reference Methods for Nanomaterials
Intensity-weighted distribution Volume-weighted distribution
Exam
ple
1Ex
ampl
e 2
Dynamic light scattering (DLS) – Comparison of different approachesPresence of nanoparticles and serum proteins in cell culture medium Analysis using mean particle size and PDI (cumulant method) difficult
Usage of complex algorithm calculating size distributions
Simultaneous detection of proteins and particles not always possible usingDLS scattered light intensity r6-dependency
Simple cumulant method “detects” proteins via increased PI, but mean sizeis error-prone SiO2 particles appear smaller than they are
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Slide title
Volume-weighted or number-weigthed concentration?
Vtennis balls!=
Vmedicine ball
xtb xmb xtb xmb
Number-weighted distribution Volume-weighted distribution
Nano?40 % below 100 nm
95 % below 100 nm
10 100 1000 10000Particle size [nm]
Developing Reference Methods for Nanomaterials
Dynamic Light Scattering vs. Nanoparticle Tracking Analysis
DLS NTAAnalysed particleproperty
Brownian motion Brownian motion
Analysis of particlevelocity
Time dependent lightscattering
Video analysis of particletracking
Calculated result Diffusion coefficient fromEinstein equation Hydrodynamic diameter
Energy-input-dependent analysis Specify specific energy input
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First results of a round-robin test
500 nm
Silica OX50
BET: 50 m²/gxBET ~ 40 nm
SOP with detailed specification of concentration, dispersion, energy input etc. 4 out of 7 partners receive similar results
Developing Reference Methods for Nanomaterials
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Slide title
Example 3: Zeta potential analysis in toxicological tests
Due to adsorptionof proteins nanoparticles changetheir behaviour in presence, i. e. of LSZ.
Proteins may act as dispersant aids xDLS remains constant.
-20
-15
-10
-5
0
5
10
15
20
2 3 4 5 6 7 8 9 10 11 12 13
pH
zeta
pot
entia
l [m
V]
tungsten carbide (WC)lysozyme (LSZ)WC + LSZ
Challenge: Analysis of zeta potentials in physiological mediaConductivity compression of electric double layer low values of zeta potential
Analysis of solid-liquid interface
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Slide title
Example 4: How to analyze a nanomaterial?
1 µm 200 µm
Nanostructured powder according to ISO/TS 80004-4:2011
Bad dispersability aggregates are micron-scaled and settle down very fast
DLS does not meet all requirements!
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Slide title
Example 4: How to analyze a nanomaterial?
Use same dispersion procedure as for nanoparticlesAnalysis by laserlight diffraction aggregates of up to 100 µm in size!
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Slide title
Conclusions
Dispersion decides, but is not yet standardized for nanotoxicological testing
“Nanomaterial” or not – sometimes a question of definition
Zeta potential - a tool for stability analysis
DLS or NTA? DLS + NTA + BET + SEM/TEM + … = results are complementary
DLS – a tool for analysis in physiological and in ecotoxicological relevant media
Particle size analysis of nanostructured materials sometimes requires devices other then DLS
As particle properties change depending on time, batch or surrounding behaviour, they always need to be investigated prior, while and after toxicological testing.