Developing Reference Methods for Nanomaterials … · Developing Reference Methods for...

Developing Reference Methods for Nanomaterials

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Date

Event title

www.nanovalid.eu

Title of Event: NanoValid Summer School, Tallinn/ Estonia

Date: June 16th, 2014

Dispersion and characterisation of nanoparticles

Partner representative name: Annegret Potthoff, Tobias Meißner

Partner organisation name: Fraunhofer-Gesellschaft (Germany)


© Fraunhofer

Wertheim/BronnbachMannheim

Karlsruhe

Pfinztal

Ettlingen Stuttgart

Freiburg

Kandern

Efringen-Kirchen

AlzenauWürzburg Bayreuth

Erlangen, Nürnberg, Fürth

Regensburg

Straubing

AugsburgWeßling

München, Garching

Freising

Prien

Holzkirchen

Bremen

Teltow

WildauPotsdam-Golm

Cottbus

Bremerhaven Hamburg

Kassel

Frankfurt

Darmstadt

Rostock

Oldenburg

Hannover

Braunschweig

Goslar

Göttingen

MünsterLemgo

PaderbornDortmund

Schmallenberg

Gelsenkirchen

Duisburg

Oberhausen

Köln

Euskirchen

AachenWachtberg

Bonn, Sankt Augustin

Kaiserslautern

Saarbrücken

St. IngbertSulzbach

Leipzig

Chemnitz

Freiberg

DresdenZittau

Magdeburg

Halle

Schkopau

Leuna

Itzehoe

Lübeck

ErfurtJena

HermsdorfIlmenau

Berlin

Sulzbach-Rosenberg

Remagen

66 institutes and independent research units

More than 22,000 staff Headquarter in München

Fraunhofer-Gesellschaft, the largest organisation for applied research in Europe

Fraunhofer Institute for Ceramic Technologies and Systems (IKTS) in Dresden

Delevopment of state-of-the-art advanced ceramic materials

Characterisation of materials Powder and suspension

characterisation


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Slide title

Topics Physico-chemical characterisation for toxicologic assessment

Relevance of nanomaterial definitions for particle characterisation

Dispersion decides

Colloid chemical stability of nanoparticles/ analysis of zeta potential

Particle size analysis Dynamic Light Scattering (DLS) Comparison to Nanoparticle Tracking (NTA)

Applications and examples Powder characterization Dispersion of nanoparticles prior to toxicological tests Proteins – natural dispersant aids How to analyze a nanostructured powder?

Conclusions


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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


5

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.


7

Materials considered in this presentation



Agglomerates, aggregates,nanoobjects


Nanoporousmaterial




Agglomerates, aggregates,

nanoparticles


Nanoporousmaterial


Independent on amount of nanoscaled material.


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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

Extrinsic propertiesSurface chargeSolubilityDispersability

Nanoparticle

Suspending media

Solid-liquid interface


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Slide title

How do they look like

Example: TiO2 P25 (Evonik)Nanoparticles form aggregates and agglomerates

Wikipedia/Nanogold

Nanostructured powders and nanocomposites Fluid nanodispersions

Example: Au dispersionNanoparticles are separated

Particle? Particle size? Particle size analysis?


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Slide title

How do they occur

Surface chemistry? Surface charge? Interactions at the interface?

Nel et al., 2009


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Dispersion decides

Prior to any analysis we have to check, what we would like to know!


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Characterisation of nanomaterials requires information about dispersability

Natural nanoparticledispersed in air

Eyjafjallajokull 2010

Oberdörster 2005

Inhalation studies fortoxicological testing

Dispersion in air Analysis after dry dispersion


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Characterisation of nanomaterials requires information about dispersability

Technical nanoparticledispersed in fluid

www.brand.de

Preparation prior to toxicologicalor ecotoxicological testing

Dispersion in liquid Analysis after wet dispersion


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Slide title

Dispersion decides

(1) Particle´s behaviour

Powders in liquids

Agglomerates or aggregates and primary particles?

Influencing factors

Kind of particle stress

Specific energy input (time, intensity)


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Slide title

Dispersion decides

(2) Particle´s behaviour in fluids

Charges at particle surfaces

Electrosteric effects

Influencing factors

pH value

Natural or artificial dispersantaids


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Slide title

Analysis of zeta potential

Colloid chemical stabilized?


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Slide title

Surface charges and zeta potential

Strongly bounded ions are fixed at particle surfaces (= inner and outer Helmholtz layer)

Loosely associated ions for charge compensation (= diffuse double layer)

Stern model describes conditions insystems with moderate ionic strength (10-4 … 10-2 mol/l).

Thickness of diffuse double layer depends on concentration and chargeof ions

Zeta potential: Difference between potential on shear plane and potentialwithin the fluid (example: negative)

diffuse Schicht

star

re S

chic

htFi

xed

laye

r

Diffuse double layer

She

ar p

lane


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Slide title

Colloid chemical stable?

Stable suspensions:suspendedor with sedimentation

Instable suspensions:flocculation orcoagulation (agglomeration)

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.


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Slide title

Electrophoretic light scattering (ELS) – measurement principle

E

FE = E . qFR = -6r v

Moving particle(negative charge)

- -- --- - - -

--

-

++

++

+

- - FEFR --

-

Shifted counter ion cloud

+

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


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Slide title

Analysis of particle sizes

Sample preparation finalized successfully


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Slide title

Analysis of particle size distributions of dispersed nanomaterials in liquids

Measurement principle Measurement range ConcentrationDynamic light scattering */ Photon cross correlationspectroscopy *

1 nm – 5 µm Up to 5 vol%

Nanoparticle tracking 10 nm – 1 µm Up to 108 particle/mLUltrasound attenuation * 4 nm – 40 µm 1 to 40 vol%Laserlight diffraction * 20 nm – 3000 µm Below 0,5 wt%Centrifugal sedimentation * 10 nm – 100 µm Below 0,5 wt%

ISO norms are available for

- sampling and sample splitting- dispersing and- *-marked measurement principles.


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Slide title

Dynamic light scattering (DLS) – measurement principle

Is 2

Is2

t

Autocorrelation function(depends on diffusion coefficient D)

Is

t

Fluctuation of scattered light intensity at detector

Laserlight

Input

Detector

Beam stopLaserlight

Output

Medium intensity

Correlator

Analyzation ofscattered light

Malvern


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Slide title

Dynamic light scattering (DLS) – measurement principle

Small particles:G1 vs time

500 1000 1500 2000Time(us)

0.2

0.4

0.6

0.8

G1

G1 vs time

500 1000 1500 2000Time(us)

0.2

0.4

0.6

0.8

G1

Large particles:

time t

time tI

I

Malvern

Diffusion velocity – highScattered light intensity – low

Diffusion velocity – lowScattered light intensity – high

Correlogram

Correlation function


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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)


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


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]


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

Diffusion coefficient fromEinstein equation Hydrodynamic diameter

Particle size range 1 nm – 5 µm 10 nm – 1 µmResults xDLS, PI;

intensity or volume weighteddistribution

Number weighted particlesize distribution,concentration

Concentration Up to 5 vol% Up to 108 particle/mLCompatibility ISO/TS 80004-4:2011 EU commission


Advantages and limitations

DLS NTALimitation due to sedimentation of large (or high-aggregated!) particles,

especially for high-density materialsGood for fluid nanodispersions and for well-dispersable nanostructured

powders and nanocomposites (narrow PSD)Few small particles very difficult todetect in presence of large particles

Small particles detectable in presenceof large particles

Detection of time stability of PSD possible (including agglomerationprocesses)

Detection of time stability of PSD should be possible

Analysis in physiological and in ecotoxicological relevant media possibleParticle concentrations as often testedin in-vitro tests

Particle concentrations as often testedecotoxicological tests


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Slide title

Applications and examples

Tools for sample preparation and particle size analysis available


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Slide title

Example 1: Powder characterisation

500 nm

Particle size= size of aggregates and primary particles= „smallest dispersable unit“

Requirements for dispersionColloid chemical stability high= high value of zeta potential dosage of dispersant necessary

Complete deagglomeration= high specific energy input dispersion by ultrasound (sonotrode)

Dispersion strategy prior topowder characterisation isdescribed in ISO 14887:2000

TiO2 P25

Comparison toBET ~ 45 m²/gxBET ~ 21 nm

Size from SEM or TEM image


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Slide title

Example 1: Comparison of results for particle size analysis

Analysis in water containing polyphosphate |ZPSmoluchowski| > 60 mV DLS measurement

Well dispersable powder, which consists of small aggregates (xDLS > xBET) Complementary results from different measurement methods

500 nm

TiO2 P25

BET ~ 45 m²/g (xBET ~ 21 nm)

Size from SEM or TEM image


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Slide title

Example 2: Dispersion – the main challenge

Dispersion of nanomaterials for toxicological testing

No standardized procedure for dispersion available!!!

Results strongly depend on sample preparation.

Recommendation for “Dispersion SOP” will be developed within NanoValid project.


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No

Yes Disperse directly in

media

Under relevant/ possible

conditions

Worst-casescenario

Acceptagglomeration

Yes

No

Smallestdispersable

unit

Disperse directly in

media

Characterisation, i. e. by DLS

Specify energyinput

Specifydispersant aid

Yes

NoPredispersion

in water

Predispersionin water

Decision of principle considerations

Origin: D4.32 (NanoValid project)


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Comparison of results

Time-dependent analysis Specify concentration of BSA, which acts as a dispersant aid

Example: TiO2 NP in PBS

Smallestdispersable unitxDLS remains constantduring testing


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Comparison of results

Example: TiO2 NP in DMEM + FBS

Increase

Smallestdispersable unit

Reducedagglomeration

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


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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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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.


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Thank you for your attention!

Contact: [email protected]

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