assessment of different electron microscopy techniques for

Assessment of Different Electron Microscopy Techniques for Particle Size Quantification of Potential

NanomaterialsPhilipp Müller1, Johannes Mielke2, Vasile-Dan Hodoroaba2, Ralf Kägi3 and Martin Ryner4

1. BASF SE, Department of Material Physics and Analytics, Ludwigshafen, Germany. 2. BAM Federal Institute for Materials Research and Testing, Berlin, Germany.

3. EAWAG aquatic research, Dübendorf, Switzerland.4. Vironova AB, Stockholm, Sweden.

NanoDefine is funded by the European Community's Seventh Framework Programme under Grant Agreement-604347

M&M 2015, Portland (OR); Session A17.03 – Paper 155

P. Müller; M&M 2015 Portland (OR), Session A17.03, Paper 155

Motivation: EC regulation

RECOMMENDATION on the definitionof nanomaterial (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.


The NanoDefine approach

• Detailed assessment and benchmark of measuring techniques to develop a sustainable, flexible, cost efficient and easy to implement solution for nano-characterization

Among several other techniques; EM is evaluated by as potential gold standard for determination of number size distributions

integral methods:• Dynamic Light Scattering (DLS)• Small-Angle X-ray Spectroscopy (SAXS)• UltraSonic Spectroscopy (USSp)• X-Ray Diffraction (XRD)• Brunauer Emett Teller surface (BET)

• Field Flow Fractionation (FFF)• Centrifugal Liquid Sedimentation (CLS)

counting methods:• Electron Microscopy (EM)• Particle Tracking Analysis (PTA)• Nano Coulter counter• Single particle ICP-MS

check www.nanodefine.eu


FEI Tecnai G2TEM/STEM800k€/$

VironovaminiTEM250k€/$

FEI MagellanFEI BAM?TSEMJeol JSM7500SEM250k€/$

Phenom G2tabletopSEM50k€/$

7 samples, 7 imaging techniques, 4 institutions

EM round robin


Samples

BaSO4

CaCO3

substances coated TiO2

Au

Ag

SiO2

mono- and bimodal calibrants

250 nm 200 nm

500 nm

500 nm

250 nm

BaSO4 (fine) MWCNT

CaCO3 (fine)

kaolin clay

coated TiO2


Sample preparationCalibrants Au, Ag and SiO2: drying of one drop of the suspension on a TEM grid

Real life material:• Dispersion in 2mg/ml SHMP solution (sodium hexametaphosphate - calgon)• 3 min ultrasonic probe treatment• 5 min ultrasonic bath treatment• 3 µl drop on TEM-grid• Removal of excess solvent by a tissue after 5 min


Approach of the round robinFind a cheap but comparable EM method for Particle size determination

manual measurement

… a pragmatic and (biased?) approach!

automated measurement usingparticle analyzer

• final version of public and free “imageJ” and “R” plugin will be released in ~2 years

• standard configuration!

10 nm 500 nm


the miniTEM processMiniTEM = BF TEM at 25 kV; low costs of ownershipEvaluation steps:1. Sample preparation for TEM (~15 min)2. Sample insertion + other manual steps (~10 min)3. Fully automated data acquisition (~2 h)4. Definition of analysis chain in the miniTEM Software (~1 h

but nearly the same protocol for all saples)5. Optional: manual optimization of results (5 min)

e.g. removing false positives

• 30 min of manual work/sample• Real- world price of 150 €/$ per sample possible

(reduction by factor 5-10) compared to TEM at BASF with manual evaluation


resultsGreen: <25% deviation from HAADF-TEM manual

Yellow: >25%<50% deviation

Red: >50% deviation

automatic evaluation of tabletop SEM images impossible

Sample bimodal silica Nano Au Nano Ag BaSO4 fine coated TiO2 MWCNT Kaolin CaCO3 fineReferenceBASF HAADF manually 13.7 19.7 2.7 283.8 185.3 11.6 119.6 160.1TEM BF BASF manually 14.3 20.0 2.8 259.4 153.1 9.2 119.9 157.9BASF particlesizer 11.1 19.1 1.9 313.5 43.0 4.2 21.0 83.0BASF particlesizer_stack 9.5 11.8 0.9 367.5 78.0 4.4 33.0 68.0TEM HAADF-STEM BASF particlesizer 11.9 18.8 2.7 268.1 16.2 7.2 28.6 87.0BASF particlesizer_stack 11.7 18.3 2.3 238.2 32.7 7.0 32.2 42.2BASF auto iTEM 12.6 18.3 2.5 490.5 68.6 14.7 77.2 155.5MiniTEM BF Vironova manually @BASF 11.4 21.4 184.7 12.1 131.4 158.2Vironova custom auto 13.7 20.5 5.5 509.4 242.5 10.9 121.9 175.3Vironova particlesizer 13.8 22.5 4.7 429.8 190.8 5.9 33.6 265.5SEM SE BAM manually #1 17.6 201.5 157.4 11.5 120.8 171.9BAM manually #2 214.0 213.0 12.0 128.0 158.0BASF particlesizer 22.8 21.2 11.0 238.0 159.8 26.9 103.3 160.8TSEM BAM man 16.1 269.8 152.9 9.6 119.7 171.9BAM particlesizer 14.7 18.4 5.1 346.7 162.8 29.4 120.7 207.6


~100 nm

BF-TEM HAADF-STEMBF-miniTEM

BF-TSEM HAADF-TSEM SE-SEM TableTop -SEM

Comparison of the techniques- nano Au


~1µm



Comparison of the techniques- Kaolin

~ 1 µm




Comparison of the techniques- CaCO3

~ 500 nm


What do we learn for a pragmatic approach?• Spherical particles work perfect for automation• Even small particles are detected by all methods• Accuracy quickly drops for particles with a diameter of a few nm

HAADF-TSEM auto HAADF-TSEM manually

Nano Ag 10 nm

Sample bimodal silica Nano Au Nano AgReferenceBASF HAADF manually 13.7 19.7 2.7TEM BF BASF manually 14.3 20.0 2.8BASF particlesizer 11.1 19.1 1.9BASF particlesizer_stack 9.5 11.8 0.9TEM HAADF-STEM BASF particlesizer 11.9 18.8 2.7BASF particlesizer_stack 11.7 18.3 2.3BASF auto iTEM 12.6 18.3 2.5MiniTEM BF Vironova manually @BASF 11.4 21.4 Vironova custom auto 13.7 20.5 5.5Vironova particlesizer 13.8 22.5 4.7SEM SE BAM manually #1 17.6 BAM manually #2 BASF particlesizer 22.8 21.2 11.0TSEM BAM man 16.1 BAM particlesizer 14.7 18.4 5.1


What do we learn for a pragmatic approach?Sample bimodal silica Nano Au Nano Ag BaSO4 fine coated TiO2 MWCNT Kaolin CaCO3 fineTEM BFBASF manually 14.3 20 2.8 259.4 153.1 9.2 119.9 157.9BASF particlesizer 11.1 19.1 1.9 313.5 43.0 4.2 21.0 83.0TEM HAADF-STEMBASF HAADF manually 13.7 19.7 2.7 283.8 185.3 11.6 119.6 160.1BASF particlesizer 11.9 18.8 2.7 268.1 16.2 7.2 28.6 87.0

• No significant difference between BF-TEM and HAADF-STEM

• HAADF-STEM has advantages if strongly agglomerated or contaminated

200 nm

coated TiO2


What do we learn for a pragmatic approach?

• Manual evaluation can deal with all imaging modes

• For SEM and miniTEMagglomeration is an even larger issue

Sample bimodal silica Nano Au Nano Ag BaSO4 fine coated TiO2 MWCNT Kaolin CaCO3 fine

HAADF manually 13.7 19.7 2.7 283.8 185.3 11.6 119.6 160.1BF manually 14.3 20 2.8 259.4 153.1 9.2 119.9 157.9miniTEM manually 11.4 21.4 184.7 12.1 131.4 158.2SE SEM manually #1 17.6 201.5 157.4 11.5 120.8 171.9SE SEM manually #2 214.0 213.0 12.0 128.0 158.0TSEM manually 16.1 269.8 152.9 9.6 119.7 171.9

HAADF-STEM miniTEMCaCO3 fine

200 nm


Issues with automatic evaluationNanoDefine Particle Sizer:• Excellent results for all materials demonstrated

(lab conditions)• Real-life drawbacks: agglomeration +

contamination

miniTEM:• Algorithm searches for unagglomerated particles• Less problems with agglomeration but possible

bias?!

1 µm

Coated TiO2 - miniTEM

100 nm

Coated TiO2 – HAADF-STEM


Discussion of uncertancies

0100200300400500

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52la

rger

# of

par

ticle

s

size [nm]

particle size histogram of bimodal silicaautomated evaluation

100 nm

• manual: ~330 particles counted• auto.: several 1000 particles counted• Still not sufficient to reflect a bimodal distribution• No false negatives for all 80 studies

significant statistical error

Potentially even larger systematic error due to preparation artifacts

Sample coated TiO2 MWCNT Kaolin CaCO3 fine

HAADF manually 185.3 11.6 119.6 160.1BF manually 153.1 9.2 119.9 157.9miniTEM manually 184.7 12.1 131.4 158.2SE SEM manually #1 157.4 11.5 120.8 171.9SE SEM manually #2 213.0 12.0 128.0 158.0TSEM manually 152.9 9.6 119.7 171.9std. dev. 22.06358374 1.1561 4.6928 6.33614

Benchmark with CRM needed;Error seems to be tolerable for threshold-based categorization


Summary• TableTop SEM

does not meet requirements for EC definition

~100 nm

TableTop –SEM (nano Au)

30 nm

TSEM (nano Ag)• SEM and

especially TSEM safely detects particles < 5 nm

• Sufficient for threshold based decisions

• Main error seems to result from sample preparation (despite dispersion protocol)

• Pragmatic approaches in evaluation should be tolerable

500 nm

500 nm

• Automation works perfect for spherical samples and is sufficient for ~50% of real life material

• No false negatives at all!


Thank you for your attention!

Funding:

The research leading to these results has received funding from the European Union’s Seventh Framework Programme(FP7/2007-2013) under grant agreement n°604347 – NanoDefine(www.nanodefine.eu)

Special thanks to:

Thorsten Wieczorek(lab work at BASF)Wendel Wohlleben(work on BET-based assesment)Thorsten Wagner (imageJ plugin + discussion)

Amusement in the poolAgglomerate of TiO2 nanoparticles as

used in sunscreen


Backup


Overview

1.Motivation – EC Regulation and the NanoDefine Project

2.Experimental section - The Electron Microscopy (EM) “round robin”

3.Results and take-home messages

4.Discussion of precision and remaining issues


Confidence interval of the medianbimodal silica Nano Au Nano Ag BaSO4 fine coated TiO2 MWCNT Kaolin CaCO3 fine

# of measureme 306 339 388 372 60 225 328 345Median 13.7 19.7 2.7 283.8 185.3 11.6 119.6 160.1upper limit CI 14.4 20.0 2.8 298.9 207.4 12.1 127.5 173.6lower limit CI 13.2 19.5 2.6 262.9 159.1 10.9 109.0 148.2

• Manual measurements on HAADF-STEM images• Despite low number of measurement: tolerable confidence interval• In all cases: clear assignment to nano/non-nano possible• All studies with a result of < 80 nm or >120 nm can be considered as clear cases


What do we learn for a pragmatic approach?Kaolin- BF TEM

200 nm

• EM cannot measure the smallest dimension of the platelets

Possible solution:• Determine Volume Specific Surface Area (VSSA) by BET• Determine the number of small dimension by EM• Modify the cutoff value by which one would define a material as nano by BET:

Irregular particleD=3 small dimensions:

VSSA cut-off = 60 m²/cm³

PlateletD=1 small dimension:


Rod / tubeD=2 small dimensions:


VSSA cutoff = 60𝒎𝒎2

𝒄𝒄𝒎𝒎3 ∗𝑫𝑫3

assessment of different electron microscopy techniques for

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