Primary particle size distribution measurement of aggregated nanoparticles
Authors: Yamamoto Kazuhiro , Fujimoto Toshiyuki , Grulke Eric A. 
Published: December 2016

Industrial applications of nanomaterials have recently been reported in many fields. The European Union (EU) announced their definition of nanomaterial in 2012. According to the EU definition, nanomaterial means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as 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. They also announced that the particle size of the primary particles in agglomerates or aggregates should be considered. For regulatory purposes, it is necessary to measure the size distribution of nanoparticles based on the particle number concentration. The European Food Safety Authority (EFSA) recommended using at least two different analytical methods to identify nanomaterials for the EU regulation, one of which should be electron microscopy [1]. Transmission electron microscope (TEM) is a most useful technique that can provide precise information on the shape and size of the primary nanoparticles. Standardization on particle size measurement is performed Technical Committee (TC) 229 of International Organization for Standardization (ISO). Scope of ISO/TC229 is standardization in the field of nanotechnologies. We have performed interlobratry comparison (ILC) test of particle size distribution measurement of titania (TiO
2
) nanoparticles. In this study, details and results of this ILC is introduced.

Participants of this ILC were 6 national research institutes including 4 national metrology instututes, and 3 companies producing nanomaterials. TEM sample preparation is very important because nanoparticles are easy to aggregate in preparation. In this ILC test, chairperson (KY) prepared TEM specimens and distributed to participants of ILC. TiO
2
nanoparticle was dispersed in the 1mg/ml of sodium hexametaphosphate (NaPO
3
)
6
) aqueous solution. TiO
2
content was 25mg/ml. Ultrasonic irradiation to TiO
2
nanoparticle dispersion was performed for 1 hour, and stable TiO
2
nanoparticle dispersion was obtained. TEM image and aggregate size by laser diffraction are shown in Figure 1(a)‐(b). Average size of aggregate was 200nm, and TiO
2
nanoparticles was well dispersed. Copper metal TEM grid with amorphous carbon support membrane was used. The support membrane surface of a TEM grid was made hydrophilic using a hydrophilic treatment device. Filter paper was placed on a hot plate that has been warmed at 100°C, and the TEM grid with the hydrophilized support membrane was placed on the top of this. 15μL of the TiO
2
dispersion liquid was collected using a micropipette, and dripped onto the support membrane on the hot plate. The TEM grid with support membrane was dried on a hot plate.

Protocol of ILC was basis on the manual tracing of primary particle shapes in aggregate, which has clear contrast and distinguishable. At least 500 particles should be counted with the image resolution more better than 0.5 nm/pixel. Max./min. Feret and area‐equivalent circle diameters (ECD) of each particle should be measured using image software. One example data of ECD distribution obtained in ILC test is shown in Figure 2. In this data, 1033 particles were counted. Median diameter (D50) is 37.5nm, and standard deviation is 12.1nm. Cumulative distribution data of ECD reported from all participants are shown in Figure 3. 5 data in 9 data agree well. We examined the measurement conditions of TEM, image pixel size, and scale calibration methods of TEM. For TEM measurements, the focusing condition or the z‐position of specimen influences strongly to size measurement. We summarize how to measure particle size correctly are; 1) the same z‐position of specimen in measurement and in calibration, 2) the same focusing condition in measurement and in calibration, and 3) using the same sized calibration standard to measured particles.