@ Annie  What would you consider the size of a nanoparticle and how should it be defined?

DLS (from Zetasizer) measures the diffusion speed of an ensemble group (many millions or billions) of particles.  Thus a protective layer (e.g. surfactant or PEG) on top of a core particle will influence this diffusion and thus the interpretation of size. Do you consider the surfactant to be part of the particle?  It certainly influences the transport and bulk (e.g. rheology) properties of the suspension.  Electron microscopy will explore the electron-rich or core of the particle - you may only be looking at a few hundred - maximum - of particles so representative sampling becomes key.  Visualization is essential and electron microscopy or AFM are the only viable routes in the nano region. 

Electron microscopy provides a number distribution whereas DLS provides an intensity distribution.  In a number distribution, every particle has equal relevance or weighting.  So the 1 nm particle is equivalent to the 100 nm particle in mathematical terms for a number distribution.  However, the 100 nm particle has a million times as much mass or volume and thus contains a million times as much value (a volume or mass distribution is equivalent to a $ distribution). In volume terms we'd look at the D[4,3] - see attached where the mass or volume weighting is important.  Unless statistically valid numbers of particles can be taken, electron microscopy is not a route to particle size distribution determination but provide essential information as to the form of the particles, degree of aggregation, agglomeration,  crystallinity versus amorphous nature and so on.  So the collected data is very different. See for example, many discussions on RG on this theme.  For example:  For example: http://tinyurl.com/mnccs95  & http://tinyurl.com/k29asgq 

DLS is very sensitive to tiny amounts of large material that's relevant in many industries (e.g. protein aggregation) - this material is unlikely to be imaged with electron microscopy.

So, you need both DLS and electron microscopy.  They provide complementary, not competitive, information. .

@ Annie  What would you consider the size of a nanoparticle and how should it be defined?

DLS (from Zetasizer) measures the diffusion speed of an ensemble group (many millions or billions) of particles.  Thus a protective layer (e.g. surfactant or PEG) on top of a core particle will influence this diffusion and thus the interpretation of size. Do you consider the surfactant to be part of the particle?  It certainly influences the transport and bulk (e.g. rheology) properties of the suspension.  Electron microscopy will explore the electron-rich or core of the particle - you may only be looking at a few hundred - maximum - of particles so representative sampling becomes key.  Visualization is essential and electron microscopy or AFM are the only viable routes in the nano region. 

Electron microscopy provides a number distribution whereas DLS provides an intensity distribution.  In a number distribution, every particle has equal relevance or weighting.  So the 1 nm particle is equivalent to the 100 nm particle in mathematical terms for a number distribution.  However, the 100 nm particle has a million times as much mass or volume and thus contains a million times as much value (a volume or mass distribution is equivalent to a $ distribution). In volume terms we'd look at the D[4,3] - see attached where the mass or volume weighting is important.  Unless statistically valid numbers of particles can be taken, electron microscopy is not a route to particle size distribution determination but provide essential information as to the form of the particles, degree of aggregation, agglomeration,  crystallinity versus amorphous nature and so on.  So the collected data is very different. See for example, many discussions on RG on this theme.  For example:  For example: http://tinyurl.com/mnccs95  & http://tinyurl.com/k29asgq 

DLS is very sensitive to tiny amounts of large material that's relevant in many industries (e.g. protein aggregation) - this material is unlikely to be imaged with electron microscopy.

So, you need both DLS and electron microscopy.  They provide complementary, not competitive, information. .

I agree with everything written above, one further point is that a light scattering technique will assume the particle is spherical and will from a diffusion coefficient calculate back to a particle diameter. This is fine if the particle is spherical or perhaps even approximately spherical, but not good if the particle is flat like a clay or rod shaped like some gold or other metal nanoparticles.

@ Paul   Agreed.  That's why some form of visualization is essential in order to understand the shape of a particle - and will require more than one number to specify for an irregular (non-spherical) material.  As stated in USP : 'For irregularly shaped particles, characterization of particle size must include information on particle shape'

Most indirect measurements (sedimentation, sieves) of particle size will have some assumption of shape (usually spherical) or will calibrate against a spherical standard (counting, electrical sensing zone) .  Most imaging techniques will provide a 2-D understanding of shape (e.g. aspect ratio) when shape is a 3-D issue.  See attached.

@Professor Luckham, @Dr. Rawle,

Thank you all for so much helping. Yet, I am not sure what exactly my iron nano is, and with the concern of magnetic could damage SEM and AFM is kind of too expensive for my experiment. If it is Fe3O4, it is allowed to analyze SEM. I can't find out how to analyze if it is Fe3O4 under such condition. That's the reason I asked. I would like to know if only with Zetasizer, can it prove it is nano-sized?

Thank you so much.

Basically yes a zeta sizer will be fine, if the size is in the correct range then you can be sure the particles in reality will be that size or smaller (if its non spherical), but note the FE3O4 particles often aggregate so if you get a reading higher that you expect (or hope for) aggregation may be occurring, where lots of small nanoparticles may aggregate into much larger entities which the zetasizer is measuring. Then you would need some type of electron microscopy. TEM would be best if they are very small, say 20 nm or less. If larger than that SEM is fine. You could also get an indication from the sedimentation rate from the Stokes equation

First 'problem' with Fe3O4 is that the long range magnetic forces overcome short range van der Waals and agglomeration followed by aggregation is inevitable unless a steric stabilizer is used (PEG or PEI of say 50 kDa) where the slipping plane is located within the polymer chain.  Second issue is the density (~ 5 g/cm3) meaning that the particles need to be < 50 nm to stay in free suspension even before aggregation - as Paul suggests, a Stokes' law calculation is helpful and mandatory here. Indeed in ASTM E2490-09 (15) I provide such a table where I've used 5.5 g/cm3 as one of the (Stokes' law) calculations for various sizes - this was a surrogate for Fe3O4.  Settling is difficult to overcome in such systems.  Microscopy is mandatory - USP - 'For irregularly shaped particles, characterization of particle size must include information on particle shape’....

@Annie,

this is the great advantage of the Zetasizer and dynamic light scattering: one can try to measure almost anything and not have to worry about breaking the system.

If your sample when measured by DLS indicates that you have nano-sized objects in your distribution then yes, you can conclude that they are real (providing that the data are of acceptable quality). As indicated by Alan, the absence of nano-sized objects would not necessarily mean that there are no nano-sized objects in your sample, because the signal from larger particles may overshadow the signal from the nanoparticles.

So my answer to your question is: Yes, it can prove that your samples contains nano-sized objects, however, it cannot rule out that it contains nano-sized objects, it can not be used to prove that the sample contains no nano-sized objects when there are larger particles in the sample.

http://www.materials-talks.com/blog/2017/01/23/intensity-volume-number-which-size-is-correct/

http://www.materials-talks.com/blog/2014/07/10/faq-peak-size-or-z-average-size-which-one-to-pick-in-dls/

http://www.materials-talks.com/blog/2014/08/05/faq-how-important-are-refractive-index-absorption-for-nanoparticles/

I still need numerical explanation of this zetasizer result that is; you can explain base on different pH for instance 5, 5.5, 6, and 7 then how do you analyse the result that is; the range of values eg positive say (+30mV) to negative charge (-30mV) of hydrochar-NZVI composite see data below:

°C mV µmcm/Vs mS/cm 25.7 -27.6 -2.166 1.25 25.7 -29.6 -2.319 1.34 25.7 -28.9 -2.266 1.38 27 -26.6 -2.162 1.76 27.1 -29.3 -2.384 1.92 27.1 -29.1 -2.361 1.99 27.8 -29 -2.393 1.31 27.9 -29.6 -2.448 1.39 27.9 -30.9 -2.555 1.43 28.4 -21.8 -1.813 3.57 28.4 -23.5 -1.954 4.09 28.5 -24.2 -2.02 4.23 28.9 -28 -2.354 1.42 28.9 -28.6 -2.411 1.52 28.9 -29.6 -2.496 1.57 °C mV µmcm/Vs mS/cm 25.7 -27.6 -2.166 1.25 25.7 -29.6 -2.319 1.34 25.7 -28.9 -2.266 1.38 27 -26.6 -2.162 1.76 27.1 -29.3 -2.384 1.92 27.1 -29.1 -2.361 1.99 27.8 -29 -2.393 1.31 27.9 -29.6 -2.448 1.39 27.9 -30.9 -2.555 1.43 28.4 -21.8 -1.813 3.57 28.4 -23.5 -1.954 4.09 28.5 -24.2 -2.02 4.23 28.9 -28 -2.354 1.42 28.9 -28.6 -2.411 1.52 28.9 -29.6 -2.496 1.57