Comparison of Particle Size (Diameter) Measured by TEM and DLS (Dynamic Light Scattering)

Dynamic Light Scattering (DLS) for Latex Standard Measurement

Dynamic Light Scattering (DLS) is a non-invasive technique suitable for characterizing the size of nanoparticles and low molecular weight molecules such as proteins and polymers. This technology measures the time-dependent fluctuations in the intensity of scattered light caused by the random motion of particles or molecules undergoing Brownian motion. The speed of this Brownian motion is measured and called the translational diffusion coefficient (D), which can be converted into a hydrodynamic diameter (D,_H) using the Stokes-Einstein equation [1-3].

DLS is an absolute technique using first principles and thus does not require calibration. However, regular instrument validation should be conducted to verify proper operation.

Spherical polymer latex is commonly used to verify correct instrument performance since it can serve as a nearly perfect spherical, monodispersed dispersion. The sphere is the only 3D shape that can be clearly described by a single figure for size, thus eliminating uncertainties associated with mean size calculations.

Polymer latex samples offer additional benefits. With a density similar to water, particles less than 1 micron remain suspended during measurement. Dispersions can be stored at room temperature and have a shelf life of several months or years.

Nanosphere 3000 series size standards [3] come with calibration certificates measured by transmission electron microscopy (TEM) traceable to NIST [4]. The standard specifications also include the hydrodynamic diameter measured by dynamic light scattering (DLS).

Nanosphere 3000 series size standards are available from 20 nm to 900 nm. The easiest sizes to measure are between 20 nm and 300 nm. Particles larger than 60 nm are large enough to provide highly reproducible results in dilutions suitable for DLS. Particles larger than 300 nm do not need consideration of angle, as smaller particle sizes start showing noticeable changes in scattering intensity with angle.

Duke Standards 2000 and 4000 series are NIST traceable size standards, which can also be used for DLS validation applications and include sizes above 1 micron.

Certified Hydrodynamic Size

The results marked on the Nanosphere bottles are certified TEM results.

DLS results (i.e., hydrodynamic size) are quoted in the provided specification sheet and are not certified values.

For all Nanosphere 3000 standards, the size accuracy by DLS should be within the specified hydrodynamic size range ±2% for samples prepared in 10mM NaCl [2,3].

Sodium chloride is used to suppress the electrical double layer. Dilution of standards in deionized water results in the double layer extending, artificially increasing the size outside of specification.

Comparison of Sizes Measured by TEM and DLS

Different measurement techniques measure different properties of particles, potentially resulting in different outcomes when interpreted as size from the measured property. A frequent question hence arises, which result is correct?

For many, ‘seeing is believing’, hence electron microscopy results are considered ‘accurate’.

Actually, samples prepared for electron microscopy are often harshly treated, and such treatment can distort soft materials like polymer lattices and alter or mask surface structures.

This could make size measurement for some types of materials like surfactant micelles impossible. In contrast, DLS measures the hydrodynamic diameter of dispersed particles in their native environment.

Any surface structure or changes in the electrical double layer impacting the Brownian motion of particles, like adsorbed polymer layers, alters the effective particle size [5].

Using a very low salt dispersant to increase surface structures or expand the electrical double layer slows Brownian motion and increases measured size. For these reasons, the hydrodynamic sizes or DLS sizes of particles that are not smooth, hard spheres are generally larger than TEM sizes.

Table: Summary of DLS measurements conducted on latex standards.

This table provides details of the latex used (part number shown in brackets), certified (#) or hydrodynamic (*) size ranges, the concentration at which latex was measured, the diluent used for preparation, the instrument used for measurements, and the obtained z-average diameter for each latex.

Results and Discussion

Table 1 summarizes the results obtained for various latex standards measured by DLS. All standards have a certified size range, and some also quote a hydrodynamic size range. The certified size range (#) was obtained using transmission electron microscopy and is traceable to NIST. The hydrodynamic size range (*) is determined by DLS.

The results demonstrate a wide range of concentrations at which latex standards can be measured. Some latex samples can be measured neatly at concentrations such as 1% w/v using the backscatter detection of the Zetasizer.

As the size of latex particles increases, issues with count fluctuations and sedimentation become significant. During DLS measurements, the intensity of scattered light fluctuates due to particle Brownian motion. Scattering intensity is proportional to sample concentration, and the number of particles in the scattering volume must remain constant during the measurement process. However, as particle size increases, the number of particles in the scattering volume decreases until severe temporal fluctuations in the number of particles within the scattering volume occur. Count fluctuations are defined as changes in the number of particles within the scattering volume during the DLS measurement process.

To avoid count fluctuations, the concentration of the sample should be increased. However, this increases multiple scattering effects, affecting obtained results. Using backscatter detection with a variable measurement position allows higher sample concentrations to be measured, preventing count fluctuation issues. Count fluctuations are typically indicated by a rising and fluctuating baseline in the correlation function.

The second issue when measuring large-sized particles by DLS is sedimentation. All particles experience sedimentation, with the rate depending on particle size and the relative density of the particles and suspending medium. For DLS, the sedimentation rate must be much slower than the diffusion rate. Larger particles diffuse slowly, making sedimentation a more significant issue.

To confirm the presence of sedimentation, check the stability of the count rate in repeat measurements of the same sample. A decreasing count rate in successive measurements indicates the presence of sedimentation, and the Zetasizer software highlights this to the user.

If viscosity does not notably increase, suspending particles in a medium with similar density can be beneficial. In this application note, samples were prepared with 13% w/v sucrose, having the same density as latex, to measure 3, 6, and 8.9 mm latexes.

The results obtained for the 3, 6, and 8.9 μm latex samples are within the expected size range using the DLS technique. They were measured at concentrations between 0.15 and 0.24% w/v. These results confirm that multiple scattering effects were minimized using backscatter detection, and count fluctuation and sedimentation did not affect the obtained results.

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References

[1] R. Pecora (1985) Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy. Plenum Press, New York.

[2] International Standard ISO13321 (1996) Methods for Determination of Particle Size Distribution Part 8: Photon Correlation Spectroscopy. ISO (International Organization for Standardization).

[3] International Standard ISO22412 (2008) Particle Size Analysis: Dynamic Light Scattering (DLS). ISO (International Organization for Standardization).

[4] National Institute of Standards and Technology, USA (www.nist.gov).

[5] R.S. Chau and K. Takamura (1988) J. Colloid. Int. Sci., 125, 266.