Nanoparticle Tracking Analysis (NTA), or particle tracking analysis (PTA), is a technique that sizes material in the sub-micron region. The basic components of the instrument that performs NTA are an injector, a fluid cell, a microscope set to 20x objective, a temperature module, a laser module, and a camera. Particle sizing utilizing NTA is calculated using the Stokes-Einstein equation:
In the equation, Dt is the translational diffusion coefficient, KB is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the carrier and dh is the hydrodynamic diameter of a spherical particle.
The instrument calculates particle size using the Stokes-Einstein equation by utilizing its components to control several factors in this equation. The fluid cell enables fluid to move through the instrument at a fixed rate. The temperature module regulates the cell’s temperature, ensuring it remains consistent. The laser module (532 nm green laser) illuminates the sample fluid and enables particles suspended in the sample to be illuminated through light scattering as the particles diffuse across the cell; particles, in this case, are described as a “minute piece of matter with defined physical boundaries” per ISO 19430.
NTA determines the particle size distribution based on the Brownian motion exhibited by particles suspended in a material. Samples that are suspended in either water or low molecular weight solvents, such as Isopropyl alcohol, are analyzed at PTL. Brownian motion is the random motion exhibited by particles when colliding with suspension medium molecules. For instance, particulate colliding with water molecules gives rise to Brownian motion. By injecting a sample at a fixed rate and illuminating the fluid cell with a laser, the instrument uses the camera attached to the microscope to capture videos of the particle diffusion under the influence of Brownian motion, which are then processed using the instrument’s software. The diffusion rate must be constant for results to be more representative of the sample itself as opposed to taking one portion of the sample and assuming that the portion is truly representative of the entire sample.
Is NTA Right for Your Sample?
By understanding how the instrument calculates particle size, we can now dive into what types of samples are suitable for this technique. As the technique’s name suggests, this instrument analyzes nanomaterial. Nanomaterial is described per ISO 19430 as a “discrete piece of material with all external dimensions in the nanoscale” with the nanoscale typically being from 1-100 nm. Nanoparticles are considered nanomaterial and fit the criteria laid out in ISO 19430. Based on internal studies performed to qualify the instrument for cGMP usage, PTL has determined that the analytical detection range of the instrument is 30 nm to 1000 nm. However, anything above 700 nm can be a challenge for NTA to analyze. In such cases, a different technique, such as Single Particle Optical Sensing (SPOS), may be more appropriate. The lower the scatter of a material (e.g. polystyrene) the lower end may be closer to 50 nm.
While materials may have different scattering abilities based on their refractive index difference from the carrier medium, there is also a size dependence on light scatter. More specifically there is a d6 proportionality of light scattered relative to the particle’s diameter (d). This means larger particles scatter more light, suggesting particles exceeding the upper end of the instrument (1 µm) should be removed if possible to prevent obscuring a measurement.
Samples must also satisfy one more requirement: they must be suspended and freely move in Brownian motion in the medium. If the sample is dissolved, no particulate will be observed as a solution will contain no visible particulate. There is a common misconception that dispersion and dissolution are interchangeable terms. Dispersed samples are not fully homogenous with the medium; when left still for a time, the particulate may settle and agglomerate. A dissolved sample is a homogenous solution that will not separate even after filtering. If the sample is placed in a medium that will dissolve it, there is nothing that can be observed, and no particles are tracked or analyzed.
Additionally, rather viscous samples can pose an issue for NTA analyses. As viscosity’s role in determining particle size is important, PTL requires an accurate viscosity value for any samples submitted. Furthermore, PTL recommends that samples submitted for NTA be less than 10 cP as particles in samples more viscous than 10 cP typically exhibit restriction in their Brownian motion.
How Concentrated is Your Sample?
A material to be analyzed by NTA must be within the studied concentration range. Based on internal studies using a polystyrene reference material, PTL has established an optimal concentration range for samples of 6.22 x 107 to 1.87 x 108 particles/mL. With concentration being a relative value used for reference for NTA, PTL has established this range produces results with an optimal degree of repeatability.
It should be noted that this range was established based on testing of a 100 nm polystyrene reference material. Thus, different-sized materials may warrant different sample concentrations. Per the guidance outlined in ASTM E2834-12(2018) Section 8.2.6, replicate analyses are recommended to determine the variance inherent to the sample as well as to assess the stability of measurements obtained on NTA. If the material is too concentrated, there may be an issue of particle crowding that may impede the instrument software from being able to track individual particles.
If a material is too dilute, little to no particulate will be analyzed resulting in a dataset with low statistical significance and more inherent variance. As a high-resolution particle counter, the NTA instrument can also analyze polydisperse materials (i.e. containing several different size populations). However, materials on the lower end of NTA’s detection limit may be obscured and not accurately represented if the material is highly polydisperse.
PTL Has the Nanoparticle Sizing Experience You Need
Particle size is a huge factor to consider in the formulation process, whether it is the formulation of new pharmaceuticals, additives to material such as asphalt, and a number of other applications. As such, it is equally as important to understand what goes into sizing particles via NTA and what factors make a sample suitable for NTA and/or that can influence the results that are obtained.
The particle sizing experts at PTL can get you on the track to analyzing your nanomaterials. Contact us today to get started!
By Victor Alvarez, Particle Characterization Chemist I.
