First principal electrophoresis zeta potential and Brownian diffusion size

Measure, what you see

With ZetaView® individual particle tracking, classical micro-electrophoresis and Brownian motion are presented as modern analysis tools. Auto-alignment and auto-focusing make the “Seeing is Believing” principle user friendly. By sub-volume scanning robust results of zeta potential and size histograms are derived from thousands of particles. In addition, particle concentrations can be determined by video frame assessment counting. Sample cell handling is reduced to a few handholds.

ZetaView®
ZetaView® - Autoscan over up to 100 subvolumes - Autofocus - instrument fits into a pilot case - anti-vibration design - Lasers from UV to red - Slide-in cell cassette
ZetaView Nanoparticle Tracking
Fig. 1: Following the movement of individual particles during a video

Features of the ZetaView®

Automation and Passive Stability

ZetaView Slide-in cell cassette
Photo: Slide-in cell cassette with sample cell carrier on top

The “AutoAlignment” procedure keeps in focus for days, even after a cell removal. The anti-vibration design enhances the video image stability. By scanning multiple sub-volumes and averaging the measurements, statistically robust results are guaranteed. Three measurement modes are selectable: Size, Zeta Potential and Concentration. Unsymmetrical cell coating by sedimentation is avoided by the orientation of the cell walls (figure 2). The cell channel is integrated in a „slide-in“ cassette (Photo), which is provided with temperature control and snap-in couplings.

Theory

The translational diffusion constant is calculated from the direct observation of Brownian motion to calculate size. From the measurement of the electrophoretic mobility zeta potential is calculated.

ZetaView® Nanoparticle Tracking Analysis (NTA)
Particle size by Brownian motion: Nanoparticle Tracking Analysis (NTA)
ZetaView® Principle of zeta potential determination
Principle of zeta potential determination

Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS)

All light scattering instruments – particle tracking included – suffer from the rapidly decreasing sensitivity below 100 nm. The lowest size level for DLS Dynamic Light Scattering is ~ 0.5 nm, for nanoparticle tracking analysis ~ 10 nm. In general the main relevant difference between DLS and NTA is the concentration range, when the concentration is too low for DLS, the ZetaView Nanoparticle Tracking Analyzer will perform the task brilliantly and fast. Conversely, the 180° DLS system is ideal for analysis of high concentration samples.

Measurement range

Depending on the sample and the instrument model, the direct tracking of particles is possible in a size range starting at 10 nm for gold and correspondingly higher for particles with less scattering power. Provided the sample is stable and does not sediment or float, the zeta potential upper size limit can be 20 µm, and for particle sizing 3 µm. 

Particle counts from video frame assessment

The particle concentration is derived from analyzing the video frames for observed particle numbers. It is normalized to the scattering volume per relevant size class. A minimum concentration of 105 particles per cm³ can be detected, the maximum is 1010 p/cm³. In volume concentration, up to 1000 ppm of 200 nm sized particles can be analyzed.

Trueness and precision

For zeta potential, the trueness is 5 mV, precision 4 mV and , instrument to instrument repeatability 5 mV. For size determination of a 100 nm standard latex suspension the trueness is 6 nm for number calculations, precision is 4 nm and, instrument to instrument repeatability 4 nm. For 100 nm particles at a concentration of 10 Million particles/mL, accuracy is 0.8 Mio/mL and precision 0.5 Mio/mL. Instrument to instrument repeatability is 1 Mio / mL. All given data are valid providing correct camera settings and sample preparation are adhered to.

The Method

The ZetaView laser light scattering microscope is sensitive to nanoparticles 100 times below the diffraction limit of ~ 1µm (fig 2). 

ZetaView® Optical Layout
Fig. 2: Optical Layout of the automated particle tracking ZetaView® Laser Light Scattering unit having “ZetaFocus” as synchronous control of the laser and microscope focus. No change of microscope optics is necessary.

Flexibility

The instrument is compact and can be carried in a pilot case. Setup is in minutes. It can be operated in a network or stand alone.

Quality of measurements

The quality of data may be affected due to bubbles or dirt in the cell. Therefore a statistical evaluation of the image quality is performed. Adequate documentation identifies these positions.

The image of the particles is focused onto the video camera. From measuring the particle velocity and direction under an applied electric field, the electrophoretic mobility and polarity are determined. With no field taken into account, only Brownian motion is detected. Electric field, temperature and conductivity are also monitored at each experiment. By scanning the cell cross section and sequencing subvolumes excellent statistical results can be obtained.

ZetaView® for zeta potential AND quality checking

The zeta potential distribution is calculated from the electrophoretic mobility results at the 2 stationary layers in the cell (ζ-layer“ / “ZP”, fig.3) or from an electrokinetic velocity profile obtained by scanning throughout the cell. The zeta potential is calculated from the Smoluchowski or Henry equation (see “Theory”). In addition to the particle zeta potential result, the curvature of a profile delivers free information on the polarity and amount of ionic coating on the cell walls. Wall coating is always present when dealing with ultrafine particles. The correctness of the electrophoresis zeta potential determination is not influenced by the wall coating.

Fig. 3: Green: ZP of Al2O3 is +50 mV, walls are cationically coated. Red: -25 mV ZP anionic polysterene, -; the walls are neutral. Blue: polystyrene -40 mV ZP, cell walls are anionic as uncoated glass usually is.

Zeta potential distributions

Fig. 4A: Zeta potential distribution of 60 nm gold particles. A: dispersed in 2 mM KCl solution (stable). B: dispersed in distilled water (unstable).
Fig. 4B: Correspondingly, the less stable sample B showing coagulated objects. .

A few examples  shall highlight the capability of the ZetaView in measuring zeta potential distributions.

A 60 nm gold dispersion was studied for size and zeta potential distribution (fig. 4). The sample is slightly bi-modal.

Curve A (red) represents gold particles in a 2m KCl environment, curve B (blue) the same sample but dispersed in distilled water.

The zeta potential distribution of curve B shows a peak near 0 mV, which is making the suspension unstable. The majority of the 60 nm particles is therefore agglomerated, which is clearly seen in the size distribution of curve B.  

Translational diffusion size distribution

Fig. 5: Size distribution (LOQ) of 20 nm BBI gold particles in distilled water.

The PMX 110 ZetaView has a lower detectable limit of 20 nm LOQ (fig. 5). For substances with lower scattering efficiency, the lower size limit is correspondingly higher.

In an example of BMBO cell derived exosomes in a pH 7 Soerensen buffer, the interdependence between conductivity, zeta potential and size distribution is demonstrated (fig.6). The conductivity is automatically monitored during the data acquisition.

Fig. 6: The interdependence between conductivity, zeta potential and particle size distribution, demonstrated with exosomes* (see text).