A basic introduction to Electrophoretic Light Scattering (ELS) for Zeta potential measurements – Q&A

Nano-bubbles are in the size range that should be measurable using the Zetasizer Nano for Dynamic Light Scattering (DLS) to obtain size and Electrophoretic Light Scattering to obtain a zeta potential measurement. However, some of the nano-bubble samples that I have been involved with contained very low concentrations of nano-bubbles so I must caution that nano-bubble measurement obtaining good results with the Zetasizer Nano family of instrumentation will be concentration dependent.

We have been very successful making measurements using Malvern’s NanoSight range of instruments. NanoSight instrumentation features prominently in nanobubble research publications. I have attached a whitepaper that discusses the measurement of nano-bubbles using NanoSight instrumentation.

In addition, another technique, measured with the Malvern Panalytical Archimedes, provides information previously unavailable to nanobubble researchers: clear differentiation between gas bubbles in solution and contaminants that may also be present in samples. This Resonant Mass Measurement (RMM) is, therefore, able to supply a clear, reproducible analysis of the concentration and purity of a nano-bubble sample.

Zeta potential measurements can be made on any emulsion or dispersion with a particle size from 4-5nm at the low end and up to 50microns at the high end. However, if the particles are large in size, let’s say 50microns and dense like silica then they will be sedimenting. So this type of sample will not provide good data. Zeta potential is not a function of size so I recommend letting the particles settle, then drawing the sample from the top supernatant of the solution. Now the sample will contain the fines from the sample and this measurement will produce good results. The surface chemistry of the finer material vs. the large sedimenting material is the same so the zeta potential will be the same.

I am also attaching a Zetasizer brochure that contains some of the specifications for the instrument.

My answer to this may be a little surprising but here goes; we live in a polar world and there are unexpected ionic interactions that occur despite our best intentions. Even the H-bonding mechanism is a source of ionic interaction. So, I believe that if you make zeta potential measurements on your samples the results will indicate that the samples are charged, with some amount of measurable zeta potential. There are truly very few chemistries that are non-ionic if any at all. You absolutely can make measurements on this type of sample and it will be interesting to see how the results fit your interpretation of the surface chemistry.

­ Zeta potential is not a function of size. Zeta potential is indicative of the chemistry at the surface or interface of the material and the solution that it is dispersed. Therefore, polydisperse sizes will not affect the zeta potential. However, if you consider a material like clay which is a flat plate-like surface; the edges are negatively charged and the flat face of the clay surface is often positively charged. In this case, alignment of plates in the electric field will determine what charge is obtained. In this case, typically clay plates stack and the edges align and move toward the electrode of opposite charge so usually, clay materials are measured as being negatively charged. Therefore, the edges are the dominant charge group studied during this measurement. Similarly, if there are charge groups that are buried below or on a porous surface, they will not typically be a factor in the zeta potential result.

Zeta potential directly measures charge stabilization. However, by combining both Dynamic Light Scattering (DLS) and zeta potential you can infer whether you have steric stability or not. Typically, you would run a set of zeta potential and size measurements vs. concentration of the steric stabilizing molecule. I’ve attached an application note that comes from a published paper showing a very detailed study of how you can use zeta potential and DLS together to identify steric stability.

Also, the one example I gave during my talk showed a zeta potential vs. polyelectrolyte concentration and DLS size vs. polyelectrolyte concentration. I was starting with a negatively charged oil emulsion and I adsorbed a positively charged polyelectrolyte. In this case, I built in both steric and electric stability so I started with a negatively charged emulsion and reversed the charge of the emulsion to cationic. In a case where I used a nonionic surface active molecule to only build pure steric stability then the zeta potential would have started negative and would have plateaued to a neutral zeta potential, close to a zero zeta potential.

It depends; are you studying metal NP assembling in vacuum, gas/air or liquid/water/solvent. If your NP’s are in water or solvent then zeta potential measurements would be very helpful and surface potential measurements could also help. You could study the zeta potential of the NP’s and the metallic particles to determine their charge. If the two different materials are oppositely charged then they will be attracted towards one another. There is probably also an optimum charge for the assembling NP’s. If these particles are too highly charged they will not want to come together and assemble evenly. Even if you are studying assembling NP’s in the air, dispersing them in water and measuring the zeta potential might still provide you with general information that might be useful. However, in this case, you will not exactly be reproducing the same conditions of your sample so I’d consider it an interesting experiment and not necessarily an absolute requirement for your studies.

Sedimentation potential is the generation of an electric field from sedimenting colloid particles. As a charged particle moves through a gravitational force or a centrifugation force, an electric potential will be induced. As the particle moves, ions in the electric double layer lag behind creating a net dipole moment due to the liquid flow. The sum of all dipoles on the particle is what causes a sedimentation potential to occur. Again, sedimentation potential is caused by a particle moving through a liquid or gas and we measure the electric field that occurs. This is the exact opposite effect of electrophoresis where an electric field is applied the particle move and we measure the velocity of the particles moving through the liquid.

This phenomenon was first discovered by Dorn in 1879. He observed that a vertical electric field had developed in a suspension of glass beads in water, as the beads were settling. This was the origin of sedimentation potential and is often referred to as the Dorn effect.

If you are truly interested in building a sedimentation potential device I suggest you read the below article which can be found in the very first volume of Langmuir. “Sedimentation potential in aqueous electrolytes”.

Wikipedia shows the below schematic of what a sedimentation device looks like and I don’t know who drew this schematic but it looks very similar to the setup that Bruce Marlow designed in the early 1980’s.

The Z-avg size is not a zeta potential measurement, it is a size measurement obtained from a Dynamic Light Scattering (DLS) intensity measurement. I am attaching another technical note that explains DLS and a note that explains the Zavg. In a zeta potential measurement, we measure the electrophoretic mobility which is the particle velocity/electric field strength. From the electrophoretic mobility, we calculate a zeta potential. It is important to keep in mind that zeta potential is the overall charge a particle acquires in a particular medium. It depends on both the chemistry of the surface and the dispersant. This is why small changes in the pH or concentration of ions in solution can lead to dramatic changes in the zeta potential. For electrostatically stabilized dispersions, the higher the value of zeta potential, the more stable the dispersion is likely to be. I cannot emphasize these points enough and I hope this helps to explain that the Zavg is a characterization of the physical size of a particle. The zeta potential is a characterization of the surface chemistry or interface of the particle.

You may also find the post on Zeta potential in salt of interest. A side effect of the lower stability near the isoelectric point IEP is often an increased average hydrodynamic size.

During the webex last week you asked; ­would you have an example of zeta potential measurement for a protein sample?­ I am attaching a couple of papers; 1.) the first is an Application Note entitled “Interactions of Bovine Serum Albumin with Aluminum Polyoxocations” 2.) a publication that is the first of a series of three papers on HIV Virus studies and 3.) a paper that was published discussing a new method we developed to help measure the zeta potential of proteins, called the Diffusion Barrier Method and includes a BSA titration and IgG measurements, also see the blog about practical details of the diffusion barrier method.

We also have a press release about Dr. Vuk Uskokovic, from the Department of Preventive and Restorative Dental Sciences, School of Dentistry at the University of California, San Francisco (UCSF). The press release points to his work which is involved in a study that aims to mimic the growth of tooth enamel in the laboratory. He published a paper entitled, ‘Zeta-potential and Particle Size Analysis of Human Amelogenins’, Dr. Uskokovic and his colleagues deliver results that suggest that: “zeta-potential may be used as a control parameter in replicating the assembly of amelogenins in vitro.”  The authors also note that: “…the meaning of the correlations established [in the paper] between zeta-potential and particle-particle attraction could be potentially applied to self-assembling proteins in general.”

The instrument is based on first principles so we do not calibrate the instrument. We measure a standard to verify that the instrument is working correctly. Malvern does provide a transfer standard to verify if the instrument is operating correctly. The transfer standard is supplied in pre-filled syringes, and has the part number DTS1235.

Zeta potential theory is based on non-overlapping double layers which mean that the sample should be diluted. Dilute measurements are the preferred way to measure zeta potential. The Zetasizer does have a low volume/high concentration zeta potential cell and the highest concentration that you should use in that cell is 40 wt%. As a rule, even with the high concentration cell, it is my preference to not make measurements above 10% but that is my personal preference. Typically, I dilute all zeta potential samples until they are optically clear, 0.01wt% or less. If I am comparing one sample to another I make sure that I dilute all of my samples to the same concentration. If pH, salt concentration or surfactant concentration is particularly important to a formulated sample then I dilute my samples to the same pH, same salt concentration, etc. By doing this I can compare differences between my samples while maintaining a very similar continuous medium as I dilute the samples. The brochure for the Zetasizer Nano family of instrumentation lists some specifications.

We measure electrophoretic mobility and calculate a zeta potential. The electrophoretic mobility is Particle Velocity/Electric Field Strength. During the measurement, we apply a Voltage and the particles move towards the electrode of opposite charge. That polarity is flipped back and forth during the measurement so the particles first move in one direction and then the other. Since the particles are moving there is a frequency shift of the light, a Doppler Shift. This is similar to how a policeman clocks you as you drive down the highway. The frequency shift of the light is directly proportional to the particle velocity. The measurement is slightly more complicated, we use an interferometric method to obtain the frequency shift of the light but without going into the optics this is how the measurement is made. For some additional details see “What is the zeta deviation?”

With a measured particle velocity and known electric field strength (Voltage/Distance between electrodes), we measure the electrophoretic mobility. We then use that value in the Henry Equation to obtain zeta potential in mV.

I am not very familiar with MXene but it is plate-like and we can measure clays which are also plate-like. Will you disperse the sample in water or organic media? If the smallest size dimension is at least 4-5nm, the measurement should be possible. SEM images of these materials suggest that the size dimensions will not be an issue for a zeta potential measurement.

The Zetasizer software has a calculation for protein charge or the charge valence in the Zetasizer software. By starting with the Henry function, f(кa) and using known size and ionic strength the protein valence can be calculated.  This tool in the software uses the Ohshima equation for monovalent salts.  The second calculation calculates protein charge from the electrophoretic mobility and Stokes radius. More details about this are outlined in a technical note that provides an overview about the Zetasizer Calculators.

In the software you can;

Select Tools-Calculators and then select the Protein Charge & f(кa) tab.

Protein Charge

The Protein Charge calculation calculates Protein Charge from the measured electrophoretic mobility and the hydrodynamic size.  The charge is calculated by the following equation:


Where:

Z = calculated Protein Charge
ζ = the zeta potential
and the other values can be taken from the above.

A colleague of mine, Kevin Mattison, also wrote a detailed explanation that he has been working on: “A Primer on Particle Sizing Using Dynamic Light Scattering“.

I have attached two application notes that will provide you with examples of the Zetasizer Surface Potential measurement. The sampling requirement for the surface potential measurement is small. Samples must be cut to fit onto the sample holders and between electrodes. The sample must be 5 – 7 mm long, 4 mm wide, not thicker than 1.5 mm. Also, the surface of the sample can be porous, like a filter or membrane but must be fairly smooth. Hopefully, this description will help you decide whether you want to consider a surface potential measurement for your samples. Also, check the blog on practical considerations for measuring surface zeta.

Measuring the Surface Zeta Potential of Silica
Measuring Surface Zeta Potential using the Surface Zeta Potential Cell

This does not sound possible to me and there are only two possible issues that I can think of that would cause this. Have the cells been used previously? It is possible that if one or both cells were not clean and contaminated the sample the zeta potential could change dramatically because of the contamination. This is why having disposable zeta potential cells is such a nice feature of the Zetasizer. If you are unsure about cleanliness then you can use a new cell.
The second possibility is that the data is not very good, what I mean by that is that it is very noisy. Therefore when you run the sample the first time you get one value and then run it a second time whether in the same cell or a different cell the second result comes out very different because the measurement is just pure noise.
In order to have good data, a good Phase Analysis result is required. The Phase Analysis result is the rawest form of the data. I’m copying a Phase Analysis graph below. Your data should have a nice clean sawtooth pattern just like below. You can also check the Quality Report to see what advice is given. I’m attaching two documents about Zeta Quality. The document titled Zeta Quality Report explains good vs. bad Phase Analysis reports. Please take a look at this it might help to explain why your data is inconsistent.

The dielectric constant for DMSO is 46.7 so it is not polar nor non-polar in my mind. You really need to calculate your own F(ka) and use that in the software. The entire Henry Eq is in the software and the software can help with the calculation.  Or you can choose to calculate your own F(ka) if that makes you more comfortable. The equation is;

K^-1 = ((e_oe_rk_BT)/(2000 e² I N))^0.5
where;
e_o = permittivity of free space             (8.8542*10^-12 C/Vm)
e_r = relative permittivity of liquid      (or dielectric constant)
k_B = Boltzmann’s constant
T = temperature in Kelvin
e = electronic charge in Coulombs
I = ionic concentration (in mol/L)
N = Avogadro’s number

Keep in mind that;
The units of k are reciprocal length
1/k = “thickness” of the electrical double layerthe Debye length
a = the particle radius
ka = the ratio of particle radius to double layer thickness

Once you calculate your ka then the below table will allow you to choose the proper F(ka) without calculating that as well.

In the software, as you set up a measurement under General Options you can select Smoluchowski, Henry, Custom or Calculated. If you click calculated and press the calculate tab you will be able to insert the dielectric constant, particle radius and ionic strength and it will calculate the F(ka) for you. Once you know your F(ka) you can use that as the Custom value for your measurements. You can also go back and Edit all your past results. You will not have to remeasure everything.

The upper concentration limit is 40wt%, however, this upper concentration is sample dependent. Your samples are already higher in concentration than the upper limit so I would do a series of dilutions until you start to get good measurements. It’s possible that with a small dilution of 40% you might get decent results but again you should try a few concentrations to make sure that you are in a stable region for the measurements.

Zeta potential theory is based on non-overlapping double layers. Therefore, dilute measurements are often preferred. However, I am attaching an application note that reports on a zeta potential study of concentrated skim milk as an example of high concentration zeta potential measurements.