Hi Choon,

Concentration can be an issue for zeta potential measurements in laser doppler electrophoresis as the light scattered by the sample is usually detected at a forward angle, and therefore the light needs to be able to pass through the sample without undergoing significant multiple scattering.

Whether you have multiple scattering occurring for zeta potential measurements is less trivial to spot than in DLS size measurements, but the frequency plot may show some noise in the baseline if multiple scattering has occurred. Additionally you could check for multiple scattering by comparing size measurements by DLS.

The Malvern Zetasizer will vary the measurement position in back scatter to reduce multiple scattering, so if an automatic measurement was performed close to the cell wall, you will have problems measuring in the forward angle. Additionally, a multiply scattering sample will typically result in a smaller size being reported, with a lower intercept to the correlation function.

There are options to measure zeta potential in concentrated systems, and Malvern Panalytical offer a high concentration cell for zeta potential, which has a shorter optical path length through the sample to allow measurements of samples up to 40% w/v in concentration.

Alternatively, dilution may be necessary, but dilution in anything other than the continuous phase of your sample may lead to destabilisation and result in a change in zeta potential. Zeta potential is a property of the particles of interest and the system they are in, so diluting in water for example will change your zeta potential as you are changing the ionic concentration and pH of the system.

You may achieve this by centrifuging or filtering some of your sample, and using this filtered dispersant to dilute another aliquot of the sample.

To add to the excellent answer above, how did you perform your dilution? In what medium?

If you had a multimolecular vesicle, then upon dilution it can turn into a monomolecular vesicle and the zeta potential in you will change. Measure the zeta potential with a constant layer of lipid, then it will be constant.

Thanks all for the inputs. The medium used is just distilled water. It is used for dilution in my case. I would wonder what I can do to measure the zeta potential if the liposome size is too broad (polydisperse) before further size reduction.

An easy check up for measurability is the turbidity of your sample. It has to be clear for reliable data.

My experience with measuring liposomes without zeta potential (around 0 mV), is a high chance of trending data (each measurement the value increases or decreases) because of aggregation and kind of electroformation of liposomes.

Measuring polydisperse samples will not provide good data, what characteristics do you want to express with the zeta potential?

Choon -- don't dilute in DI water. Dilute in the mother liquor to preserve the ionic environment of the original particles. Dilution in DI water is not 'just dilution' - it washes out all the stabilizers and ionic content that preserve the stabilty of the original system. And the ZP must change as well. In the international standards a dilution ladder is recommended so that artifacts such as multiple scattering and particle-particle interactions can be examined. That dilution should be in the same background material as the original liquid - the mother liquor.

"...any way to know if a sample is too concentrated?"

Using a size measurement would be an ideal way to see if multiple scattering is important in your sample. As a very simple check for multiple scattering: If you can read a newspaper through the sample (i.e. looking through the cuvette your are usingsample filled part of the ).. then you should be OK. in other words, if the sample is clear enough that one can see through it, it is single scattering. If it's blurry or cloudy, this indicates the beginning of multiple scattering.

Thanks Ulf Nobbmann for the recommendation!

Hi Goh, do you have zetasizer in your lab?

Sometimes dilution might affect properties of the sample and change zeta potential. There is only one justified way to perform this dilution - by using equilibrium supernatant. In this case the interfacial equilibrium between the surface and the bulk liquid would be maintained and zeta potential would be the same for all volume fractions of particles in the suspension. When the diluent is known (as is the case for a chemical formulation), additional diluent can be prepared. If the diluent is unknown, equilibrium supernatant is readily obtained by centrifugation.

"The original sample has a zeta potential of -0.414 mV while the diluted one has a value of -2.37 mV. Even though both values indicate that the system is stable" is an odd statement. Such zeta potentials would indicate an unstable system IF electrostatic charge is the only stabilizing mechanism.

At some point, too much dilution will simply lead to you measuring noise which the instrument will happily convert to a nonsense zeta potential. You have to be cautious of the graphical results that an instrument may give.

You don't specify your instrument. If it is one that uses PALS then it should show you a "phase plot" from which you can visually assess the level of noise in the result.

One thing to try is to change the voltage applied to the sample and see if the calculated zeta potential remains constant or if it changes. In the latter case, it would support the possibility that you are simply getting a nonsense value.

Regarding multiple scattering, you can also shine a laser pointer through your sample (preferably in a vial rather than a cuvette). If the beam is clear and there's no glow around it then you are probably at a safe dilution. Depending on the instrument, you may also be able to look down into the cuvette when it is in the instrument and look for a similar effect.

Finally, measure the dispersion liquid and see what you get. Also try the laser pointer trick to see if there's anything else scattering.

John.

Hi John, thanks for your advice. I worry about over-dilution as mentioned. As I'm measuring milky liposomal solution, it is difficult to judge for the scattering problem using laser pointer. What's your recommendation for voltage to be applied in this case?

Hi Choon,

If the sample is milky then it is probably too concentrated and should be diluted. Can you look inside the sample cell when it is in the instrument and the laser beam is on? If so, if beam looks narrow and the rest of the sample is clear then the dilution is probably acceptable. If the entire sample glows then it is too concentrated.

Regarding voltage, it depends on many factors. The two most important are the geometry of the sample cell (is it a simple cuvette with 'dip' electrodes or the folded capillary cell that Malvern provide?) and the electrolyte concentration of your sample. For low electrolyte (1mM or less) and a traditional electrode configuration then 2 to 5V will probably be a good starting point. What instrument are you using?

John.

Hi Choon,

I have just started analysing with zeta-sizer have no experience regarding to your question but I have another question :)

How do you know that your sample is stable or not?

Thanks

Hi Hanan,

Colloidal systems may be stabilized using either electrostatic or steric interactions between particles.

If you are replying in electrostatic stabilisation, Zeta potential measurement may indicate stability if your sample presents a strongly positive or negative Zeta potential. People often quote a Zeta potential of greater than 30mV in magnitude for stability however this may not apply to all samples.

Zeta potential is a property of a material and it's surroundings, and will vary with ionic and pH changes.

Alternatively, you may study stability by measuring particle size as a function of time, with consistent results indicating stability.

http://www.materials-talks.com/blog/2012/01/17/dispersion-stability-what-is-important/

Further from my concern for dilution, anyone has an idea to what extent should we dilute the sample?

A good starting point is to achieve somewhere in the region of 10^8 particles per milliliter. You need to know the mass or volume fraction of particles in your dispersion and their size so you can estimate the number concentration of particles. These numbers don't need to be too precise. Make the diluted dispersion and a series of two-fold dilutions of that dilute dispersion to get a range of concentrations. Some instruments will indicate a "count rate". You can use this figure to help. Note, the dilution requirements for zeta potential are the same as for sizing for a given instrument. Be sure to dilute your sample with the same liquid composition as the particles are already dispersed in - or a close as possible.

Thanks John. That is very helpful!

Dilution in DI water would change your zeta potential as it changes ionic strength. You have to take into account concentration and pH factors that affect ionic strength. I have better results with a 1:8 dilution from my original milky sample of micrometric liposomes but diluted in the original PBS medium.

I recommend David's answer

Best Regards

David E. Bautista-Erazo Hello.I diluted my liposomal sample too much I guess. It was reached 820ul from 20ul sample by PBS. Strangely, I found Zp value of -30 mv, which is supposed to be neutral. Also, its graph did not appear. Do you think the problem is their low concentration?

Fariba Fahmideh Mahdizadeh If you are using a Zetasizer Nano, please post your dts file for the measurement here. This is by far the quickest and most reliable way to try to give you an accurate answer to your problem. If you don't know how to do this, please ask.

You mention using PBS buffer. It is possible that the instrument (if a Zetasizer Nano) would have selected the so-called "monomodal" analysis mode instead of "general purpose". Monomodal mode only gives a mean numerical result (and a "phase plot" that you can view. General purpose can provide a distribution (i.e., a zeta potential plot).

Fariba Fahmideh Mahdizadeh Thank you. As I suspected, the instrument chose the monomodal mode which is why there is no distribution shown. This is probably due to the conductivity of the sample that it detected. The results look acceptable.

I also notice you specified the DTS1060C folded capillary cell. You may want to check that it is not the DTS1070. The one you specified was discontinued by Malvern.

John Francis Miller I really appreciate it. Do you have any idea why the Zp value is quite high? -30mv. Since I have capsulated my active molecule in a zwitterionic liposome, I expected a neutral system.

Fariba Fahmideh Mahdizadeh how is the process of the separation of the unencapsulated compound done? if you use centrifugation maybe some of the unencapsulated compound has precipitated over the bilayer, changing the zeta potential (like some sort of absorption?)

David E. Bautista-Erazo Actually since this molecule was hydrophobic and not soluble in water, for this type of molecule I expected its non-entrapped compounds were already separated by getting stuck behind the polycarbonate membrane. In fact, I skip the purification step for the capsulation of this type of molecule.

Hi,

According to the following statement:

"The charge density of nanoliposomal surface and the binding affinity of various ions to the lipid vesicles can be determined by measuring a parameter called “zeta potential” (ZP). The ZP of a nanoliposome is the overall charge that the nanovesicle acquires in a particular environment or suspension medium [1, 19]. In another words, ZP is the charge that develops at the interface between a particle’s surface (e.g. nano- liposome surface) and its liquid medium. This parameter, which is measured in Mil- liVolts (mV), may arise by any of several mechanisms. Among these are the dissociation of ionogenic groups on the vesicle surface and the differential adsorption of solution ions into the surface region. The net charge at the surface of the nanoliposome affects the ion distribution in the surrounding region, increasing the concentration of counter-ions near the surface. Consequently, an electrical double

layer is formed in the region of the nanovesicle-liquid interface (Fig. 3)...."

Source:

Probing nanoliposomes using single particle analytical techniques: effect of excipients, solvents, phase transition and zeta potential

M Danaei, M Kalantari, M Raji, HS Fekri, R Saber, GP Asnani, ......

Heliyon 4 (12), e01088

It should be noted that:

In simple words: Zeta potential is a property of the particles and the medium they are present in. Therefore, diluting with an aqueous solution, for instance, will change the zeta potential since the ionic concentration and pH of the system will change.

M. R. Mozafari Thank you so much. As I diluted them by PBS and pH meter showed, pH was still 7. Considering this parameter, I am still wondering an increase in negative charge from 0 to -30mv is due to the ionic concentration.

"Therefore, diluting with an aqueous solution, for instance, will change the zeta potential since the ionic concentration and pH of the system will change."

Well, yes and no. If that aqueous solution has the same ionic composition as that of the sample you are diluting, it is unlikely to change. If the sample is in PBS buffer and you are diluting with PBS buffer, it is unlikely that the observed zeta potential will change significantly.

It is good practice to measure the zeta potential as a function of dilution under constant ionic conditions anyway.

Dilution with the same ionic conditions may result in a change in observed zeta potential when dealing with highly concentrated dispersions as far as particle concentration is concerned (e.g., >10%) where the ionic composition of the bulk fluid contains dissociated ions from the particles themselves.

One final comment - what is the right dilution for your particles in the first place? If your liposomes are intended for an injectable medical product then, preferably, all characterization should be done at that particle concentration. If it is too opaque for light scattering, then the particles should be diluted with the same vehicle that they are dispersed in. If that is not possible, then a vehicle with similar properties should be used. Either way, measurements should be made at a range a dilutions to confirm/deny the presence of a change of zeta potential due to dilution. When diluting, the samples should be given sufficient time to reach any new equilibrium. i.e., if you dilute a sample and measure it immediately, you may get an erroneous result because the distribution of ions etc near/at the liposome/liquid interface has not equilibrated.

John Francis Miller Great explanation. Sure. I examine a set of different concentrations to conclude it.

Fariba Fahmideh Mahdizadeh Is it possible that some of your drug is entrapped within the membrane and headed to the exterior (something like an anchor) and thus changing a bit the "expected" zeta potential. This could happen during sequential passages through the membrane.

David E. Bautista-Erazo You are perfectly right. This is what I just noticed. It is because of the compound's location where is near the headgroup and alters zp value. Thank you again.