when there is no change in droplet size (individual droplets or agglomerates) nano emulsion will not phase separate due to diffusion is faster then phase separation by gravity.

You should be aware that testing under different conditions tests different instability phenomena to different degree. No destabilization during only storage at elevated temperature tells you that Ostwald ripening is not a dominating destabilization process. In case of o/w emulsions freezing provokes coalescence, i.e. mostly indicates vulnerability against coalescence. As Alan pointed out this maybe the mechanism of bioactivity.

If stability is an issue in your case, then prepare self nanoemulsifying drug delivery system (SNEDDS). But stability point of view, nanoemulsion is unstable system. So you have to justify instability in the terms of freeze thaw. It should not be skipped.

To me your statement 'the emulsion that produced highest biological activity was unstable' seems perfectly logical.  If the system was perfectly stable then no material could be released from it.

Respected Alan,

Instability as in, there is phase separation. That makes my formulation unfit to bring it into a product.

Greetings Himanshu,

Be it self emulsified or energy driven emulsified, stability is based on interfacial surface interaction, right? And yes. Nanoemulsions are unstable. And practically, we are not going to store a mosquito repellent at -4 degree. So, is it necessary still? 

Hi Abdul:

If the freeze-thaw method for nanoemulsions stability testing is the industrial or scientific standard, you should carry out the tests as established. In this way, you can really compare your results with the ones in the litterature. It is true that it is quite unlikely the mosquito repellent will be stored  at - 4ºC but, in any case, increasing the stability at this temperature does not seem to me difficult, it is just below freezing point. 

Try to find a way to reduce the freezing point of the aqueous phase (cryogenic protectant? electrolyte? glycerol?). This might solve the problem.

Emulsions are thermodynamically unstable. In time, the different phases of the emulsion will separate. Creating stable emulsions of any kind is very important in most practical applications. Tests such as freeze-thaw, centrifuging, storage at 50oC, 25oC and other temperatures, from one month to a year are routinely conducted to make sure the emulsion will be stable. While phase separation is easy to visually observe, quite often other phenomena such as droplet size, viscosity, color and appearance changes could be happening as a function of the thermal history of the emulsion. These changes should be follows as well as they could be related to the performance of the emulsion such as it biological activity. 

when there is no change in droplet size (individual droplets or agglomerates) nano emulsion will not phase separate due to diffusion is faster then phase separation by gravity.

You should be aware that testing under different conditions tests different instability phenomena to different degree. No destabilization during only storage at elevated temperature tells you that Ostwald ripening is not a dominating destabilization process. In case of o/w emulsions freezing provokes coalescence, i.e. mostly indicates vulnerability against coalescence. As Alan pointed out this maybe the mechanism of bioactivity.

Hi Maria,

Trying electrolyte can be theoretically eliminated as the ions will disturb the volatile compounds I have isolated from the plant extracts (in aqueous phase). Am using ethanol as co-surfactant. So may be I will try replacing ethanol with glycerol as you suggested.

Thanks Ma'am.

Hi Ramiro,

I agree with your comment. As a matter of fact, nano emulsions are more unstable than micro emulsion due to increased surface area. But as Alan said, an emulsion should be unstable to exhibit its biological activity. So, I will work on the most possible stable emulsion formulation and get back to you people.

Thanks for sharing your Knowledge.

Hi Titus,

Coalescence is what at least I expect. But the phases get drastically separated on thaw. What can be mechanism behind that?

Thanks in advance

expectation and reality may differ. Further change to 45°C and back to freezing may play a role. Very complex things may happen. Coalescence stability might change at higher temperature. In freezer you will have a large temperature gradient inside the sample. Further as you mention ethanol now, which is cosolvent not cosurfactant, by freezing water ethanol concentration in continuous phase will increase and may lead to destabilization.

Hi Abdul, if you wish for a more in-depth knowledge on emulsions stability under freeze-thaw conditions, read the publications of Dominique Clausse and her collaborators. This is the group I referred to when I was working on this topic.

Thanks Maria.

Will refer them