Dear Sir. Concerning your issue about the separation of phospholipid monolayer from bilayer. I think the following below links may help you in your analysis:

http://www.ajofranklin.com/caroline/papers/LenzP_Langmuir_2004.pdf

https://hal.inria.fr/file/index/docid/211013/filename/ajp-jphys_1989_50_12_1535_0.pdf

https://etd.ohiolink.edu/rws_etd/document/get/kent1322501214/inline

Thanks

I am not quite sure what you are asking, so I will reply to two different questions. 

In general separating phospholipid bilayer and monolayer VESICLES is not a problem because VESICLES made of phospholipid monolayers do not exist for normal phospholipids, since the reason vesicles form is that the hydrophobic tails can be shielded from water in a bilayer. This does not happen with a monolayer, so a monolayer vesicle with normal phospholipids is impossible. Only way you can make monolayer phospholipid vesicles is to use synthetic bola lipids (i.e. lipids with two polar headgroups at the opposite ends of the lipid). You can have a phospholipid monolayer covering an oil droplet, but then it is an emulsion, not a vesicle, and you cannot have bilayer on an oil droplet. 

It might be that you are actually asking how one can know if the vesicles are unilamellar or oligolamellar, i.e. how you can know if your vesicles have only one bilayer or if they have more than one bilayer stacked. Then the answer to your question depends on what you want to know. If you want to know the numbers of vesicles with one, two, three, etc. bilayers, then you need to use cryo-TEM. If you just need to check that you do not have a significant oligolamellar population, then you can apply a small mole fraction (no more than 1 %) of an NBD-labelled lipid in your vesicles, then check the vesicle size distribution with dynamic light scattering, calculate the expected numbers of the NBD lipids in the inner and outer leaflets of your vesicle population, then apply dithionite to reduce NBD and its abolish fluorescence, the fast initial fractional decrease corresponds to the fraction of NBD lipids on the outer leaflet of the outmost bilayer, so you can check how far this deviates from the expected. You can also get a rough estimate from the avegage size. E.g. if the mean diameter of you vesicle is 100 nm and your bilayer thickness is 4 nm, then the external radius is 50 nm and the internal 46 nm, the areas (4*pi*r^2) are approx. 31400 nm^2 for the external leaflet and 26600 nm^2 for the internal leaflet, so that you would expect roughly 54 % of your probe to be in the external leaflet and 46 % in the internal leaflet. If the initial, fast decrease in the fluorescence of your NBD-labelled lipid is much less than 54 %, then you will know that you have significant population of oligolamellar vesicles.

Hi,

You mean separating them, like by centrifugation?

Or distinguishing between bilayers and monolayers?

Dear Juha,

The hydrophobic section of an amphiphile can be in contact with air or oil hence resulting in the formation of monolayer systems such as micelles. 

Dear M.R., it is a monolayer, but it is not a "vesicle". Please read the question and the answer. 

The question says:

"phospholipid monolayered vesicles" 

Yes, so it says. Air/water (or gas/fluid) interface monolayer is not a vesicle.