Dear Muhammad Husnain Ahmad ,

Thanks for showing me the procedure. As I suspected, the protocol indicates “After evaporating the chloroform solvent, PA was dissolved in culture medium and dispersed via 10 min of sonication in a water bath to form micelles.” So, unlike what you described in your question, not ‘simply’ dilute. Looking at your ‘stock’ solution (it depends on the concentration) it looks indeed somewhat too cloudy, but batch sonication is at higher concentrations certainly not fully transparent (but should render a homogeneous solution). According to https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229452/ the used pH 6.5 you have to use according to the protocol you used might be pretty critical.

I suggest two things (provided that you indeed evaporated the chloroform):

-Change pH of your stock solution to at least 7.0-7.5 (with more real charge the PA will/can behave as a ‘detergent’ and forms micelles/vesicles)

-Try a as low as possible concentration of your stock (let’s say mw= 700 g/mol (depending on the exact type you have). Where 7 gram in 20 mL which will give you a 0,5 mM = 500 μM solution

Tip: perform sonication preferably at low temperature (use cold water or add some ice first). Hope this helps.

Best regards.

Try hot plate evaporation method. Usually phospholipid and chloroform is mixed together and evaporated by hand shaking method. Once evaporated, thin layer will be formed which can be diluted by adding warm water by maintaining the temperature 30°C.

In my case till this step solution was not clear until I added warm mixture of ethanol and propylene glycol.

Thanks Rob Keller Your guidelines helped me a lot to conduct my experiment smoothly