[Nam]

Well, I am trying to separate cholesterol from a lipid mixture and skimming through one of the references which mentioned about TLC method. And I don't really get the part where it said "[...] develop the TLC in 65:25:4 methanol, water, chloroform." Just wonder how can I use that ratio in preparing my solvent (mobile phase). Is that the percent ratio where each of the substance take up in total respectively? but since they are not add up to a 100, I assume it is something else

[discodermolide]

It's not a "percent ratio". Just take 65mL of methanol, 25mL of water and 4mL of chloroform. Give them a good mix and use it.

[fledarmus]

What Discodermolide said. These are proportions - the recipes can be read as

65 parts methanol
25 parts water
4 parts chloroform

It doesn't really matter what measurement you decide to use for the parts.

[Nam]

Thank you. That really help.