Hi everyone,
I am following a protocol for extracting lipids from liver tissue for a cholesterol assay. The protocol calls for adding a 2:1 chloroform:methanol mixture, homogenizing, and rotating at 4C overnight. The next day, KH2PO4 is added and the mixture is split into three phases. The lower phase contains the lipids as well as chloroform, which must then be evaporated by drying in a stream of nitrogen.
I do not have access to inert gas, so I was looking for an alternative to this step. I read somewhere that an air stream can be used if butylated hydroxytoluene is added to prevent oxidation. I don't have access to a speed vac either. Could I just evaporate the chloroform in the hood for a few hours without compromising the samples?
Thanks !
You can use a rotary evaporator (40°C maximum temperature of the bath)
SpeedVac concentrators will be best option for rapidly removing organic solvents from lipids extracted using these solvents, especially when you have large number of samples (this is based on my own experience).
Hi Beth!
If you are going to measure ONLY cholesterol, you don't need a speedvac, rotary evaporator or even nitrogen stream.
Just add 0.1% of butylhydroxytoluene to your samples, and let them evaporate overnight in a fume hood. It works perfectly, and the samples will not suffer.
But, again, you can do that if you are planning to quantify cholesterol only. Do not even bother to measure fatty acids, phospholipid composition or other lipid species. It won't work.
Best luck,
Sandra
Drying lipids in hood in no way is recommended, even for cholesterol as oxidation of lipids will take place. If some one is planning or doing lipid research, must have either speed vac/nitrogen evaporator. Unsaturated fatty acids as well as other lipids will be lost. Just buy 22:6 Fatty acid expose little over time to air and see the decrease in level.
As you want to look for cholesterol I do not see any problem if you evaporate the chloroform by which ever way. Of course you might oxidize unsaturated fatty acids, so what? you are looking for cholesterol.
Even if you don't have a SpeedVac or Rotavapor, you can simply use a regular vacuum pump to strip off the solvent. What you need is a clean hose ( rubber ), an vent and a glass trap immersed in ice cold water .
Hi. The cholesterol is a unsaturated lipid that is prone to oxidation in the same manner of the fatty acids, producing oxysterols (named COPs); the oxysterols, in some circumstances, can reach the 10% of total cholesterol. Therefore, be careful in how you dry the lipids.
Sure main purpose is to dry in absence of oxygen so lipid oxidation is avoided
you can dry the lipids on rota vapour drying using low pressure that would be easy and fast
https://www.youtube.com/watch?v=8xg6vRdmTUg
check this simplest and easiest method to create inert atmosphere during lipid extraction to prevent oxidation, i used it, it is very effected.
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