Aim: isolation of phospholipids from egg yolk by performing TLC of this sample. Introduction: Thin layer chromatography is a simple, versatile, inexpensive technique. The separation on thin layer is affected by adsorption or partition. It has several advantages over paper chromatography that it provides sharper and faster separation and has higher sensitivity.
Requirements: silica gel, D/W, glass plate, sample, hexane diethyl ester, glacial acetic acid, capillary, iodine chamber oven etc.
Principle: thin layer of a stationary phase is formed on a suitable tube structure such as glass or a plastic plate. Since the layer is too thin, the movement of the mobile phase across the layer generally by simple capillary action is rapid as the mobile phase moves across the layer from one edge to other opposite side it transfer any solvent phase on the layer at rate determined by their distribution co-efficient has the stationary and mobile phase. Here the stationary phase is silica gel & solvent system is mobile phase.
Procedure: take 30 gm of silica gel add some amount of D/W init then stir it on till the bubble have been removed in it. Add some amount of D/W in it. It is needed to make a uniform layer of silica gel on the glass plate. Once the layer has been prepared the plates are dried over night to leave. The coating as stationary phase next day activate the plates at 80-100°c in the oven before loading the sample.[then load the sample] with the capillary. Then put this sample loaded plate into the saturated solvent chamber to run the sample. After some take the plate from the solvent chamber & allow it to dry. For the detection many methods are used for examination of plates under U.V of light which shows the position of U.V absorbing of flurescent compounds then plates are subjected to the I2 vapour for detection purpose.
Sample preparation: •
egg yolks are collected from 2 eggs eg gs & mixed well.
•
Add 30 ml of cold acetone & allow it to slame for 15min.
•
•
•
Many phosphor lipids are insoluble in acetone so it precipitates out where as triglycerides; steroid, pigment etc. dissolve acetone in it. The ppt is collected by centrifugation at 300-400 rpm for 15 min & washes it 3-4 times with cold acetone till the supernatant is clear & colorless. The ppt is now exlirated with 250ml 2 50ml of chloroform: methanon mixture in 2:1 for 3-4 times at room temperature .exlirated is evaporated to dryness by keeping in oven.
•
•
After drying the residue it taken in small volume of petroleum ether & phospholipids are reprecipited by addition of cold acetone (50-60ml). The ppt is collected dried and sterol & can be used as a sample by dissolving in a minimum volume of chloroform: methanol in 2:1.
Result:
Aim: isolation of phospholipids from liver tissue & perform TLC of liver tissue with phospholipids sample.
Introduction: Thin layer chromatography is a simple, versatile, inexpensive technique. The separation on thin layer is affected by adsorption or partition. It has several advantages over paper chromatography that it provides sharper and faster separation and has higher sensitivity.
Requirement : chicken liver, tissue homogenate , motor pestle, silica gel , D/W, glass plate ,capillaries, I2 chamber , chloroform , methanol etc.
Principle:
thin layer of a stationary phase is formed on a suitable tube structure such as glass
or a plastic plate. Since the layer is too thin, the movement of the mobile phase across the layer generally by simple capillary action is rapid as the mobile phase moves across the layer from one edge to other opposite side it transfer any solvent phase on the layer at rate determined by their distribution co-efficient has the stationary and mobile phase. Here the stationary phase is silica gel & solvent system is mobile phase.
procedure : take 30 gm of silica gel add some amount of D/W init then stir it on till the bubble have been removed in it. Add some amount of D/W in it. It is needed to make a uniform layer of silica gel on the glass plate. Once the layer has been prepared the plates are dried over night to leave. The coating as stationary phase next day activate the plates at 80-100°c in the oven before loading the sample.[then load the sample] with the capillary. Then p ut this sample loaded plate into the saturated solvent chamber to run the sample. After some take the plate from the solvent chamber & allow it to dry. For the detection many methods are used for examination of plates under U.V of light which shows the position of U.V absorbing of flurescent compounds then plates are subjected to the I2 vapour for detection purpose.
Sample preparation from chicken liver:
•
Weight 5gm of chicken liver & cut it into small pices.
•
Add 50ml of chilled water & prepare 10% of homogenate.
•
Take 50ml of in a different beaker & add equal volume of hexane.
•
Stir it well & settle it for 10 mins while 2 layer separations is observed.
•
Discard hexane layer carefully.
•
Collect the pellet in a different beaker adds equal volume of chloroform: methanol mixture.
•
Mix it for 10-15 mins. Centrifuge it at 10000 rpm for 10-15min.
•
Collect the upper layer in a different backer.
•
To the pellet & equal volume of CH-Cl2-CH2OH mix.
•
Again centrifuge it collect the supernatant in the beaker. Do this 2-3 times.
•
Collect the upper layer in a different beaker, lower layer also in a different beaker.
•
Use the lower layer for TLC by concentrating it using it in different solvent system.
Result:
