- Nebulizer spray bottles
- Hot plate @ 200°C
- (TLC) plates (Whatman K6)
- Glass pasteur pipets
- Spray chamber
- TLC tanks
- Iodine vapor tank
- Chloroform (B&J)
- Methanol (B&J)
- Deionized Water
- Ammonium Hydroxide
- Phosphorus spray
- Ninhydrin spray
- Prepare TLC Solvent Mixtures.
- 65:25:4 (v/v/v) chloroform : methanol : water – Mix 650 ml chloroform, 250 ml methanol, and 40 ml deionized water in a 1 liter graduated cylinder.
- 65:25:4 (v/v/v) chloroform : methanol: ammonium hydroxide – Mix 650 ml chloroform, 250 ml methanol, and 40 ml ammonium hydroxide in another 1 liter graduated cylinder.
- Prepare TLC Migration Tanks.
- Line the sides of the TLC tank with filter paper.
- Wet the filter paper and cover the bottom of one TLC tank with approximately 1cm of the chloroform:methanol: water solvent mixture.
- Prepare another TLC tank as described above and cover the bottom of it with approximately 1 cm chloroform:methanol:ammonium hydroxide solvent mixture.
- Spotting and Migration of TLC Plates.
- Place two clean TLC plates on the hot plate silica side up for approximately three minutes to reactivate silica. Remove TLC plates from heat.
- Collect a sample of product in appropriate solvent.(no more than 50 mg/ml) with a glass pasteur pipet. Spot sample 1/4 inch from the bottom of each TLC plate.
- Place a TLC plate in each solvent tank. Make sure the solvent does not touch product sample. Replace lids on top of tanks.
- Remove TLC plates from tanks when 1/4 in from top. Place TLC plates on hot plate to remove excess solvent.
- Spraying and Interpreting TLC Plates.
- Place TLC plates in iodine vapor1 tank for a minimum of 5 minutes. Place on hot plate to remove excess iodine.
- Place TLC plates in spray chamber. Completely spray TLC plates with ninhydrin2 spray. Place TLC plates on hot plate for one minute. Read TLC plates for any ninhydrin positive material/product.
- Place TLC plates in spray chamber and spray with phosphorus3 spray. Place TLC plates on hot plate for 30 seconds. Read TLC plates for phosphorus positive material.
- Dip plates in water and place them on the hot plate to determine if any contaminants are present as a visible wet spot. Repeat dip once.
- Allow the plates to remain on the hot plate until the sample disappears or shows a charred appearance.
- Mangold, H.K., (1961) J. Am. Oil Chemists’ Soc. 38, 708.
- Mangold, H.K. & Malins, D.C., (1960) J. Am. Oil Chemists’ Soc. 37, 383.
- Sims, R.P.A. & Larose, J.A.G. (1962) J. Am. Oil Chemists’ Soc. 39, 232.
- Skipski, V.P., Peterson, R.F. & Barclay, M., (1962) J. Lipid Res. 3, 467.
- Ellingson, J.S. & Lands, William E.M., (1968) “Phospholipid Reactivation of Plasmalogen Metabolism”, Lipids, 3, 111-120.