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Thin Layer Chromatography (TLC) Analysis of Phospholipids

Apparatus

  • Nebulizer spray bottles
  • Hot plate @ 200°C
  • (TLC) plates (Whatman K6)
  • Glass pasteur pipets
  • Spray chamber
  • TLC tanks
  • Iodine vapor tank

Reagents

  • Chloroform (B&J)
  • Methanol (B&J)
  • Deionized Water
  • Ammonium Hydroxide
  • Phosphorus spray
  • Ninhydrin spray

Procedure

  1. Prepare TLC Solvent Mixtures.
    1. 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.
    2. 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.
  2. Prepare TLC Migration Tanks.
    1. Line the sides of the TLC tank with filter paper.
    2. Wet the filter paper and cover the bottom of one TLC tank with approximately 1cm of the chloroform:methanol: water solvent mixture.
    3. Prepare another TLC tank as described above and cover the bottom of it with approximately 1 cm chloroform:methanol:ammonium hydroxide solvent mixture.
  3. Spotting and Migration of TLC Plates.
    1. Place two clean TLC plates on the hot plate silica side up for approximately three minutes to reactivate silica. Remove TLC plates from heat.
    2. 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.
    3. Place a TLC plate in each solvent tank. Make sure the solvent does not touch product sample. Replace lids on top of tanks.
    4. Remove TLC plates from tanks when 1/4 in from top. Place TLC plates on hot plate to remove excess solvent.
  4. Spraying and Interpreting TLC Plates.
    1. Place TLC plates in iodine vapor1 tank for a minimum of 5 minutes. Place on hot plate to remove excess iodine.
    2. 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.
    3. 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.
    4. 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.
    5. Allow the plates to remain on the hot plate until the sample disappears or shows a charred appearance.

References

  • 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.