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Detection Methods for Thin Layer Chromatography

Detection of Phosphorus

Equipment & Reagents

  • Fume Hood
    • Nebulizer – 250 ml, 20/40 Joint
      • Mfg – Kontes
    • Catalog # – K4225300250
      • Mfg – Chemglass
  • Catalog # – NC9755621
  • Stirring hot plate
  • Magnetic stir bars
  • Assorted glassware
  • Molybdenum VI Oxide – Aldrich Catalog Number – 22,181-3
  • Molybdenum metal – Aldrich Catalog Number – 20,382-3
  • Sulfuric acid
  • Deionized water

Procedure

  1. Preparation of Reagent 1.
    1. Dissolve 40.1g of molybdic anhydride in 1L of conc. sulfuric acid with heat and stirring overnight.
    2. Cover the beaker or flask with plastic film (Saran wrap) and secure with rubber band to prevent uptake of moisture.
    3. Produces a clear, light blue solution.
  2. Preparation of Reagent 2.
    1. Dissolve 1.78g of molybdenum metal using 500mL of Reagent 1 with heat and stirring overnight.
    2. Cover the beaker or flask with plastic film (Saran wrap) and secure with rubber band to prevent uptake of moisture.
    3. Produces a dark blue solution.
  3. Preparation of Phosphorus Spray.
    1. To 120mL of deionized water add with stirring: 40mL Reagent 1 40mL Reagent 2
    2. Use extreme caution while adding reagents to water.
    3. Important! Always add reagents to water, not water to reagents. Adding water to sulfuric acid solutions can be explosive.
    4. Thoroughly mix the solution, allow to cool, and pour into a nebulizer.
  4. Application of Phosphorus Spray.
    1. In a fume hood, spray TLC plates with molybdate solution until plate is covered with reagent.
    2. Heat plate slightly until spots completely develop.
    3. Phosphorus containing compounds will appear as blue spots on white background. Additional heating will char unsaturated compounds.

References

  • Ellingson, J.S. & William E.M. Lands (1968) Phospholipid Reactivation of Plasmalogen Metabolism, Lipids, pp. 111-120.

Detection of Amines

Equipment & Reagents

  • Nebulizer
  • Assorted glassware
  • n-Butanol
  • Ninhydrin reagent – Acros Catalog Number – 41572-0250
  • Deionized water

Procedure

  1. Preparation of Ninhydrin Spray.
    1. Saturate n-butanol with water by shaking 200mL of n-butanol with 200mL of deionized water in a separatory funnel. Discard the water phase (lower phase).
    2. Dissolve 200mg of ninhydrin reagent in saturated n-butanol with stirring.
    3. Produces a clear, light yellow solution.
    4. Thoroughly mix the solution and pour into a nebulizer.
  2. Application of Ninhydrin Spray.
    1. In a fume hood, spray TLC plates with ninhydrin solution until plate is covered with reagent.
    2. Heat plate until spots completely develop.
    3. Primary amine containing compounds will appear as pink spots on white background.

References

  • Skipski, V.P., Peterson, R.F., & M. Barclay (1962) J. Lipid Res. 3:467.