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SOP: Detection of Antioxidants by Paper Chromatography

GIFSQ — Global Institute for Food Safety & Quality

Principle

The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and the gallates — propyl (PG), octyl (OG) and dodecyl (DG) — are extracted with methanol, separated by ascending paper chromatography on oil-impregnated paper with 65% methanol, and detected as blue spots with phosphomolybdic acid and ammonia fumes. The antioxidants are identified from their Rf values.

Apparatus required

  • Water bath (50 °C)
  • Whatman No. 3 chromatographic-grade filter paper (20 × 20 cm)
  • Jar containing a small dish of concentrated ammonia

Reagents

a. 95% methanol.

b. Calcium carbonate.

c. 10% solution of olive oil in ether (for impregnating the paper).

d. 65% methanol (developing solvent).

e. 1% phosphomolybdic acid in acetone.

f. Concentrated ammonia.

g. Standards of BHA, BHT, PG, OG and DG.

Procedure

Extract 10 g of sample with 25 ml of 95% methanol at 50 °C in a water bath for 15 min, shaking the samples during extraction; remove the upper layer with a pipette

↓

Make a second extraction similarly; combine the extracts, add a small quantity of calcium carbonate, shake, and filter; concentrate the solution if necessary

↓

Dip Whatman No. 3 chromatographic-grade filter paper (20 × 20 cm) in a 10% solution of olive oil in ether and allow it to dry

↓

Spot the sample and the standards on the paper; run the chromatogram (ascending) using 65% methanol as solvent until the solvent layer has run 12 cm; remove the paper and air dry

↓

Dip the paper in 1% phosphomolybdic acid in acetone and allow it to drain; expose the paper to fumes of ammonia in a jar containing a small dish of concentrated ammonia

↓

Antioxidants appear as blue spots; identify them from the Rf values below

Rf values (as printed):

  • BHA: 0.33
  • BHT: 0.00
  • PG: 0.78
  • OG: (illegible in the available text — consult the printed book)
  • DG: 0.11

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.