Estimate the β-carotene content of the given vegetable by column chromatography and colorimetry (demonstration).

1. 1 · Aim. To estimate the β-carotene content of the given vegetable, per 100 g fresh weight.
2. 2 · Principle. Acetone extracts the pigments; they are moved into petroleum ether. On an alumina or MgO column, β-carotene (least polar) elutes first. Its absorbance at about 450 nm is compared with a standard (Beer's law).
3. 3 · Requirements.
   • Acetone, petroleum ether, anhydrous sodium sulphate, adsorbent, standard β-carotene.
   • Mortar and pestle, separating funnel, column, volumetric flask, colorimeter.
4. 4 · Procedure.
   • Grind 5 g sample with acetone until colourless.
   • Partition into petroleum ether; dry with sodium sulphate; concentrate.
   • Run the column; collect the first yellow-orange band; make up to 50 mL.
   • Read at about 450 nm with the standard and a blank.
5. 5 · Observation. [Table: Solution | Concentration | Absorbance] Blank 0; standard 2.0 µg/mL, 0.50; sample eluate, 0.45.
6. 6 · Calculation. (0.45 ÷ 0.50) × 2.0 = 1.8 µg/mL; × 50 mL = 90 µg in 5.0 g; = 1800 µg per 100 g.
7. 7 · Result. The given sample contains ___ µg β-carotene per 100 g (fresh weight).
8. 8 · Precautions.
   • Dim light; work quickly; read soon after elution.
   • Flammable solvents: no flames, fume hood, gloves and goggles, pipette filler.
   • Do not let the column run dry.

Scoring tip: Write the formula before putting in numbers, and give the unit (µg per 100 g) in the result.