Experiment #: 5
Title: Column Chromatography – Separation and Isolation of Pigments from Spinach Leaves
Purpose:
To demonstrate the use of column chromatography, to isolate and analyze the different pigment
classes found in spinach leaves. The goal is to separate and identify carotenoids and chlorophylls
using column chromatography and assess the efficiency of the separation through thin layer
chromatography (TLC).
Procedure:
• Preparation of Pigment Extract:
o Tear 3.5 grams of fresh spinach into small pieces.
o Add spinach to a mortar w/ sodium sulfate (drying agent) and sand
(macerating agent).
o Grind mixture using a pestle until a dry, free-flowing green powder is formed.
o Transfer the powder to a flask and extract pigments w/ 10 mL acetone.
o Repeat extraction w/ an additional 10 mL acetone, then decant the liquid.
o Wash remaining powder w/ more acetone to recover additional pigments.
o Evaporate excess solvent using a warm water bath and stream of dry nitrogen gas.
• TLC Pre-Spotting:
o Using a microcapillary, apply a small amount of raw spinach extract to TLC plate.
o Allow spot to dry for later analysis.
• Column Chromatography:
o Load concentrated extract onto a prepared alumina column using hexane as
a carrier.
o Open stopcock to allow sample to enter stationary phase.
o Begin elution w/ hexane to isolate yellow carotene band.
o Collect carotene fraction in a separate Erlenmeyer flask.
o Switch mobile phase to a 70:30 hexane-acetone mixture to begin eluting
green chlorophylls.
▪ If needed, increase the polarity further to 80:20 acetone-methanol
to improve movement.
o Collect green fraction in a second Erlenmeyer flask.
• Evaporation and Reconstitution:
o Evaporate solvents from both fractions using warm water bath and dry nitrogen.
o Reconstitute pigments in a few drops of acetone
• TLC Analysis:
o Spot each pigment fraction (carotenoid and chlorophyll) on a TLC plate
w/ multiple applications.
o Develop the TLC plate in a 70:30 hexane-acetone chamber.
o Compare results to a plate spotted w/ the original spinach extract.
Data/Results/Calculations:
• Mass of extracted pigments:
o Carotenoids: 50 mg → (50 mg / 3500 mg) × 100% = 1.4%
Title: Column Chromatography – Separation and Isolation of Pigments from Spinach Leaves
Purpose:
To demonstrate the use of column chromatography, to isolate and analyze the different pigment
classes found in spinach leaves. The goal is to separate and identify carotenoids and chlorophylls
using column chromatography and assess the efficiency of the separation through thin layer
chromatography (TLC).
Procedure:
• Preparation of Pigment Extract:
o Tear 3.5 grams of fresh spinach into small pieces.
o Add spinach to a mortar w/ sodium sulfate (drying agent) and sand
(macerating agent).
o Grind mixture using a pestle until a dry, free-flowing green powder is formed.
o Transfer the powder to a flask and extract pigments w/ 10 mL acetone.
o Repeat extraction w/ an additional 10 mL acetone, then decant the liquid.
o Wash remaining powder w/ more acetone to recover additional pigments.
o Evaporate excess solvent using a warm water bath and stream of dry nitrogen gas.
• TLC Pre-Spotting:
o Using a microcapillary, apply a small amount of raw spinach extract to TLC plate.
o Allow spot to dry for later analysis.
• Column Chromatography:
o Load concentrated extract onto a prepared alumina column using hexane as
a carrier.
o Open stopcock to allow sample to enter stationary phase.
o Begin elution w/ hexane to isolate yellow carotene band.
o Collect carotene fraction in a separate Erlenmeyer flask.
o Switch mobile phase to a 70:30 hexane-acetone mixture to begin eluting
green chlorophylls.
▪ If needed, increase the polarity further to 80:20 acetone-methanol
to improve movement.
o Collect green fraction in a second Erlenmeyer flask.
• Evaporation and Reconstitution:
o Evaporate solvents from both fractions using warm water bath and dry nitrogen.
o Reconstitute pigments in a few drops of acetone
• TLC Analysis:
o Spot each pigment fraction (carotenoid and chlorophyll) on a TLC plate
w/ multiple applications.
o Develop the TLC plate in a 70:30 hexane-acetone chamber.
o Compare results to a plate spotted w/ the original spinach extract.
Data/Results/Calculations:
• Mass of extracted pigments:
o Carotenoids: 50 mg → (50 mg / 3500 mg) × 100% = 1.4%
