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Paper Chromatography of Plant Pigments in Spinach Leaves

Experiment video

What you will learn

Separate and identify photosynthetic leaf pigments (Chlorophyll a, Chlorophyll b, Xanthophyll, and Carotene) from fresh spinach extract using ascending paper chromatography.

Materials needed

Fresh Spinach Leaves g
Fresh Spinach Leaves g
₹50
What is it?
Whatman No. 1 qualitative cellulose chromatography paper sheets and strips.
Specifications
Paper Grade
Whatman No. 1 pure cellulose
Pore Size
11 μm
Thickness
0.18 mm
Capillary Rise
130 mm / 30 min (water)
Chromatography Paper (Whatman No. 1, 20 cm cm strip)
Chromatography Paper (Whatman No. 1, 20 cm cm strip)
₹95
What is it?
Chromatography solvent system: Petroleum Ether (b.p. 60-80°C) and AR Acetone in 9:1 volume ratio.
Specifications
Petroleum Ether
Boiling range 60 – 80 °C AR grade
Acetone
Anhydrous AR grade ≥ 99.5%
Ratio
9 parts Pet Ether : 1 part Acetone (v/v)
Volume
200 mL pre-mixed
Extraction Solvent: Acetone (analytical grade) mL
Extraction Solvent: Acetone (analytical grade) mL
₹140
What is it?
Gas-tight cylindrical glass chromatography developing chamber with ground flat rim and airtight glass lid.
Specifications
Height
250 mm
Internal Diameter
60 mm
Lid
Ground glass disc with suspension hook
Material
Heavy-wall borosilicate glass
Developing Solvent: Petroleum Ether and Acetone (9:1 ratio v/v) mL
Developing Solvent: Petroleum Ether and Acetone (9:1 ratio v/v) mL
₹185
What is it?
Standard laboratory apparatus used in Paper Chromatography of Plant Pigments in Spinach Leaves.
Specifications
Category
Chemistry Apparatus
Grade
CBSE / NEP 2020 Laboratory Standard
Mortar and Pestle with fine washed sand set
Mortar and Pestle with fine washed sand set
₹230
What is it?
Standard laboratory apparatus used in Paper Chromatography of Plant Pigments in Spinach Leaves.
Specifications
Category
Chemistry Apparatus
Grade
CBSE / NEP 2020 Laboratory Standard
Capillary tube / fine micropipette
Capillary tube / fine micropipette
₹275
What is it?
Standard laboratory apparatus used in Paper Chromatography of Plant Pigments in Spinach Leaves.
Specifications
Category
Chemistry Apparatus
Grade
CBSE / NEP 2020 Laboratory Standard
Chromatography Developing Jar with airtight lid
Chromatography Developing Jar with airtight lid
₹320
What is it?
Standard laboratory apparatus used in Paper Chromatography of Plant Pigments in Spinach Leaves.
Specifications
Category
Chemistry Apparatus
Grade
CBSE / NEP 2020 Laboratory Standard

Tick items as you collect them. Tap + to see the product, specifications and buy options.

Stay safe

Keep the sample spot as small and concentrated as possible (< 3 mm diameter).
Do not let the pigment spot immerse directly into the developing solvent pool.
Keep chromatography jar strictly closed and undisturbed during solvent ascent.
Work in a fume hood or away from open flames as petroleum ether and acetone are volatile and flammable.

Steps

0 of 7 steps done

1Grind fresh spinach leaves with 10 mL acetone and a pinch of sand in a mortar to obtain a concentrated dark green extract.
Observation
Grind 10 g fresh spinach leaves in mortar with 5 mL acetone and pinch of sand to extract concentrated dark green chlorophyll.
Safety
Acetone and petroleum ether are extremely flammable volatile solvents; keep away from any open flame or sparks.
2Cut Whatman chromatography paper into a strip 20 cm × 3 cm with a pointed V-shape at the bottom.
Observation
Cut Whatman No. 1 chromatography strip (20 cm × 3 cm); draw pencil baseline 2 cm from bottom notch.
Safety
Handle chromatography paper only by edges with clean dry forceps to avoid finger oil contamination.
3Draw a faint pencil baseline 2 cm above the pointed edge.
Observation
Using fine glass capillary, spot pigment extract on baseline; dry with gentle breath; repeat 10 times to build dense concentrated dark spot.
Safety
Keep spot diameter under 2-3 mm for maximum resolution of separated bands.
4Using a fine capillary tube, apply a tiny drop of pigment extract on the center of the baseline and let dry.
Observation
Suspend strip vertically inside development jar containing petroleum ether : acetone (9:1) solvent, dipping 5 mm below spot.
Safety
Ensure spotted pencil line is strictly ABOVE the solvent reservoir level.
5Repeat spotting 8-10 times over the exact same spot until a dark, concentrated green dot is formed.
Observation
Solvent climbs by capillary action; watch four distinct pigment bands separate progressively up the paper.
Safety
Keep development chamber tightly sealed with greased glass lid during development.
6Pour 10 mL of developing solvent into the jar and suspend the paper so the tip dips into solvent while the spot remains 1 cm above the liquid.
Observation
When solvent reaches 10 cm, remove strip; instantly mark solvent front; measure band centers and calculate Rf values.
Safety
Dry chromatogram in chemical fume hood away from heat sources.
7Cover the jar tightly; allow the solvent front to ascend 15 cm. Remove strip, mark solvent front, and calculate Retention Factors: Rf = distance traveled by pigment / distance traveled by solvent.

Tap + on a step to see what you will observe and any safety tips.

Record your observations

Pigment Band Identity	Visual Color of Band	Distance Traveled (cm)	Solvent Front Distance (cm)	Calculated Retention Factor (Rf)
Chlorophyll b	Olive / Yellow-Green	1.8 cm	10.0 cm	0.18
Chlorophyll a	Blue-Green	3.5 cm	10.0 cm	0.35
Xanthophyll	Bright Golden Yellow	6.2 cm	10.0 cm	0.62
Carotene (β-Carotene)	Vibrant Orange-Yellow	9.5 cm	10.0 cm	0.95

Tap a highlighted cell to write your own observation.

Result and conclusion

Distinct color bands separated on Whatman paper strip: olive green (Chlorophyll b), blue green (Chlorophyll a), yellow (Xanthophyll), and orange-yellow (Carotene) with distinct Rf values.

Rf = (Distance traveled by pigment solute) / (Distance traveled by solvent front)

Observation	What it means
Differential Adsorption & Partition	Carotene is highly non-polar and hydrocarbon-soluble, traveling almost with the petroleum ether front (Rf = 0.95).
Polarity Order Revealed	Polarity sequence: Chlorophyll b (most polar, strong H-bonding to cellulose, Rf = 0.18) > Chlorophyll a (0.35) > Xanthophyll (0.62) > Carotene (least polar, 0.95).
Distinct Band Separation	Four razor-sharp pigment bands separate cleanly across 10 cm solvent migration without smearing or tailing.
Solvent Front Calibration	Marking solvent front pencil line immediately upon removing strip prevents under-estimation of Rf due to rapid solvent evaporation.

Chromatographic Retention Factor (Rf) vs Pigment Hydrophobicity

Sample readings. Replace with your own results.

Experiment photos

Before the experimentDuring the experimentResult