Separating Spinach Pigments Using Leaf Chromatography
Materials needed:
spinach, mortar and pestle, petroleum ether, acetone, test tube, test tube rack, beaker, scissors, pencil, ruler, TLC (Thin Layer Chromatography) plate, pipette, capillary tubes
Experiment date: 12/15/23
Written by: Sophia Han
Step 1 (Preparation)
Cut thin slices of spinach leaves and grind it until the pigment is extracted, adding 5mL of acetone (solvent for pigment extraction) in the process.
Match the size of the TLC plate to the test tube the chromatography will take place by cutting it with scissors (if there's no TLC plates available, paper can also be used).
Make an eluent by mixing petroleum ether with acetone in the ratio of 7 to 3 (7 for the petroleum ether and 3 for the acetone) and keep it in a beaker.
Step 2
Mark the baseline with a pencil (make sure to use a pencil, not a pen, as it may affect the results) 1cm from the bottom of the TLC plate and use a capillary tube to add a small spot of the spinach pigment on the starting point. Repeat the process of adding the pigment and drying until there's enough sample on it.
Add the eluent in a test tube and put in the TLC plate (the starting point should not touch the eluent- it shouldn't be dipped in it).
Step 3
The TLC plate can be held using a pencil (refer to the pictures of the experiment on the page). Cover the top of the test tube with parafilm so the eluent cannot evaporate away.
Step 4
Observe the changes as the eluent gets absorbed into the TLC plate.
When the eluent reaches the solvent front of the TLC plate, take it out and calculate the Rf value using a ruler.
Discussion
Results:
Although the starting point is marked by the green spinach pigment, as the eluent is absorbed into the TLC plate, different colors (in ours, green was at the bottom, yellow in the middle, and orange in the upper side) can be seen on the plate.
Explanation:
The different colors signify the various pigments present in the spinach leaf.
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Green- chlorophyll (gives leaves their color and absorbs light energy; is essential for photosynthesis of plants)
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Yellow- xanthophyll
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Orange- carotene
Together, xanthophyll and carotene make up carotenoids that transfer light energy to the chlorophyll for photosynthesis.
Rf value:
The Rf value (retention factor) value is the ratio of the is the ratio of the distance travelled by substance to the distance travelled by solvent (i.e. substance that dissolves a solute). Because of this, the Rf value is always between 0 and 1, and the closer it is to 1, the more soluble the substance (in this case, the pigment) is in that specific solvent.
This value can be calculated by dividing the distance travelled by substance by the distance travelled by solvent (both the distances should be measured from the baseline of the TLC plate).
Our TLC plate was 15cm long, and because we measure the distances from the baseline, the distance travelled by the solvent was 13cm (similarly to the baseline, the solvent front was 1cm away from the top of the plate).
When we round the numbers up to the nearest tenths, the distances of the substances were...
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Green/chlorophyll: 10cm
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Yellow/xanthophyll: 11cm
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Orange/carotene: 12cm
Their Rf values:
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Green/chlorophyll: 10/13 ≈ 0.77
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Yellow/xanthophyll: 11/13 ≈ 0.85
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Orange/carotene: 12/13 ≈ 0.92
Hence, carotene travelled the farthest distance out of the three pigments and is therefore the most soluble (smallest molecular size) with the biggest Rf value while chlorophyll travelled the shortest distance and is the least soluble (largest molecular size) with the smallest Rf value.