University of Pittsburgh at Bradford
Science In Motion
Bio/Chem Lab 014
THIN LAYER CHROMATOGRAPHY OF VEGETABLES
Introduction:
Thin layer chromatography (TLC) is one type of chromatography. In TLC, the stationary phase is
a thin layer of adsorbent particles (silica gel) attached to a solid plate. A small amount of sample is
applied (spotted) near the bottom of the plate and the plate is placed in the mobile phase (70:30 hexaneacetone solvent). This solvent is drawn up by capillary action. Separation occurs as each component,
being different in chemical and physical composition, interacts with the stationary and mobile phases to a
different degree creating the individual bands on the plate. The retardation factor, Rf value, is used to
characterize and compare components of various samples.
Rf value = distance from origin to component spot
distance from origin to solvent front
The pigments in vegetables, flowers and leaves can be separated and identified by using thin-layer
chromatography. Green pigments, known as chlorophylls, serve as the main photoreceptor molecules of
plants. Carotenoids, yellow pigments, aid the plant in the photosynthesis process. In addition,
xanthrophylls are contained in the chloroplasts which can be isolated and identified using
chromatographic techniques.
Pigment
Color
Rf value
carotene
yellow-orange
0.93
pheophytin a
grey
0.55
pheophytin b
lt. grey (may not be visible)
chlorophyll a
blue-green
0.46
chlorophyll b
green
0.42
xanthophylls
yellow
0.41
xanthophylls
yellow
0.31
xanthophylls
yellow
0.17
0.47-0.54
Purpose:
The purpose of this laboratory experiment is to isolate the pigments from spinach and carrots by
using thin layer chromatography.
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Equipment/Materials:
TLC silica gel plates
chromatography chambers
spinach
shredded carrots
hexane
acetone
capillary tubes
70:30 hexane-acetone solvent
mortar
pestle
thin stemmed pipets
test tubes
Safety:


Always wear an apron and goggles in the laboratory.
Wear gloves when handling the TLC plates.
Procedures:
1. Obtain a spinach leaf and tear it into small pieces. Place the spinach pieces in a mortar.
2. Add 2 pipet bulbs of acetone and carefully grind the spinach and acetone together with the pestle.
Continue grinding until the acetone becomes dark green in color. More acetone may be added to
compensate for evaporation.
3. Using a thin stemmed pipet, extract the acetone and place it in a small test tube. Be careful not to
extract the small pieces of spinach.
4. Add 1 pipet bulb of hexane to the acetone extract in the test tube.
5. Mix the contents of the test tube by using a clean thin stemmed pipet to repeatedly withdraw a sample
and then immediately dispensing the sample back into the test tube.
6. Allow the two layers to separate.
7. Repeat steps 1-6 using shredded carrots.
8. With a pencil draw a line approximately 1 cm from the short edge of the TLC plate. Mark the line
with two x’s approximately 1/3 from either side. (*Wear gloves!!)
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9. With a capillary tube draw up a sample of the upper hexane layer from the test tube containing the
spinach sample and spot the sample on one of the previously marked x’s on the TLC plate. Be sure to
label the spot at the top of the TLC plate.
10. Reapply the sample to the same place at least 3 times or until the spot is clearly visible (the more the
better).
11. Repeat steps 10 and 11 using the carrot extract.
12. Fill the chromatography chamber to a depth of approximately 0.5 cm with the 70:30 hexane-acetone
mobile phase.
13. Place the TLC plate in the chromatography chamber with the sample spot toward the bottom. Be sure
the sample spot is above the level of the solvent. Close the chamber.
14. Allow the plate to remain undisturbed until the solvent reaches to within 1 cm of the top.
15. Remove the plate from the chamber and immediately mark the solvent front using a pencil.
16. Measure and record the distance from the spotting line (origin) to the center of each spot and from the
spotting line to the solvent front.
17. Lightly circle the individual bands with a pencil after the plate dries because the pigment will
eventually fade.
18. Identify each component (spot).
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Name_______________________________
Name_______________________________
Period______________________________
Date________________________________
THIN LAYER CHROMATOGRAPHY OF VEGETABLES
STUDENT EVALUATION
Data Table:
Distance solvent moved from the spotting line (origin) __________________________
Spinach
Color of spot
Distance moved
Rf value
Identity
Carrot
Color of spot
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Distance moved
Rf value
Identity
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Calculations:
Calculate the Rf value for each spot observed.
Questions:
1. Explain the color of the vegetable based on the results of the chromatography.
2. Which pigments, if any, were present in both vegetables? Explain.
3. Why was acetone used?
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