CHROMATOGRAPHY OF SPINACH & Autumn Leaves LAB
Name __________________
Background - The primary pigment of photosynthesis is Chlorophyll a. This is the pigment that acts as
the main receptor for light energy. The other pigments are called accessory pigments, as they gather light
from different wavelengths and pass the energy to Chlorophyll a.
Chromatography is a method of separating substances from each other to identify them. In this activity
you will be experimenting with a technique called chromatography which will allow you to visually
demonstrate that the pigment in leaves is a combination of several different colored pigments. In this
activity you will use paper chromatography discover the photosynthetic pigments in spinach leaves and
determine their relative Rf value. Rf value is the relative distance each pigment travels in the solvent.
Pigments that are very soluble in the solvent will travel farthest and have a high Rf value; Pigments that
are less soluble will travel less far and have a lower Rf value.
This technique is useful in that it can separate and identify the various components of mixtures, such as
those contained in plant pigments. A pigment is a substance that absorbs light at specific wavelengths,
chlorophyll is one of these pigments. Its green-yellow in color is due to the absorption of red, orange, blue,
and violet wavelengths and the reflection of the green and yellow wavelengths.
If the conditions are identical, the relative distance moved by a particular compound is the same from one
mixture to another. This is why chromatography can be used to identify a compound. The actual
identification requires a simple calculation as shown below:
The Pigments you will be looking for are CHLOROPHYLL a, which is blue green and the main
photosynthetic pigment, and the accessory pigments CHLOROPHYLL b, a yellow-green pigment;
XANTHOPHYLL, a yellow pigment; and CAROTENE, a yellow-orange pigment
When separated by chromatography, Carotene is the most soluble and travels furthest, followed by
Xanthophyll, chlorophyll a and finally chlorophyll b.
Rf = distance moved by compound from original spot divided by the distance moved by solvent from
original spot. It is important to remember that several factors can influence the reliability of the Rf value,
these include humidity, temperature, solvent, pigment extract
preparation, and the amounts of the material present. Values
are comparable only when the extracts are prepared in the
same way and the chromatograms are prepared identically
and developed together in the same container.
Chromatography is an experimental process
that scientists use to separate out the
constituents (i.e. molecules, chemicals,
minerals, pollutants, etc.) in a mixture. Once
separated, the individual pieces can be
identified, analyzed, purified, and/or quantified.
The stationary and mobile components that are
used depend on the solution to be examined
and the goals of the project. The separation of
the different constituents in the mixture
depends on their various attractions to the
stationary component.
Materials –
Alcohol/Acetone
(Use caution, do not
breathe vapors)
Spinach leaves
Chromatography paper
quarter
Scissors
Pencil
Metric ruler
Flask
stopper
Procedure – Preparation of the Paper chromatography strip
1.
Cut chromatography paper strip. Use scissors to cut off the two bottom corners of the strip.
[See the diagram below] TRY TO MINIMIZE TOUCHING THE STRIP. IT MAY INTERFERE
WITH THE CHROMATOGRAPHY. Hold strip by edges.
2.
Measure 2 cm from the bottom of the paper strip. Lightly draw a pencil line across the strip.
Do Not use ink
3.
Place the leaf over the pencil line and Roll a quarter or
other coin over the line Repeat until there is a very dark
green line over the pencil line at least 15 times, Change leaf
to different position each roll.
4.
5.
6.
7.
8.
9.
10.
Place solvent in flask, only a small 1 cm high amount.
Slowly lower the chromatography paper into flask. DO NOT allow pigment to touch solvent
Cover with lid or stopper
Let solvent rise until it reaches the mark at top of paper. KEEP WATCH!
STOP just before solvent front reaches top
Remove chromatogram and place on paper towel. Replace lid on solvent flask. DO NOT discard.
Immediately draw a pencil line across the top of the strip where the solvent front got to before
it dries. See diagram below.
11.
Next draw a pencil line at the top of each pigment, showing how far that pigment traveled.
12.
Measure the total distance traveled by solvent from the starting line to the top in mm. Record
on first line of Data table.
13.
Measure the distance each pigment traveled (migration distance) from origin in mm. Record on
data table.
14.
On Data sheet, draw each migration line on chromatogram copy, use ruler and place lines
according to the measurements you recorded in table.
15.
Label and attach your chromatogram paper, in the space provided.
16.
Label the pigments on your diagram, as well as the loading line (start) and solvent front (finish)
17.
Calculate the Rf value for each pigment. Record on Data Table.
Rf value is the relative distance each pigment traveled.
Rf = Distance Substances traveled
Distances Solvent traveled
Example:
Solvent front traveled 6.0 cm
Pigment 1 traveled 5.5
Rf pigment 1 = 5.5/6.0 = .92
Ex. 2
Rf Pigment 3 = 3.0/6.0 = .5
Spinach Leaf
Autumn Leaf
Calculate your Rf values
Show your work:
Data Table
Chromatography of plant pigments
Band Number
Solvent
Name of
Pigment
N/A
Color of
Pigment
Migration
distance (mm)
Rf value
1.0
1 (top)
2
3
4 (bottom)
1. What reference numbers (Rf) did you calculate for chlorophyll a and b on your
spinach strip? How do they compare to the values given The reference numbers (Rf) for
the chlorophylls are:
0.28 = chlorophyll a
0.18 = chlorophyll b
Use these values to figure out which chlorophyll you have.
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2. Which pigment is the MOST soluble in the solvent? Explain your answer.
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3. Which pigment is LEAST soluble in the solvent? Explain your answer.
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4. Which pigment band is the widest? Hypothesize why.
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5. Which pigment band is the most intense (darkest)? Hypothesize why.
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6. Which autumn leaf color is not present in the spinach? What pigment causes this color?
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7. How could Rf values be useful when comparing chromatograms from other types of leaves?
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8. If you could go back and redo this lab, what mistakes would you correct? If none,
identify areas where someone could make an error. Explain.
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9.What kind of professional out there would need to use a skill such as chromatography?
(forensics? chemist? etc.) Why?
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