LAB Chromatography of Spinach - nnhsbergbio.pbworks.com

NNHS - Berg © 2012

BIO 611 Paper Chromatography of a Spinach Leaf Lab

Background Information:

Paper chromatography is a process that uses
special filter paper to separate and identify the different
substances in a mixture. Chromatography means “to write
with color.” The substances in the mixture dissolve in the
alcohol and move up the paper. Substances are
separated by their weights. The heavier substances move
up the paper more slowly, while the lighter substances
move up the paper more quickly.

Plants may contain a number of pigments. The following chart shows a very brief list of
pigments that may arise in a plant:

Pigment Class Pigment Name Pigment Color
Porphyrin Chlorophylls Green
Carotenoid Carotene Yellow, orange, red
Lycopene Yellow, orange, red
Flavonoid Xanthophylls Yellow
Favone Yellow
Flavonol Yellow
Anthocyanin Red, blue, purple, magenta

Materials: a test tube & test tube rack
Solvent (92% petroleum ether and 8%
colored pencils
acetone) tape
spinach leaf
pencil
chromatography paper ruler
paperclip

NNHS - Berg © 2009

Procedure:
1. Obtain a strip of chromatography paper. Be sure to handle the paper by the edges,
taking care to touch them as little as possible. Oils from your fingertips can interfere with
the migration of pigments up the paper.

2. Use a ruler to measure and draw a fine pencil line about 2-3 cm from one end. This line
is the “origin.”

The “solvent front” is the position of the liquid solvent on the
chromatography paper at any given time. The solvent will
gradually move from the bottom toward the top of the paper,
carrying dissolved pigments with it. Stop the chromatogram
before the solvent front reaches the top of the paper. Mark
the location immediately with a pencil. You will use this
distance to calculate the Rf value.

Using a pencil mark the location of each pigment at the time
the chromatogram is stopped.

Using a pencil mark the origin (where you applied the pigment
extract) before you begin the chromatogram.

3. Place the edge of the spinach leaf over the pencil line indicating the origin. Using the
edge of a coin, gently press on the spinach leaf to create a single green line over the
origin. You want this line to be thin but concentrated with the pigment from the spinach
leaf. Therefore, repeat this edging process carefully about 4-6 times allowing the paper
to dry in between each edging. Be sure not to press too hard or you will poke a hole
through the paper!

4. Label the top of a test tube with your name. Bring the test tube, a stopper, and your
chromatogram to your teacher. Your teacher will add 2-5 mL of solvent into your test
tube. Immediately place your chromatogram into the test tube with the origin end facing
down. Make sure that the level of the solvent is below the origin on your chromatogram
– you do not want to submerge the origin in the solvent! Once the chromatogram is in
the test tube, stopper the test tube to prevent evaporation of the solvent and fumes from
escaping.

WARNING: The solvent is a mixture of petroleum ether and acetone. This solvent
is highly flammable and can be dangerous if inhaled. Take care to avoid inhaling
the fumes as much as possible. Keep clear of any flame, spark, or other ignition
source! If a fume hood is available, your set up will be left under the hood.

5. Check the chromatogram frequently to observe the movement of solvent and pigment
up the chromatography paper.

6. When the solvent front is within 2-3 cm from the top of the paper, remove the
chromatogram. Use a pencil to quickly mark the location of the solvent front. Allow the
chromatogram to air dry by an open window or under a fume hood for 3-4 minutes.
Then trace and label the pigments you observe with a pencil. (These pigments will fade
over time.)

7. Using colored pencils, draw the results of your chromatogram in the data table.

8. Using a ruler, measure the total distance traveled by the solvent. Record data.

9. Using a ruler, measure the total distance traveled by each pigment. Record data.

10. Calculate the Rf value for each pigment using the following formula:

Since the fastest molecules will travel the greatest distance, or to the highest point
along the strip, the relative distances can be measured, and the flow rate (migration) of
the molecules (Rf) can be calculated by using the following formula:

Rf = _Distance pigment traveled__
Distance solvent traveled

Name: ______________________________________ Date: ___________ Block: ____

Paper Chromatography of Spinach – ANALYSIS SHEET

Data: Use colored pencils to draw your observations
Filter Paper

Before Paper
Chromatography

After Paper
Chromatography

Total Distance the Solvent Traveled: ___________ mm

Color of Pigment Distance Traveled Rf value
(mm)

Analysis Questions:
1. Did the leaf you test contain different pigments? Refer to your results to support your
answer.

2. Why did the separation of pigments in the spinach extract occur as it did? (i.e. How
does paper chromatography work?)

3. Why do you think leaves are green even though other pigments are present?

4. Based on what you have learned in this lab, explain why leaves tend to change color in
autumn in New England.

5. Why is it important to stop the chromatogram before the solvent front reaches the top of
your chromatography paper? What would happen to your chromatogram if you let it run
too long?

6. In some cases, two or more pigments may overlap each other on the chromatogram,
making it difficult to isolate and identify them. What are some things you could do to
modify the method to improve pigment separation (distance between pigments)?