Pigments in Plant Leaves Photosynthesis Lab




     Can pigments be extracted out of leaves?   Photosynthesis is the conversion of solar energy by plants into several forms of chemical energy.  Photosynthetic productivity is the carbon balance of a plant over a time period and depends on the following external environmental factors: light availability, water (including air humidity), nutrient availability, carbon dioxide availability, and temperature. Photosynthesis begins when light is absorbed by pigments in the plant cell. One technique for separating and identifying these pigments is paper chromatography. In paper chromatography, solvent moves up the paper carrying with its dissolved substances – in this case plant pigments.  The pigments are carried along at different rates because they are not equally soluble in the solvent and are attracted in different degrees to the paper. Many green leaves contain pigment colors that are not seen until autumn because they are hidden by the chlorophyll.  A few plants have leaves that are red, orange, or yellow all year long. In this investigation, paper chromatography will be used to determine what differences exist in the plant pigments of various colors of leaves.  You will also determine which leaves or which parts of leaves contain the chlorophyll necessary to carry out photosynthesis.  The hypothesis is that if pigments are extracted from a leaf or leaves, then the solvent in which the pigments are exposed to will separate the various plant pigments on paper.











Spinach Leaf (i.e. utilize a pre-packaged bag)

Field Leaf (i.e. from a neighboring tree)

(2) 12.5 cm Filter Paper

(1) 250 mL Beaker

Coin (quarter)

Large Paper Clip

Metric Ruler


Pencil (not pen/marker)







Petroleum Ether (~9 mL / partners)

- include: 100 mL beaker

                  25 mL graduated cylinder

Acetone (~1 mL / partners)

- include: 100 mL beaker







  1. Using one of the filter-papers, place a ruler along the edge of the paper so that 7 cm’s can be measured across the bottom of the circle. With a pencil, draw a line and then using scissors, cut along the line so that you now have a straight bottom cut on the filter-paper.                     


  1. Using a pencil, place a “dot” 1.5 cm from the bottom of the cut side of the filter paper. Do this 2-3 more times by choosing a different starting point to measure along the bottom long cut side.  Thus, you will have 3-4 “dots” 1.5 cm’s from the bottom cut side at different points.


  1. Using a pencil, draw exactly a 10 cm long line called a “Loading Line” by using the 3-4 “dots” created earlier as a guide to help make the loading line parallel to the bottom long cut side (see diagram):


  1. Place the “spinach” leaf blade under the filter paper.  Using a ruler to guide you along the line, place the coin on the filter paper along the edge of the ruler. Gently yet firmly press and roll the coin on the pencil line so that you’re crushing the leaf and making a colored line on the opposite side of the filter paper just behind the pencil line without tearing the paper. Turn the filter paper over and perform the same step on the opposite side.


  1. Collect solvent solution by using the graduated cylinder to measure first, ~9ml of petroleum ether and then by using an eye dropper to measure ~ 1ml (or 1 dropper full) of acetone for a ~ 10mL of solution. Add all of the solution to your 250 mL beaker.


  1. Make a cylinder of the filter paper by using a large paper clip to hold the ends together at the top (not near loading line).  Lower the paper cylinder into a beaker containing solvent flat side of paper down.  You can put the paper clip side against the inside of the beaker to help the cylinder stand up right better. The solvent will begin to move up the paper and cause the pigments to move as well within seconds.


  1. Do not disturb the beaker for 5 minutes, or until the solvent is about 1 cm from the top of the paper.


  1. After 5 minutes or the solvent is about 1 cm from the top of the paper, remove the paper and unfold the paper and place the paper flat on the table (solution should evaporate).


  1. Allow the filter-paper cylinder to dry. Some possible colors and the pigments they represent are:  


            yellow / orange = carotenes

            faint yellow = xanthophyll

            bright green = chlorophyll a

            yellow-green = chlorophyll b








  1. Starting from the “loading line” and with a pencil, place a “dot” on the paper at 1 cm increments up to 4 cm’s total.  Thus, you should have a “loading line” with 4 “dots” newly made and a 5th “dot” to represent the “front line” (see diagram):


  1. Repeat steps 1-9 for the “Field Leaf”


* Complete the questions on the data/result sheet provided.

* Clean and put away all materials used in the lab as instructed.




Result/Data Sheet



“Spinach” Leaf

Area Between Lines

Color(s) Observed (if any)

Probable Pigment(s) (if any)

loading line to 1 cm line

(loading line to 1st dot)




1 cm to 2 cm line

(1st dot to 2nd dot)




2 cm to 3 cm line

(2nd to 3rd dot)




3 cm to 4 cm line

(3rd dot to 4th dot)




4 cm line +

(4th dot to 5th dot (Front Line))





“Field” Leaf

Area Between Lines

Color(s) Observed (if any)

Probable Pigment(s) (if any)

loading line to 1 cm line

(loading line to 1st dot)




1 cm to 2 cm line

(1st dot to 2nd dot)




2 cm to 3 cm line

(2nd to 3rd dot)




3 cm to 4 cm line

(3rd dot to 4th dot)




4 cm line +

(4th dot to 5th dot (Front Line))





Answer the following questions: 2 pts each


1.      What is the name of the technique for separating and identifying pigments called?


2.      What was the purpose of the solvent?


3.      How are the pigments separated (carried up the paper) at different rates?


4.      How many pigments were separated in from the spinach leaf?


5.      What were the names of the pigments that were separated from the spinach leaf?


6.      What color(s) (R,O,Y,G, B, I, or V) are mainly “absorbed” by the spinach leaves?


7.      Photosynthesis requires “primarily” what molecule to absorb and transfer energy?


8.      What benefit(s) would there be for having carotenoids such as carotene and xanthophyll?   


9.      Would you agree that accessory pigments were in the spinach leaves along with chlorophyll?


10.  How did the “spinach” leaf compare to “field” leaf?



Conclusion Paragraph:  10 pts


Write a proper conclusion paragraph by “restating” the problem question and hypothesis below and then answer the remaining questions (see bullets below). You may need to review the beginning of the lab; such as, the background information paragraph. When you are done, you should have “one” indented paragraph summarizing the activity.


Do not include any personal pronouns: “I, we, our, etc”….. in the paragraph

Do not include the word “it” anywhere in the paragraph.

Do not have fragmented sentences in the paragraph.


- ie Start off by writing… The problem question was that ….

- ie Start off by writing… The hypothesis was that …

- ie Start off by writing… The hypothesis was …

            - ie Start off by writing… An unforeseen event was …

- ie Start off by writing…  An improvement that could be made for this lab would be …


      * A springboard question is a “question” that you make up based on the problem

                question or this activity.  Do not answer this question; but simply state it as the last   

                sentence in your conclusion paragraph with a question mark (?) at the end.  The  

                question is meant to “springboard” other thoughts that the reader could investigate

                having done this lab which is an inventive yet effective way to end a lab activity.)


Conclusion Paragraph Here: (write clearly)