College of Arts and Sciences
Visayas State Unversity
Visca, Babay City, Leyte
Name: Millama, Engene O. Course & Year: BS Biotech-2 Lab Schedule: W (1-4) Group 5 Date Performed: Dec. 12, 2012 Date Submitted: Jan. 9, 2013
Separation of Photosynthetic Pigment by Paper Chromatography
There are different types of components in plant pigments. The most important and abundant chemical pigment found in plants is chlorophyll. This pigment exists in two forms; chlorophyll a and chlorophyll b. Chlorophyll a, being the main photosynthetic pigment, has a primary purpose to convert light energy to chemical energy used by the plant itself. Chlorophyll b absorbs light in a region of the spectrum apart from the dominant chlorophyll, and transfers the energy it produces to chlorophyll a. Along with chlorophyll b in transferring their energy produced to the dominant chlorophyll, two other pigments that are found in plants are carotenes and xanthophylls, which are orange and yellow respectively. Since chlorophyll is such a dominant pigment in green plants, this domination hides the color of the carotenes and xanthophylls in the leaves. This causes most plant leaves to appear green most of the time. During the autumn, however, the chlorophyll starts to break down, causing the carotenes and xanthophylls to show their bright red, orange and yellow colors. These brilliant colors can be separated another way. This different technique, known as paper chromatography, separates mixtures in a liquid into individual components. The process of chromatography separates molecules because of the different solubilities of the molecules in a selected solvent. In paper chromatography, paper is marked with an unknown, such as plant extract, is placed in a developing chamber with a specified solvent. The solvent carries the dissolved pigments as it moves up the paper. The pigments are carried at different rates because they are not equally soluble. A pigment that is the most soluble will travel the greatest distance and a pigment that is less soluble will move a shorter distance. The Retention Value (Rf value for short) of each pigment is calculated to establish the relative rate of migration for each pigment. This value represents the ratio of the distance a pigment traveled on the chromatogram relative to the distance the solvent front moved. Scientists use the Rf value of a sample to identify the molecule. Any molecule in a given solvent matrix system has a uniquely consistent Rf value.
* To understand the process of chromatography and to separate a mixture of photosynthetic pigments extracted from leaves. * To separate the pigments from a leaf by paper chromatography. * To identify a mixture by separating it into the different compounds. * To compare pigments found in different leaves to see similarities and differences.
The materials used in this experiment were microcapillary tubing, 100mL Erlenmeyer flask, and ruler, three pieces of paper chromatogram and mortar and pestle. The specimens that were used were corn (Zea mays), gumamela (Hibiscus rosa-sinensis) and San Francisco (Codiaeumvariegatum). There were five steps that are involved in the pigment extraction and separation. First step was to place 1.0 gram each of freshly grounded leaves of the above specimens in 5-10 mL of acetone in the test tubes. Stopper the tube and must be shaked intermittently and stand the tube for about 10 minutes. Second, the three strips of chromatography filter paper was cut by 5.1 cm x 15.3 cm for each type of extract then by using a pencil, a mark or spotting line approximately 2 cm from the bottom of the paper was drawn lightly. Touching the paper apart from the edge was refrained because proteins and oils from fingers may interfere with the development of the chromatogram. Third, using a microcapillary tubing or pipette, a drop of...