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 pigment extract was placed on the marked line and was air dried then the process was repeated until ten to fifteen drops of extract has been added to the original spot. Then the filter paper was suspended so that it extends into the solvent but the pigment spot was not immersed in the solvent which is carbon tetrachloride and a scoop of anhydrous sodium sulfate. Finally the slvent was observed as it ascends past the spot moving with any soluble pigments upward with it and each pigment migrates upward at its own rate, independently of any other pigment molecules present resulting in a series of color bands or spots vertically spaced at different levels on the paper strip. The distance any pigment moves upward is specific for that pigment under a given set of conditions: yellow-orange = carotenes
2 yellow bands = xanthophylls
blue-green = chlorophyll a
yellow-green = chlorophyll b
The RF value must be also computed. This is the ratio determined by dividing the distance traveled from the baseline by the solute and by the baseline traveled by the solvent and here is the equation: Rf=distance traveled by solutedistance traveled by solvent
Figure 1. Chromatogram of Zea mays Figure 2. Chromatogram of Figure 3. Chromatogram of qqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqqq Hibiscus rosa-sinensis Codiaeum variegatum Table 1. Chromatography results of Zea mays
Each pigment has its own affinity for solid matter and its own degree of solubility in polar and non-polar solvents. Because of these differences, pigments can be separated by a paper chromatography. The term chromatography (from the Greek chrom, “color” and graph, “to write”) was first introduced in 1906 by the Russian botanist Michae Tsweet. Paper chromatography separates a mixture of compounds based on their different affinities for a stationary phase (paper, gel or other material) and their differential solubility in a moving phase (a suitable liquid solvent).
In our case, the stationary phase is the absorbent chromatography paper and the mobile phase is the mixture of the solvents: carbon tetrachloride and a scoop of anhydrous sodium sulfate. In this laboratory, we have observed how the particular affinities and solubilities of the various photosynthetic pigments (carotenes, xanthophylls, chlorophyll a, chlorophyll b) from the chloroplast of three different plants (Zea mays,Hibiscus rosa-sinensis, Codiaeum variegatum) cause them to migrate up a piece of chromatography paper at different rates.
In order to make the technique more scientific rather than a mere interpretation by sight, what is called the Retention Value (Rf value for short) was applied in chromatography. Rf value was defined as the ratio of the distance traveled by solute to the distance traveled by the solvent in the paper chromatography. Rf value also determines the solubility of a solute which in this lab were the photosynthetic pigments in a particular solvent which is the mixture of carbon tetrachloride and a scoop of anhydrous sodium sulfate.
Two solvents were used in the process because different pigments will be soluble in one solvent but not on others and better separation of pigment bands will result if a combination of solvents is used. If the Rf value is nearer to or equal to one it was said that the solute or the pigment is more soluble in the solvent. In all three different plants used, the Rf value of the pigment carotene is equal to 1 which means that out of all the pigments present carotene was the most soluble. The order of solubility from most soluble to least soluble were as follows based on tables 1, 2, 3:
carotenes > xanthophylls > chlorophyll a > chlorophyll b
* Paper chromatography proved to be an accurate method of separating and observing the various colors of plant pigments. * The pigments dissolved in the solvent and migrated upward. They were observed and their migration distances was measured & recorded. * The Rf value of each pigment was determined by dividing its migration by the migration of the solvent. * It was determined that 4 pigments were present in the original spot carotene, xanthophyll, chlorophyll a, and chlorophyll b. * Carotene was the most soluble, while chlorophyll b was the least soluble.
* (Introduction to botany) Murray W. Nabors * (Introductory botany: plants, people and the environment) Linda R. Berg * http://biologyjunction.com/chromatography_of_simulated_plan.htm