Name_____________________________________________Per______________Date______________
Spinach Chromatography Lab Objectives: Understand the use of chromatography in identifying compounds. Perform chromatography to identify the pigments of green leaves. Calculate the R f values of individual pigments of green plants. Study the relation and use of plants pigments in photosynthesis. Background: All life on Earth depends on the process of photosynthesis. Photosynthesis is a process that plants, cyanobacteria, and algae use to convert CO 2 and water, in the presence of light energy, into sugar. Photosynthesis requires the presence of special pigments that can absorb the energy of light. A pigment is a substance that absorbs light of a particular wavelength. Its color depends upon the color of light that if reflects. For example, a green substance appears green because it reflects green light and absorbs all other colors of light, especially red and blue. The most important plant pigments in photosynthesis are the “chlorophylls”. Green plants contain both chlorophyll a and chlorophyll b. In addition to chlorophyll, the leaves of many green plants also contain one or more other pigments, including “carotenes”, which are orange, and “xanthophylls”, which are yellow. The presence of these other pigments is masked by the abundance of chlorophyll during most of the year. The pigments in plant cells can be separated from one another by a technique known as chromatography. Chromatography is a technique for separating and identifying substances in a mixture, based upon their solubility in a solvent. It is one of the most valuable techniques chemists and biochemists use to determine ingredients that give flavor or scent, analyze environmental pollutants, identify drugs in urine, and even separate proteins that can identify evolutionary relationships. The name chromatography is derived from the Greek words “chroma” and “graph”, which mean “color writing”. Chromatography was invented in 1910 by a Russian botanist, Mikhail Tswett, who used it to separate plant pigments. When a dye mixture is placed on a strip of chromatography paper and placed into a solvent solution, the individual substances in the mixture will migrate up the chromatography paper at different rates. The rate of migration is based upon the adsorption capacity of the chromatography paper and the solubility of the sample in the solvent. As the solvent moves up the chromatography paper strip substances in the mixture that are soluble in the solvent are carried along up the strip. On the other hand, the substances that are more attracted to the chromatography paper than to the solvent stop moving and form bands or spots along the paper strip. After chromatography, each band or spot on the chromatogram can be assigned a Relative Mobility Factor (Rf ) which is R
Adapted from Colors of Autumn Lab by Neo/Sci
Name_____________________________________________Per______________Date______________ characteristic of the specific substance that is associated with it. The R f is a ratio of the distance a substance travels to the distance the solvent travels. It is calculated as follows: R f = Distance solute traveled (D unknown ) Distance solvent traveled (D solvent ) The R f value for a particular substance is constant with a consistent set of chromatographic conditions. Therefore, substances can be identified according to their R f value when compared to a known standard. In this investigation, you will separate the individual pigments involved in photosynthesis. Prelab Questions: R
R
R
R
Answers should be in complete sentences
1. What is the basic importance of chromatography? 2. What does D unknown signify? 3. What does D solvent signify? 4. What does the R f value stand for? R
5. How do you think that the R f value is useful to scientists? R
Adapted from Colors of Autumn Lab by Neo/Sci
Name_____________________________________________Per______________Date______________ Procedure: 1. Using a pencil, draw a faint line across each paper strip, just over the pointed end. 2. Carefully pour 2 mL of the “chromatography solvent” into your test tube. 3. With a quarter, roll a portion of a spinach leave onto the pencil line along the paper strip. 4. Carefully place each paper strip into a test tube with the “chromatography solvent” so that the tapered end of the paper strip just touches the bottom. Adjust the height of the strip so that the solvent does not touch the sample line. 5. When the solvent reaches approximately 1cm from the top of the vial, carefully remove the chromatography paper strip and allow it to air dry. 6. Observe the various plant pigment colors that have separated on the chromatography paper strip and record your results in Table 1. 7. Using a pencil, mark the location of each pigment color as well as the solvent. Take the following measurements of each chromatogram: a. The total distance the solvent moved up the paper (D solvent ) ‐ measured from where you placed the line of spinach pigments (the starting point) to where it ended its climb up the paper (the ending point). b. The distance each pigment traveled (D unknown ) ‐ measure from the pigment’s starting point to the ending point for each individual color. 8. Record your measurements in Table 1 and calculate the R f value for each pigment identified. 9. The chromatography paper strips may be taped or stapled to your lab sheet once they have dried. R
Table 1: Solute color Orange (carotene) Yellow (xanthophylls) Light green (chlorophyll a ) Green (chlorophyll b)
D solvent (cm)
Adapted from Colors of Autumn Lab by Neo/Sci
D unknown (cm)
R f = D unknown /D solvent R
Name_____________________________________________Per______________Date______________ Postlab Questions: Answers should be in complete sentences
1. How many pigments were you able to identify on your spinach chromatogram? 2. What purpose does the chromatography paper have in this experiment? 3. Do some extra investigating to answer the following question: Green pigments are essential for photosynthesis to take place. How do you think that a Japanese maple tree, which only has red leaves, can carry on photosynthesis?
Adapted from Colors of Autumn Lab by Neo/Sci
