Chromatography Lab CER Introduction The primary pigment of photosynthesis is Chlorophyll a. This is the pigment that acts as the main receptor for light energy. The other pigments are called accessory pigments, as they gather light from different wavelengths and pass the energy to Chlorophyll a. Chromatography is a method of separating substances from each other to identify them. In this activity you will use paper chromatography to discover the photosynthetic pigments in spinach leaves and determine their relative Rf value. Rf value is the relative distance each pigment travels in the solvent. Pigments that are very soluble in the solvent will travel farthest and have a high Rf value; Pigments that are less soluble will travel less far and have a lower Rf value. The pigments you will be looking for are CHLOROPHYLL a, which is blue green and the main photosynthetic pigment, and the accessory pigments CHLOROPHYLL b, a yellow-green pigment; XANTHOPHYLL, a yellow pigment; and CAROTENE, a yellow-orange pigment When separated by chromatography, Carotene is the most soluble and travels furthest, followed by Xanthophyll, chlorophyll and finally chlorophyll b. Objective   Use paper chromatography to separate leaf pigments Identify the photosynthetic pigments in the leaves and determine their Rf values Procedure – Preparation of the paper chromatography strip 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Cut a chromatography paper strip from the coffee filter. Make a rectangular strip about an inch wide and the length of the filter's diameter. Measure 2 cm from the bottom of the paper strip. Lightly draw a pencil line across the strip. (Do not use ink pen) Place the leaf over the pencil line and roll a penny or other coin over the line marked in pencil. Repeat 10 15 times, until there is a very dark green line over the pencil line. Change the leaf to a different area on the leaf for each roll to maximize the amount of pigment deposited. Add approximately 1 cm of isopropyl alcohol to a cup. Prepare your pencil and filter set-up. You'll want to tape the filter paper to the pencil so that when the pencil is placed over the cup, the filter paper barely touches the isopropyl alcohol, but the pigment does NOT touch the isopropyl alcohol. Measure how far this needs to be taped by estimating from the outside of the cup. Once prepared, slowly lower the chromatography paper into the cup and rest the pencil on the rim of the cup. Cover the top of the cup with plastic wrap to prevent evaporation as much as possible. Let isopropyl alcohol rise until it reaches about 3/4 of the way up the filter paper. KEEP WATCH! Remove chromatogram and place on paper towel. Immediately draw a pencil line across the top of the strip where the solvent front got to before it dries. Next draw a pencil line at the top of each pigment, showing how far that pigment traveled. Measure the total distance traveled by solvent from the starting line to the top in mm. Record on first line of Data table. Measure the distance each pigment traveled (migration distance) from origin in mm. Record on data table. In your BILL, draw each migration line on chromatogram diagram, use ruler and place lines according to the measurements you recorded in table. Use colors to recreate the chromatogram on your paper, filling in the appropriate colors. Label the pigments on your diagram, as well as the loading line (start) and solvent front (finish) Calculate the Rf value for each pigment. Record on Data Table. Rf value is the relative distance each pigment traveled. Rf = Distance Substances traveled / Distances Solvent traveled Example 1: Solvent front traveled 6.0 cm Pigment 1 traveled 5.5 Rf pigment 1 = 5.5/6.0 = .92 Example 2: Rf Pigment 3 = 3.0/6.0 = .5 📘 Data Collection Band Number Name of Pigment Solvent N/A Color of Pigment Migration Distance (mm) Rf Value 1 (Top) 2 3 4 (Bottom) 📘 Analysis questions – Answer the following questions in your BILL. Be sure to use part of the question in your answer so when you refer back to your responses, they make sense! 1. What factors are involved in the separation of the pigments? 2. Name each of the pigments you were able to identify and describe their roles in plant cells. 3. Which pigment traveled furthest? Why? Which pigment traveled least? Why? 5. How does the amount of each pigment explain why leaves are certain colors? 6. How could you use paper chromatography to determine if the leaves of 2 different plants had the same or different pigments? Your Submission for the Lab CER: a. Consider the experimental objective and how it relates to the process of photosynthesis. What role do plant pigments play in the process of photosynthesis?    Make a CLAIM to answer this question. Support your claim with SPECIFIC EVIDENCE (data collected, observations, etc.) from the experiment you conducted. Include and describe at least one photo of an observation from the lab activity as part of your evidence. Then back up your claim and evidence with REASONING - what scientific concepts/principles connect the evidence to your claim? b. Submit your response in the text box below. All information in your CER must be in your own words. If any sources other than your BILL are used, they must be cited properly in APA or MLA format.