2. Overview of the Experiment Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the pigments in leaves with paper chromatography. I will be able to understand the process of paper chromatography and the differences in plant pigments. I will also be able answer the question: “Why do leaves change color in Autumn?”

3. Introduction: Background : Leaves are green because of the chlorophyll pigment that you can see. It is involved in the process of photosynthesis. –However chlorophyll is not the only pigment. –Chromatography is the process that will separate out all of the pigments. Purpose: to know that sunlight is captured in the chloroplasts where the chlorophyll and other pigments are found.

4. Lab Setup: Materials: 1 beaker 1 beaker 1 piece of chromatography paper 1 piece of chromatography paper Washer or coin for transferring pigment onto paper Washer or coin for transferring pigment onto paper Leaf material Leaf material Rubbing Alcohol(just enough to cover the bottom of the beaker) Rubbing Alcohol(just enough to cover the bottom of the beaker) Ruler Ruler

5. Procedure: 1.Obtain a piece of chromatography paper, a piece of plant leaf and a washer. 2.Place plant leaf over the pencil mark on the chromatography paper and rub the washer over the top of the plant leaf.

6. Procedure Continued: 3. Make sure to get a nice green line transferred to the paper without tearing the paper. 4.Fold the paper length wise so that it will stand up in the beaker. 5.Obtain a beaker with rubbing alcohol, return to your table and stand the chromatography paper up in the beaker. We will allow it to run for at least 10 minutes.

7. Make sure tip of paper is touching solvent Make sure green line is ABOVE solvent

8. What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Quantify ComponentsMixture

9. Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass. Terminology: Differential – showing a difference, distinctive Affinity – natural attraction or force between things Mobile Medium – gas or liquid that carries the components (mobile phase) Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)

10. Simplified Definition: T he molecules migrate, or move up the paper, at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper. Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. T he molecules migrate, or move up the paper, at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper. Explanation: Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase solubilizes the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Definition of Chromatography

11. Uses for Chromatography Chromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample

12. Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product

13. Illustration of Chromatography Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ----------------Insoluble in Mobile Phase Black Red Yellow MixtureComponents Separation Stationary Phase Mobile Phase

15. Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) Types of Chromatography

16. Gas Chromatography Used to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below. Paper Chromatography Can be used to separate the components of inks, dyes, plant compounds (chlorophyll), make- up, and many other substances Liquid Chromatography Used to identify unknown plant pigments & other compounds. Thin-Layer Chromatography Uses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances. Examples of Chromatography

17. Chorophyll a is the primary photosynthetic pigment in plants. A molecule of chlorophyll a is located at the reaction center of photosystems(The photosystem is where the light reactions take place inside the thylakoids in plants).

18. Other chlorophyll a molecules, chlorophyll b, and the carotenoids (including caratones and xanthophylls) capture light energy and transfer it to the chlorophyll a at the reaction center. Side Note: Carotenoids also protect the photosytems from the damaging effects of ultraviolet light.

19. Factors that affect movement of molecules along chromatography paper 1.___________of molecule in solvent used (better it dissolves… farther it will move) 2. _____________of molecule (bigger molecules move slower) 3. _____________ of paper used (Attraction of molecules to paper) 4. __________allowed to run Solubility Size Surface Time

20. PLANT PIGMENTS. http://members.aol.com/diaspore/vhouse/biology/projects/photosynthesis/woida/meaning_of_light.html#question_1

21. Examples:

22. PLANT PIGMENTS Carotene is orange yellow Xanthophyll is lemon yellow Chlorophyll a is blue green Chlorophyll b is yellow green.

23. Measuring the Distance

24. Why Do Leaves Change Color? Extracting Pigments From Chloroplasts

25. Why Does This Happen? The green chloroplasts mask the other pigments. They are always there. We just can’t see them until cool weather approaches and photosynthesis decreases. Without food production, the green disappears and the masked pigments appear. Eventually the leaf falls.

26. Substance/ Pigment Distance MovedRf Value Solvent (clear)------------ Carotenes (yellow and orange) Lutein (gray) Chlorophyll a (blue- green) Xantholphylls (yellow) Chlorophyll b (yellow-green)

27. Conclusion Individual 1. Which pigment seems to be the most abundant judging by the darkest? 2. Which pigment had the highest Rf factor and what does this mean? 3. Do you think the results would be the same if you used a different kind of green leaf and why?

28. Conclusion at your tables Write these down after talking with your group and I will randomly call on some groups so be ready!!! 4. Why do leaves look green when there are so many other colors in them? 5. Why do leaves change colors in fall? What do you think is happening.