Bio 3465 - Lab 7 - Plant Pigments., Isolation _ Separation

Bio 3465 - Lab 7 - Plant Pigments., Isolation _ Separation...

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BIO 3465 – Plant Physiology Laboratory 7 PALNT PIGMENTS: ISOLATION & SEPARATION Goal : Plants contain many pigments that are used for many purposes such as photosynthesis, animal interaction, antioxidants etc. The goal of today’s lab is to extract and separate plant pigments so that chlorophylls and accessory pigments can be visualized while learning the basic principles and the technique of Thin Layer Chromatography (TLC) for separation of plant metabolites. Additionally, you will learn how to find the absorption spectrum of a chemical compound using the spectrophotometer. INTRODUCTION In photosynthesis, plant pigments play a major role in absorption of light energy. This energy is being used to oxidize water while reducing NADP into NADPH and to create a proton (H + ) gradient across the thylakoid membrane. Energy of the proton gradient is used to generate ATP from ADP and inorganic phosphate (P i ). NADPH and ATP will then be used to reduce CO 2 in the carbon (dark) reaction to produce carbohydrates. The major pigments found in plants are chlorophyll a and b, carotenoids, xanthophylls, anthocyanins and betains. While chlorophyll a and b function as major light harvesting pigments, carotenoids and xanthophylls function as accessory pigments to harvest light energy that cannot be harvested by chlorophylls. The colors of pigments vary depending on their chemical structure (Figure 1): chlorophyll a - blue-green; chlorophyll b - yellow-green; xanthophylls- yellow; carotene- orange-yellow. Different pigments have the ability to absorb light energy at different wavelengths of the electromagnetic spectrum (Figure 2) and the absorption spectrum of a particular pigment is a (A) (B) (C) Figure 1 : Structures of Xanthophyll (e.g. Zeaxanthin) (A), Chlorophyll (B), and Carotene (C).
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characteristic of that pigment. Thus by looking at an absorption spectrum of a purified pigment one can easily determine identity of the pigment. Ability of different pigments to absorb light energy at different wavelengths gives the plants the opportunity for maximum use of light energy they receive. Thin Layer Chromatography Thin Layer Chromatography (TLC) is a technique that is used to separate mixture of compounds into individual components. In this technique, a mixture of compounds (e.g. plant extract) is allowed to move along with a solvent on a solid surface (in this case silica gel). Different compounds in the extract have different affinities toward the solvent and the solid surface. Thus, when all the compounds move in one direction on the solid surface, they will move at different speeds separating each compound from the other on the solid surface. The separation efficiency depends on the
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This note was uploaded on 11/09/2011 for the course BIO 101 taught by Professor Pesthy during the Fall '07 term at Texas State.

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Bio 3465 - Lab 7 - Plant Pigments., Isolation _ Separation...

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