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Lab Session 6

IUG, spring 2015TMZ

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Analysis of pigments and oxidation processes in photosynthetic organisms

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1. Photosynthetic Organisms

They contain an operative system that can selectively trap the visible light and use it in production of energy in form of ATP:This operative system includes:

a. Chlorophyllsb. Phycobilinsc. Carotinoids

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2. Animal and plant cells

• In mitochondria of animal and plant cells , the energy is produced in a similar system (respiratory chain), where there are specific enzymes responsible for the production of energy

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In plants…

A number of high actively enzymes called oxidases is observed:

1. Peroxidases2. Polyphenoloxidases

Such enzymes are involved in oxidation of secondary metabolites

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Color reactions for plant pigmentsA. extraction of pigments from nettle leaves

PrinciplePigments such as chlorophyll and others have

the property to dissolve in hot ethanol Reacting with ethanol, chlorophyll is

converted to ethylchlorophyllide (an ester in which the phytol is replaced by the ethanol)

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Procedure

1. Grind thoroughly 0.5g of dried nettle leaves in a mortar with 5 ml of ethanol.

2. Transfer the pulp to a test tube and heat in water bath at 90-100 c, until ethanol boils.

3. Filter the contents into a clean test tube. The filtrate contains chlorophyllide, chlorophyll, xanthophylls, and other pigments (it has a grean color and it exhibits an intense red fluorescence on irradiation with UV light.

4. Store the filtrate in a small stoppered flask for further experiments.

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B. Separation of pigments according to Kraus

• PrincipleDifferent pigments have different solubility in petroleum ether.Chlorophyll and chlorophyllide are soluble, while xanthophyl insoluble

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Procedure

1. Transfer 10 drops of the pigment filtrate to a test tube and add an equal volume of petroleum. Mix thoroughly with shaking.

2. Note the difference in color of the pigments contained in the two immiscible solvents.

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C. Precipitation of chlorophyll

• Principle• Chlorophyll ester groups are able to form a

water-insoluble barium salts of chlorophyllide a & b on saponification with barium hydroxide solution

Supernatant liquid has a yellow color due to the presence of carotene and xanthophyll.

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Procedure

1. Transfer 10 drops of filtrate to a test tube and add 20 drops of barium hydroxide soln.

2. Mix thoroughly with shaking, and note the changes observed in this reaction.

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D. Reduction of chlorophyll with ascorbic acid

• PrincipleAscorbic acid can reduce chlorophyll that forms a yellow color in a reduced form state.

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Procedure

• 1. Transfer 10 drops of filtrate to a test tube and add 2 drops of distilled water, and several crystals of ascorbic acid.

• 2. Place the test tube for 3-5 min in a water bath at 70-80 c. Note the change in color.

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E. Formation of Pheophytin from chlorophyll

• PrincipleHydrochloric acid can bind to Mg2+ of chlorophyll, to produce a Pheophytin that has an olive-brown color.

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Procedure

1. Transfer 5 drops of filtrate to a test tube and add 1 drop of hydrochloric acid soln.

2. Note the change in color.