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Alm : To separate plant pigments through paper chromatography., , 1, Requirements • Chromatographic paper (Whatman No.1- It is fine quality Glucose paper), a_cohol,, acetone, pestle and m~rtar, Ether : Acetone= g : 1, green leaves (e.g., spinach), capillary, glass Jars, scissor5,, pencil, scale, filter paper., , Prlnclple of chromatography :, , Paper chromatography is monodimensional ascending chromatography in which pigments move upwa rd5 in, one direction only. Thus pigments move to different distances, depending on their solubility in sol~ent syste~. On, a special chromatographic paper, pigments are adsorbed on chromatographic paper and rise· with the capillary, action. Solvent system components vary in density and move with different rates due to wick action through, chromatography paper. Heavy components move slow than lighter component. Thus pigments are separated due, to differential solubility in solvent system and different rate of mobility of solvent system components., . Four major photosynthetic pigments of chloroplast are-chlorophyll a, chlorophyll b, carotenes and Xanthophylls. Pigments absorb solar radiations at different 11.'s of visible spectrum for photosynthesis., , Theory:, , =, , Distance travelled by the compound, Distance travelled by the solvent, , Chromatography : It is a technique used to separate molecules of different substances present together in J, a solution according to the rate they travel. It was discovered in 1906 by a Russian Botanist Michael Tswett., ', Principle : Separation of substances takes place according to the rate they travel along a chromatographic, p~per or column of a~sorbe~t material. Such a preparation is called chromatogram. When a solution contain ing, different substances Is applied to chromatogram and allowed to flow, molecules of different substances travel, through medium at different rates and are separated. R,value for each compound can be calculated as, R, ', , Types of chromatography : Depending upon the n:1edium of chromatogram, chromatography is of fou r, types-paper chromatography, column chromatography, thin layer chromatography and partition chromatography., , Procedure :, Preparation of chromatographic chamber _, , 1. Take a strip of chromatographic paper., 2. Draw a line 1 cm away from one end of chromatographic paper., 3. Take a glass jar. Put a little amount of solvent (Ether, acetone solvent in ratio Eth ., ., the jar and cover it or chromatographic chamber can be used., ', er · acetone = 9 : 1) 1n

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preparation of pigment extract :, 4. Take few spinach leaves and crush them with pestle and mortar by adding BO% acetone little by little., s. Filter the material and collect the green coloured extract in a test tube., 6. Put a drop of green extract on already prepared chromatographic paper with the help of a capillary., 1. Allow the drop to dry and again put a drop. Repeat it 3-4 times., a. Place the chromatographic paper, Whatmann No-1 in glass jar in such a way that It does not touch the, sides of glass jar and is only 1/2 cm dipped in solvent., 9. Keep the set up undisturbed tlll solvent rises in the chromatographic paper. (It may take about 40, minutes in chromatographic chamber.), 1o. Take out paper and allow It to dry., 11 . Mark the different components with pencil and label., 12. Cover the chromatogram with black paper to prevent photo oxidation., Identify pigments on the basla of colours, Colour, Pigment, 1., greenish yellow, Chlorophyll b, 2., Bluish green, Chlorophyll a, 3., yellow, Carotene, 4., Orange, Xanthophyll, , Observations :, , l, , ---IV, , Ill, II, I, , Ch romatographlc paper, , ~, , ......, ......, , ..., , Carotene, Xa nthophyll, , ,., ', , Chiorophyll-a, Chiorophyll-b, , le\_, , Ja r, Loading marl<, , -, , s olvent, , Paper chromatography., Calculations :, Distance travelled by compound of pigment band I from loading spot = ........ ., Distance travelled by compound of pigment band II from loading spot = ...... ... ., , Distance travelled by compound of pigment band Ill from loading spot = ......... ., Distance travelled by compound of pigment band IV from loading spot = .........., Total distance travelled by solvent, R, _ Distance travelled by compound of pigment bond, Total distance travelled by solvent., Similarly calculate R, value fo r each pigment band., Result : Following result should be present., Colour of spot, , For green pigment band, For bluish green pigment band, For yellow pigment band, For yellow orange pigment band, , R, value, 0-45, 0 -65, 0 -7 1, 0 -95, , Inference, , Pigment is chlorophyll •b', Pigment is chlorophyll 'a', Pigment is xanthophyll, Pigment is carotene.