>| AY) ~Chromatography Syllabus \ (} Separation of pigments from extracts of leaves and flowers by paper chromatography and determination of Ry values. i) Separation of constituents present in an inorganic mixture containing two cations only (constituents having large difference in Ry values to be provided.) Ar EXPERIMENT E-t To separate the coloured components (pigments) from extract of leaves and flowers by ascending paper chromatography and find their Ry values. Theory : The extract of leaves and flowers contains a number of coloured organic compounds called (pigments) which are soluble in organic solvents. A spot of the extract is made near one end of the strip of the filter paper. It is suspended in a jar containing a suitable solvent (generally a methanol ot acetone) so that the lower end of the strip dips in the solvent, The pigments travel with different speeds with the moving solvent (a mobile phase) on the paper and form separate spots. Distance travelled by the solute from the reference line R= y= Distance travelled by the solvent from the reference line pparatus : Gas jar, glass rod, filter paper (Whatman No. 1) strip, jar cover, fine capillary tube or syringe. laterials : Flowers/leaves, acetone, HCI, dis- tilled water. es =m \\ Solvent Front—_| Lid Procedure © Crush flowers/leaves in a mortar with the help of a pestle and transfer the paste into a test & tube. | 4 | = Glass rod to ——— | Hang paper © Add small amounts of methanol or acetone in Separated spots <1 the crushed material. Cork the test tube and of differen} shake it well. Filter it and collect the filtrate in consituents —— a test tube and cork the test tube. This is the ® Reference line extracts of leaves/flowers. «© Drawa reference line about 3 em from one end of the filter paper (Whatman No. 1) strip (2-5x2-Sem) with a pencil. © Put a drop of flower/leave extract on the reference line with the help of a fine capillary tube syringe. Dry the spot in air. Repeat 2-3 times putting every drop on the same spot. © Put about 20 mL of solvent (10 ml acetone, 2 mL HCI, 10 mL water) in a gas jar and suspend the filter paper with the help of a glass rod in the gas jar in such a way that the pencil line and the spot remains about 2 cm above the solvent and the lower end of the paper dips in the solvent as shown in Fig E-1 © Cover the jar with a lid and keep it undisturbed till the solvent rises 12-15 em above the original line. : Tre the filter paper out of the jar and mark the solvent front with a pencil. Dry the filter paper and put a mark with pencil in the centre of each coloured spot obtained, Measure the distances travelled by the solvent and each coloured spot from the original line. © Record your observations as shown in the Table. }—Jar #—* 25