Experiment - 20 Aim: To separate the pigments present in leaves and flowers by paper chromatography and determine their Rf values. Theory: Paper chromatography is essentially a partition chromatography. In paper chromatography the stationary phase is paper. Paper contains 22% of water molecules absorbed on about 78% of cellulose. ‘The separation of the components of the mixture takes place by partitioning of the components between the stationary phase and mobile phase. The mobile phase travels through the paper by capillary action. Based upon the ways the solvent travels on the paper there are three types of chromatography 1, Ascending Paper Chromatography 2, Descending Paper Chromatography 3. Circular Paper Chromatography‘The distribution occurs in a definite ratio which represents the characteristic distribution coefficient of the solution. ‘The Rr coefficient ration is given by _ Distance travelled by the pigments from the point of application «Distance travelled by the solvent from the original distance Different substances possess different Rr values. Rr depends upon a number of factors. + Nature of the substance + Nature of the solvent + Temperature + Presence of impurities + Quality of the filter paper If the compound is coloured, it cam be easily located on the chromatographic paper. if the substance is colourless, however, a reagent may be used to treat it, which gives it a characteristic colour. ‘The name developer is given to this reagent. Iodine is the most frequently used paper chromatography developer. Glass rod Paper clip Jar — Strip of filter paper ‘Spot of ink Strip of filter paper Line drawn by pencil Water Spot of ink (a> (b) Materials Required: 1, Whatman’s filter paper 2. Extract of leaves and extract of flowers3. Chloroform /acetene 4, Methanol/Acetone 5. Rubber cork fixed with hook in the centre 6. Glass jar 7. Rubber cork fixed with hook in the centre 8. Test tubes 9. Distilled water 10. Petroleum ether Procedure: 1. Take the whatman filter paper and draw a line with the help of a pencil above 4cm from one end. 2. Grind the leaves and flowers in a motor and transfer the paste into a test tube. 3. This crushed material add acetone or methanol, shake well and filter the mixture. 4. The filtrate is collected in a test tube for performing experiments. 5. Using a capillary put one drop of the filtrate on the filter paper and allow it to dry. 6. Now hang the filter paper in a jar containing 20ml of petrolem. ether and chloroform. 7. Keep this jar till the mobile phase rises up to 2/3th of the length of the paper. 8. Remove the filter paper from the jar mark the solvent front. 9. Outline the spots with the help of pencil and make the filter paper to dry. 20. Measure the distance between the solvent front and the ceatre of different spots in relation to the reference line as indicated 11, Determine the number of pigments in the leaves and flowers extract. 12. Using the expression, calculate the Rr value of different spots. Observations and Inference: S.No Name ofthe Colour Distance Distance Rr Values extract and of the travelled by travelled colour spot the spotfrom by the =a/xX the original solvent Hne.{A} from the original Hine (&)1 Chlorophyll Green 3cm 12cm 0.25 2 Xanthophyll Yellow 4cm 12 cm 0.33 3 Carotene Red 10 cm i2em 0.83 Precautions: 1, Always make use of a fine capillary tube. 2. Do not allow spots to spread while spotting the test solution on the paper. 3. Use the capillary finely drawn to place the spot on the paper. 4. Do not disturb the jar once the experiment is set as long as the chromatogram is developed. 5. Before developing the spots, make the paper strip perfectly dry. 6. Carefully handle organic solvents. Experiment - 21 AIM : Separate Cu” and Ca ions Present in the Given Mixture by Using Ascending Paper Chromatography and Determine their R; Values: Apparatus Gas jar, glass rod, filter paper strip (What man No. 1 filter paper), jar cover, fine capillary tube.Chemicals Requirement Sample solution containing cobalt (11) and nickel (7) ions, acetone, concentrated aqueous ammonia, Rubeanic acid spray reagent. Procedure 1. Take a What man filter paper strip (20 x 2 em) and draw 4 line with pencil above 3 cm from one end. Draw another Hine lengthwise from the centre of the paper as shown in Fig. Chromatographic paper Original line about 4 om above the edge Point to put the spot of the mixture Fig. Spotting of the mixture. 2. With the help of fine capillary tube, put a drop of the mixture of yed and blue inks at the point P. Let it dry in air. Put another drop Fea' he same spot and dry again. Repeat 2-3 times, so that the spot is rich in mixture. 3. Suspend the filter paper vertically in a gas Jer containing the srtont (eluent) with the help of glass rod in such « way that the pencil ne (and the spot) remains about 2 em cabove the solvent jewel (50% alcohol + distilled water}. 4. Cover the jar and keep it undisturbed. Notice the rising solvent along with the mixture of red and bine inks. After the solvent has Sisen about 15 cm you will notice two different spots of blue and red colours on the filter paper. 5. Take the filter paper out of the jar and mark the distanes that the solvent has risen on the paper with a pencil. This is called the solvent front. 6. Dry the paper. Put pencil marks in the centre of the blue and red spots. 17, Measure the distance of the two spots from the original line and the distance of the solvent from the original line.8. Calculate the Rewalnes of the bine and red inks by using the formula : ; We eacetrevlled by the Ble o rod ink fom the point of applceto ae =e travelled by the solvent from the original ine After elution and drying, place the paper in a large, dry, covered beaker containing a smaller beaker of concentrated aqueous ammonia. After Shout two minutes, remove the paper and spray it on both sides with ccbeanic acid reagent. Allow it to dry. Nickel becomes visible as blue we band while cobalt becomes visible as yellow orange band. Evaluate Rrvalues of the two ions. Observations and Calculations: Colour ofthe | Distance Distance Rvalues = A/K spot travelled by travelled by different solvent (%) component (A} Black 4.0 em 5.3om 0.75 component {Ca} | Yellow 45cm 5.3cm 0.85 component {Ca?"} The above experiment can be carried by using a mixture of (@} Tron (11) and cobalt (1) (i) Iron (i) and nickel (H} Precautions: 1, Always make use of a fine capillary tube. o pereue altow spots to spread while spotting the test solution on the paper. 3, Use the capillary finely drawn to place the spot om the paper, S De aot disturb the jar once the experiment is set as long es the chromatogram is developed. §. Before developing the spots, make the paper strip perfectly dry. &. Carefully handle organic solvents.