CHROMATOGRAPHY A PROJECT IN CEHMISTRY. Chromatography includes various experimental techniques Designed to separate mixture of

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1 CHROMATOGRAPHY A PROJECT IN CEHMISTRY Chromatography includes various experimental techniques Designed to separate mixture of compounds. Chromatography involves a stationary and a mobile phase. Separation takes a place because of parting between the sample compound in the moving and stating phase the general classifications of chromatography methods is based on the nature of these phases. Table : 1-Chromatography Methods Mobile phase stationary phase Method Gas Solid/Liquid gc Gas Solid gsc Liquid Solid Liquid, Solid Chrom. Gas Liquid glc Liquid Solid TLC Liquid Paper Paper

2 In all the chromatography techniques the solutes to be separated migrate along a column (or as in paper or thin layer chromatography, the physical equivalent of a column), and of course the basis of separation lies in different rates of migration for the different solutes. We may think of the rate of migration of a solute as the result of two factor one tending to move the solute and other to retard it the tendency of solutes to absorb on the solid phase retards their movement while their solubility in the moving phase tends to move them along. A slight difference between two solutes in the firmness of their absorption and in their interaction with the moving solvent become the basis of separation when the solute molecules repeatedly distribute between the two phase over and again throughout the length of the column. Perhaps 80% of all chemical compounds are better handled in solution than in gas phase; thus, liquid chromatography is potentially more important than gas chromatography. We shall first examine the phase distribution process that are involved in the common form of liquid choromatography,

3 Absorption Imagine a solid material with a clean and dry surface. Now if this surface is exposed to a fluid gas pure liquid, or solution there is generally a tendency for molecules of gas solvent or solute to interact with the surface the phenomenon of higher concentration of any molecular species at the surface than in the bulk of a solid (or a liquid) is known as adsorption. If the solid material is very finely divided or is highly area, than the extent of adsorption may be appreciable. The various molecular force i.e.., van derwaals forces (dipole dipole, dipole induced dipole and Landon forces are responsible for adsorption. If a solution containing a solute is placed in contact with a solid adsorbant then that solute will distribute itself between two phase i.e., the surfacer and the solvent. The position of their equilibrium is determined by the affinity of the surface for the solvent and the affinity of the solvent for the solute. Some typical adsorbents used as packing material in the column are carbon black. Alumina, AI 2 O 3, silica gel, calcium carbonate calcium sulphate talc sugar and powdered cellulose. The elution power of a solvent is a function of its polarity end of most adsorbents such a aluminum oxide and magnesium silicate of its hydrogen bonding ability. Few common solvents used for elution in the increasing order of their polarity are : petroleum either carbon tetrachloride benzene chloroform ethyl acetate, 1 butanol,ethonal, Water.

4 EXPERIMENT 1 (A) AIM To separate the different compounds present in grass (or spinach) extract by paper chromatography. Theory It was considered at first that paper chromatography was simply a from of liquid liquid partion. The hydrophile cellulose fibres of the paper can bind water. (A filter paper that appears dry may actually contain a large percentage of water, say 20% or more by weight). Solutes then were portioned between this water and the moving immiscible organic solvent. It was soon realize however that this model was too simple. Separations were obtained where the moving phase was miscible with water or in some casewhere it was itself an agueous solution. Thus although liquid liquid partion may indeed play a role liquid-liquid partion may indeed play a role in some cases the mechanism of paper chromatography is often more complicated than that. Interaction between solutes and the cellulose support are probably involved for example adsorption and hydrogen bonding carboxyl and other ionizable groups are introduced into cellulose during the pulping and bleaching operations of paper manufacture and hence the paper may also act as an ion exchanger.

5 In ascending paper choromatography the paper is suspended from thetop of the chamber so that it dips into the solvent at the bottom and the solvent creeps upt he paper by the capillary action. In the descending from the paper in anchored in a solvent through migrates downwards by capillary assisted by gravity. Material White filter paper (whatman s filter paper) pestle and mortar, test tubes, beaker (100 ml) or crystilling dish for support for filter paper chromatography tank or jar test piprette (drawn out to a fine point), scissors supply of grass or spinach, acetone or ethonal or toluene.

6 Procedure 1. Cut a handful of grass (or spinach) into small places with scissors. Gring the piece with enough acetone or Ethan Sal to produce 2 to 3 ml. of tree liquid in mortar and then decant the green liquid into test-tube. The extract should be as concentrated as possible. 2. Take a circular filter of dismeter of roughly 10 cm. and place it on the top of a beaker or crytallising dish or other support. 3. Using a tear pipette or a capillary tube, place a drop of the extract I the centre of the paper. When it has stopped spreading and another drop and repeat until three or four drop have been added. 4. Allow for a minute or so for the last drop to finish spreading and then add more drops one at a time actone or ethanol the teat pipette. 5. Well separated green and yellow bands will develop as the coloured substances travel to wards the edge of the paper.

7 A better sepraton of the mixture can be obtained in the following manner but it will take rather longer to Obtained in the following manner but it will take rather longer to obtain the result. 1. Cut a strip of filter paper choosing its length and breadth according to the height and diameter of the chromatograph tank and jar. Strip should be long enough to fit easily almost to the bottom of the container and to fold about 1 cm. over their lip. The strip should ideally be 2.3 cm. wide. 2. Using a test pipette or capillary place a drop of the extract about 4 cm. From one end of threr filter paper. Put at least 3-4 drops of the extract. 3. Add enough acetone or ethonal to the container to cover the bottom of the filter paper but not enough to reach the spot. 4. Place the filter paper in there container with the spot end facing down. Be sure that solvent does not touch the spot. 5. Observe for few minutes. When the solvent has almost reached the top of the paper. Remove the paper and put a pencil mark where the solvent stopped. 6. Let the paper dry and observe the chromatogram from coloured spots.

8 7. The topmost orange band is xanthophylls and the green band is chlorophyil. There are two types of chlorophyil : chlorophyil a and chlorohhyil b : carotene is also present. 8. Repeat the experiment by changing solvent to a less polar one. e.g. toluene and observe the difference. A spot for carotene may also be visible if toluene is used as the eluent (developing solvent) 9. Spot are often characterized by their R f Retension factor) value. An R value is the ratio of the distance moved by solute to the distance that the solvent front moved during the same time. 10. Tabulate the values for different spots i.e., for different plant pigments.

9 EXPERIMENT 1 (B) AIM Separation of grass extract using blackboard chalk. Materials Grass extract ( for preparation, see previous experiment). Small dish stick of blackboard chalk (plain white not the dustless or yellow coloured varieties). Procedure Place a little grass extract in a small dish and make stick of black-board chalk stand on the end in the extract. The liquid will be draw up the chalk piece and various coloured bands of the various components will be formed on the chalk piece, The various components of the extract are absorbed on the chalk piece with different strength. Thus they chalk piece with different strengths. Thus they separate as the solvent moves up the chalk column.

10 EXPERIMENT 1 (C) AIM Theory Separation of plant pigments by thin layer chromatography (TCL). Thin layer chromatography (TLC) can be used to separate small quantities with simple apparatus. TLC is usually an analytical adsorptive technique, although some times it involves partition. The adsorbant, usually silicii acid or silica gel or some times alumina or other material, is spread in a thin layer on a glass plate or other insert support. The adsorbant is usually activated by heat after application. The material to be separated is applied in small spots on the adsorbant. The plate is placed in a chamber and the material eluted with a solvent mixture. TLC is often used to determine the optimum adsorbant and elute for column chromatography. MATERIALS Glass strip (25 cm * 3 cm), wheat starch, silicic acid, beaker (100 ml.), stirring rods, oven, chromatography solvent (acetone/ethanol and / or toluene), grass or spinach extract, chromatography chamber or jardistilled water.

11 PROCEDURE Preparation of thin layer strips ; 1. Heat 80 ml. of water and 1 g. of wheat starch on a water bath at 65 to 70 c; stir the mixture until the wheat starch dissolves. 2. Allow the mixture to cool to room temperature and slowly add, with vigorous stirring 40 g of silicic acid. When approximately half the silicic acid has been blended with the mixture, add another 25 ml. of water and continue to stir in the rest of the silicic acid. 3. Pour the mixture on to the glass strips. Use a stirring rod to spread the slurry while gently tapping the slides. The glass sstrips with the thin layer of adsorbants should be handled very carefully so that the adsorbant layer is not disturbed. 4. Allow the glass strip to stand for 25 to 30 min. Place them in an oven at 110 c for 2 hours. Just look for the formation of bubbles and other defects on thin layer.

12 CHROMATOGRAPHY OF PLANT PIGMANTS 1. As soon as plates are removed from the oven, these should be spotted. Spot the solution of plant pigments 2 cm. From the end of the glass plate. Add 4-5 drops and allow the solvent from each drop to evaporate before adding the next. 2. Take the developer as pure solvent or mixture of solvents in a convenient developing chamber. (A mixture of isooctane and ethyl either in 10 : 2 : 2 : ratio is the best choice) 3. Place the slide so as to stand it nearly in the chamber. The solvent should under no condition, touch the spots. 4. In about an hour the solvent will have climbed to the top of the strip, separating the different the different coloured components as it does so. 5. Note the colours of each of the spots and their distance from the origin. Tabulate the R values. SEPARATION OF OTHER PLANT PIGMENTS (OPTIONAL) Repeat the above procedure on crushed tomato pulp, carrot scrapping or geranium flower petals.

13 EXPERIMENT 1 (D) AIM THEORY Separation of plant pigment, using column liquid chromatography. Once the optimum adsorbant and eluant has been determined, using thin layer chromatography, large quantities are conveniently separated by coloumn chromatography. In this form of chromatography, a long glass coloumn is loaded with adsorbant like alumina. The mixture is pipetted on to the top of te adsorbant bed. The liquid reservoir is positioned and the folw of the mobile phase is started. The effluent solution from the reservoir. Different components in the mixture move with different speeds and get separated. MATERIALS Glass coloumn (2 cm. in diameter and 32 cm, to 45 cm. in length, or a burette), glass wool, sand, petroleum either, alumina, grass extract, iso-octane ethyl, funnel.

14 PROCEDURE Preparation of a chromatography coloumn : 1. In a coloum (or burrete) place a small amount of glass wool and close the bottom (stop-clock) of the coloumn. 2. Fill the coloumn with petroleum either. 3. Add about 1.2 cm of clear sand and align the coloumn parallel to the earth s gravitational pull. Smoothen the surface of the sand by gently tapping the coloumn with a piece of tube. 4. Place a stemless funnel at the top of the column. 5. Slowly add about 110 g. of alumina to the coloumn through the funnel. While the alumina is going into the coloumn, tap the coloumn gently with rubber tubing. Simultaneously, allow the pettoleum either to flow slowly out of the column. However, maintain the level of either well above the tip of the settling alumina by adding more petroleum either if necessary. 6. When all the alumina has been added, fill the coloumn to within 3 cm. of the top with petroleum either and close the stop-clock.

15 7. Slowly and few grams of sand so that the top of the aluminais not disturbed. 8. Seal the top of the column with a cork stopper until you actually want to do the separation experiment. This ends there preparation of chromatography column. CHROMATOGRAPHY OF THE PLANT EXTRACT 1. Take the extract solution and pour on the top of the column. 2. In the meantime, be ready with eluant i.e., a mixture of iso-octane, acetone and ethyl either in the ratio 10:2:2, respectively. Better take the solvent in a separting funnel. 3. Place the separating funnel over the coloumn and open the stop-clock of the separating funnel. Adjust the flow of the solvent so that an almost continuous stream of drops flows rapidly from the bottom of the column. 4. Elute with the solvent till there is a clear separation of coloured bands on the coloumn bed. 5. Different separate coloured bands can be collected separately in small tubes.

16 6. Alternatively, a coloured photographs of the separation band on the coloumn can be taken can be fixed on the note book. 7. Compare the result obtained here that in thin-layer chromatography. 8. Similarly, other plant pigments can be separated by repeating the experiment with tomato soup, carrot scrapings or geranium flowers..pa

17 AIM To separate the different components of chlorophyll present in grass (or spinach) extract by paper chromatography and to find their R f values. Theory It was considered at first that paper chromatography was simply a form of liquid-liquid partion. The hydrophile cellulose fibres of the paper can bind water. (A filter paper that appears dry may actually contain a large percentage of water, say 20% or more by weight). Solutes then were portioned between this water and the moving immiscibvle organic solvent. It was soon realize however that this model was too simple. Separations were obtained were the moving phase was miscible with water or in some case where it was itself an agueoussolution. Thus although liquid-liquid parion may indeed play a role liquid-liquid partion may indeed play a role in some cases the mechanism of paper chromatography is often more complicated than that. Interaction between solutes and the cellulose support are probably involved for example adsorption and hydrogen bonding carboxyl and other ionizable groups are introduced into cellulose during the pulping and bleaching operations of paper manufacture and hence the paper may also act as an ion-exchanger.

LAB #6 Chromatography Techniques

LAB #6 Chromatography Techniques Objectives: To learn how to story board a procedure Explain how a chromatograph of pigments is formed from both paper and thin layer chromatography. Isolate and identify