PURPOSE: To separate the pigments of spinach by Column Chromatography. To analyze Column Chromatography fractions by Thin Layer Chromatography.
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1 LAB 1: CHROMATOGRAPHY OF SPINACH Thin Layer and Column Chromatography PURPOSE: To separate the pigments of spinach by Column Chromatography. To analyze Column Chromatography fractions by Thin Layer Chromatography. SAFETY CONCERNS: Always wear safety goggles. Handle and dispose of broken glass safely. Avoid inhalation of solvent fumes. Acetone and Hexane may be harmful for pregnant women. Acetone and Hexane are flammable so do not use them near open flames. READING: Mohrig, et al; Techiniques in Organic Chemistry; W.H. Freeman and Co.; TLC; introduction, preparation & development, visualization techniques, Choosing a Developing Solvent Liquid Chromatography (introduction, preparation & development; Sources of Confusion) CHROMATOGRAPHY: Introduction: Most samples of matter are impure mixtures of two or more substances. Chromatography is a widely used experimental technique for the separation of a mixture of compounds into its individual components. The word chromatography means "separation of colors" but today chromato-graphy is used for both colored and colorless substances. The separation process is based on the fact that porous solids adsorbs different substances to different extremes depending upon their polarity. The term Adsorption refers to the adhesion or stickyness of a substance to the surface of another substance, as opposed to the term absorption which refers to a substance penetrating into the inner structure of another substance. A mixture to be separated is first applied to an immovable porous solid (like paper, or alumina, or fine silica sand) called the stationary phase. The components of the mixture then get washed along the porous solid by the flow of a solvent called the mobile phase. The mobile phase can be liquid (as in column, paper, or thin-layer chromatography) or it can be a gas (as in gas chromatography). Each component of a mixture to be separated will be attracted differently to the porous stationary phase depending on its polarity and the polarity of the stationary phase chosen. Remember that Like attracts Like. If the stationary phase is polar then polar components will be attracted or stick more to it but non-polar components will move across the surface easily. If the stationary phase is nonpolar then nonpolar components will be more attracted to it and the polar compounds will move along more quickly. Likewise, if the mobile phase or solvent that is washing over the components of a mixture is polar then it will attract polar components of the mixture and carry them along easily, leaving the nonpolar components behind or moving slow. A non-polar solvent will attract and carry along the non-polar components of a mixture but leave the polar substances behind or moving slow. As the mobile phase (solvent) moves through the porous stationary phase by capillary action, it "pulls" along the molecules of the mixture to be separated at different rates. Because of the different polarities of the molecules the components have different attractions to the mobile and to the stationary phases, and therefore do not travel at the same speed through the stationary phase. This leads to a separation of the various molecules. The simplest types of chromatography, paper and thin-layer, will be used in this experiment. Other chromatographic methods, including column chromatography, gas chromatography (GC), and high performance liquid chromatography (HPLC) are used extensively in chemistry and related fields such as medicine. In medicine, chromatography is used to separate and identify amino acids and proteins in mixtures. Chromatograms of blood samples will sometimes reveal the presence of foreign proteins associated with certain diseases. Law enforcement agencies sometimes require chromatographic analysis of urine specimens from suspected drug addicts. CH242 Lab 1: Chromatography of Spinach (W15) 1
2 Thin-Layer Chromatography: In Thin-layer chromatography the stationary phase is a thin coating of adsorbent material, called the sorbent, on a sheet of glass, plastic, or metal. The TLC sheet is suspended vertically in an eluent and the eluent travels up the sheet. Common sorbents include silica (SiO 2, very pure, finely ground sand), alumina (Al 2 O 3, also used in abrasives, ceramic materials, and dental cement), cellulose (similar to very pure, finely ground wood fibers). In order to separate the substances of a mixture, the substances must have different R f values. By carefully choosing an eluent and a sorbent, it is usually possible to find a combination that will separate the mixture. A tiny drop of the mixture to be separated is placed on the plate near the bottom of the paper. A lightly drawn pencil line marks the location of the spot. This location is called the origin. The paper is suspended vertically in the mobile phase, a solvent or eluent. The eluent could be water or alcohol, or a solvent solution made form several reagents whose proportions are chosen to enhance their ability to "pull" along some substances in the mixture being separated better than others. We want each chemical in our mixture to have different attractions to the solvent so that they will travel at different speeds and be separated. The origin must be above the surface of the eluent. The eluent rises up the paper by capillary action. When the eluent reaches the origin, the components of the mixture rise at different rates. The container must be covered to prevent evaporation of eluent. The chromatogram must be removed from the eluent before the eluent reaches the top of the paper. As the substances in the mixture rise up the paper, they spread out and the spots become larger. For this reason, the original spot should be as small as possible, less than 5 mm in diameter. If too much material is applied to the small spot, the spot may develop a long "tail." If too little material is applied to the spot, the color of the spot may be too faint to see as the spot enlarges while moving up the paper. Trial-and-error and experience help the experimenter obtain both a small spot and one with the proper amount of material. Substances can be identified by the heights they reach on the completed chromatogram by calculating R f (rate of flow or retention factor) values. The R f value is a constant for a given substance under the same experimental conditions. The R f value may be calculated from the following equation. The R f value itself is unitless. R f = Distance of the center of the sample spot from the origin Distance of the solvent front from the origin Figure 6.1 shows the finished chromatogram of substance A, substance B, and a mixture containing substances A and B. To determine the distance traveled by each component measure the distance from the origin to the center of the migrated spot. If the spot is large with a "tail," measure to the "center of gravity" or densest concentration of the spot. R f (substance A) = 3.1 cm = 0.28 R f (substance B) = 8.5 cm = cm 11.2 cm Solvent front Origin (With original sample spots) A B A&B & A B A&B & 2 Figure 6.1 Typical finished chromatogram of two substances. CH242 Lab 1: Chromatography of Spinach (W15)
3 Once the R f value is known, the substance can sometimes be identified by comparing its R f value with those reported in the literature. To check the identity of an unknown substance, it is usually necessary to run a chromatogram of a known sample simultaneously with the unknown. Paper Chromatography: Paper chromatography is almost identical to thin layer chromatography however the stationary phase is a sheet of absorbent paper, such as filter paper. TLC offers advantages over paper chromatography in that TLC can be performed with a variety of sorbents and gives better separation of mixtures with less spreading of the spots Liquid Column Chromatography: Column chromatography is similar to thin layer chromatography however the stationary phase is a column of adsorbent rather than a layer stuck to a backing. The advantage of column chromatography over paper or TLC is that it can be performed on a larger scale for the purpose of purifying samples. Vegetable Pigments: Deeply colored vegetables such as spinach contain a mixture of pigments including Carotenes (1 spot) (yellow-orange) Pheophytin a (gray, may be nearly as intense as chlorophyll b) Pheophytin b (gray, may not be visible) Chlorophyll-a (blue-green, more intense than chlorophyll b) Chlorophyll-b (green) Xanthophylls (possibly 3 yellow spots) When exposed to air the chlorophyll pigments are slowly oxidized to form brown-colored products. The pigments are nonpolar and do not dissolve in water, a highly polar solvent; that's why grass stains are so difficult to launder from clothing. The pigments do dissolve in acetone, a common solvent found in fingernail polish remover. IN THIS EXPERIMENT: In this experiment we will isolate the pigments of spinach by column chromatography and then analyze the chromatography fractions by Thin Layer Chromatography. The eluents for the chromatography of these pigments will be variations of hexane, acetone, and methanol with increasing polarity. (Traditionally a solvent called Ligroin was used instead of hexane. Ligroin is a non-polar solvent similar to gasoline, mineral spirits, or painter's naphtha--it is a mixture of hydrocarbons with a boiling point range of C sometimes called petroleum ether. A 2:1 ligroin-acetone eluent mixture has polarity that gives a good separation of the spinach pigments and could be used as an alternative.) The eluent mixture must be free of water--one drop of water would considerably change the polarity of the mixture. Resources: Pavia; Introduction to Organic Laboratory Techniques CH242 Lab 1: Chromatography of Spinach (W15) 3
4 PROCEDURES: ACTIONS: I. ISOLATION OF SPINACH PIGMENTS: A. Extraction of pigments 1. Use a mortar and pestle to grind about 0.5 grams of cut or torn fresh 1 (or 0.25g frozen) 2 spinach leaves (avoid stems or thick veins) with 1 ml acetone 3 until the leaves have broken into particles too small to be clearly seen With a Pasteur pipet, transfer the mixture to a centrifuge tube. 3. Rinse the mortar and pestle with 1.0 ml cold acetone and add it to the remaining mixture in the centrifuge tube. 4. Centrifuge the mixture (be sure to balance the centrifuge) to allow the sediment to fall to the bottom. Using a Pasteur pipet, transfer the liquid extract to another centrifuge tube with a tight fitting cap. 5. Add 2.0 ml of hexane to the extract. Cap and shake to mix. 6. Add 2.0 ml of water and shake thoroughly with occasional venting. 7. Centrifuge the mixture to break the emulsion. 5 Remove the bottom aqueous layer with a Pasteur pipet and discard. 8. Dry the remaining hexane layer (which contains the dissolved pigments) by filtering it through a column 6 containing anhydrous sodium sulfate 7 into a dry test tube labeled E for extract. 9. Flush the drying column with an additional 0.5 ml hexane to extract all the pigments from the drying agent. 10. Evaporate the solvent by placing the test tube (E) in a warm water bath (40-60 o C) in a hood. Redissove the residue in 0.5 ml hexane. 11. Stopper test tube (E) and set aside in the dark until you are ready to run the alumina chromatography column. NOTES: 1 The crushing helps break cell walls and free the pigments from the cells. 2 Fresh leaves are preferred but if frozen spinach must be used then thoroughly dry thawed leaves by pressing between paper towels. 3 CAUTION: The solvents, Acetone and Hexane, used in this experiment are flammable. No flames should be present! 4 If too much acetone has evaporated add an additional mls. 5 The emulsion usually appears as a cloudy green layer in the middle of the mixture. 6 Into a 5 ¾ inch Pasteur pipet put a plug of cotton and tamp it into position using a glass rod. Add about 0.5 g of powdered or granular anhydrous sodium sulfate, and tap the column with your finger to pack the material. Clamp the column vertical. Column for drying extract. 0.5g Anhydrous sodium sulfate Cotton 7 Water will attract to the anhydrous sodium sulfate and be pulled away from the organic solvent. 4 CH242 Lab 1: Chromatography of Spinach (W15)
5 B. Column Chromatography 1. In preparation have ready: 4 tubes of chromatography solvents of increasing polarity: A ml hexane B. 6.0 ml 70% hexane 30% acetone solution (by volume) C. 6.0 ml acetone D. 6.0 ml 80% acetone-20% methanol solution (by volume) 5 dry test tubes for sample collection numbered Prepare a chromatography column 8 packed with about 1.25 g of alumina. 9 Read and understand the following procedures before continuing. The procedure takes about 15 minutes. If using a column without a stopcock then once started it cannot be stopped until all the material is eluted from the column. 3. Using a Pasteur pipet, slowly 10 add about 3.0 ml of hexane to the column and let drain to completely moisten the alumina. Collect any eluent in test tube #1. Drain the excess hexane until the level of hexane just reaches the top of the alumina. 11 If necessary, add more hexane. 4. When the hexane has drained to the top of the column add about half (0.25 ml) of the dissolved pigments from tube E Continue collecting the eluent in tube 1. Just as the pigment solution penetrates the top of the column, add 1 ml of hexane and drain until the surface of the liquid has reached the alumina. 6. Add about 4 ml of hexane. If the yellow band (carotenoid pigments) begins to separate from the green band (chlorophyll pigments), continue to add hexane until the yellow band passes through the column. If the yellow band does not separate from the green band, change to the next more polar solvent (70% hexane-30% acetone) 13 Collect the yellow band in test tube #2. 7. When the yellow band is through and the eluent becomes colorless again start collecting in tube #3. 8. Add several mls of the next more polar solvent. 13 If the green band moves down the column continue to add this solvent to elute the green band. 14 Collect the green band in tube #4. 9. When there is little or no green color in the eluent, place test tube #5 under the column and stop the procedure. 10. Place the carotenoids (tube #2), the chlorophylls (tube #4), and the remaining extract (tube E) in a warm water bath (40-60 o C) just until 15 the solvent has evaporated. Increasing polarity of solvents 8 If preparing a column from a Pasteur pipet push a loose plug of cotton into the pipet with a glass rod. Add the measured amount of alumina while tapping the column gently with your finger to reduce air pockets. Continue tapping to ensure that the alumina is tightly packed. 9 Use activated alumina from EM science, No. AX0612-1); Particle sizes mesh; material Type F-20. Dry overnight at 110 o C and store in a tightly sealed bottle. 10 Add the solvent slowly to avoid disrupting the level surface of the alumina. 11 The top of the column must not be allowed to run dry. It is essential that the liquid level not be allowed to drain below the surface of the alumina at any point during the procedure. 12 Stopper and save the remainder of the pigment in a dark place for use with TLC later. 13 When changing solvents, do not add the new solvent until the last solvent has nearly penetrated the alumina. 14 If the green band does not move or if a diffuse yellow band begins to move, change to the next more polar solvent. Change solvents again if necessary. 15 Do not allow any of the tubes to continue heating after the solvent has evaporated. CH242 Lab 1: Chromatography of Spinach (W15) 5
6 C. Thin Layer Chromatography 11. Obtain a 10 cm x 3.3 cm silica gel 16 TLC sheet. 12. With a pencil 17, draw a faint line (the origin line) lightly & carefully 18 across the bottom of the silica gel sheet about cm from the bottom edge. 13. Carefully 18 keeping at least 0.6 cm from the edges make 3 small vertical pencil marks on the origin line as shown 19 to indicate the place where the spinach pigments will be applied. 12. Redissolve the pigments in tubes 2, 4, & E by adding two drops of 70% hexane-30% acetone to each of the tubes. 13. Dip a clean capillary tube into each sample tube. Apply each pigment to the corresponding mark at the origin of the silica gel TLC sheet by quickly touching the capillary tube to the sheet. Hold the capillary tube at right angles to the sheet. Do not scrape off the adsorbent with the capillary tube. Allow each spot to dry 20 for 30 seconds and repeat the application about 15 times or until dark Prepare a dry 22 chromatography chamber with 70% hexane-30% acetone eluent mixture. 15. Lower the TLC sheet into the test tube making sure that the origin on the TLC sheet stays above the surface of the hexane-acetone eluent mixture and keeping the plate from coming in contact with the filter paper liner. 16. Cover the TLC chamber and allow it to sit undisturbed until the eluent front is 1-2 cm from the top 23 of the TLC sheet. 17. Use forceps to remove the TLC plate and with pencil immediately mark the position of the solvent front As soon as the plate is dry lightly outline all visible spots with a pencil and make note of the colors Draw a diagram of the chromatogram but tape 26 the original in your notebook as well. 20. Label all spots as A, B, C, D, etc., calculate their R f values, and identify the pigments by their colors Disposal: Place the excess Hexane-acetone in the "Waste Organic Solvent" container. 16 Whatman Silica Gel Plates No Handle carefully by the edges to avoid flaking. Do not touch the surface. 17 Do not use a pen as the ink may run in the chromatography solvent. Pencil lead is graphite, a form of carbon and will not dissolve or run in the organic solvents used. 18 Make your pencil marks very carefully so as not to flake off the silica adsorbent. 19 Indicate origin spots for tubes 2, 4, & E. 20 A drying period is necessary to ensure small spots. 2 4 E & & 21 The repeated application is necessary to ensure sufficient material is applied. 22 The hexane-acetone mixture must stay dry--we don't want water added to the mixture. 23 Do not allow the eluent to reach the top of the sheet. 24 You must draw the line quickly before the eluent disappears. Hexane and acetone solvent evaporates very rapidly and soon you will no longer be able to see the position of the eluent front. 25 Outline and observe the spots soon after the plates have dried as some pigments may change color when exposed to air & light. 26 Attach the chromatogram to paper by completely covering it with transparent tape to prevent the silica from flaking off. 27 In the crude extract the following components should be visible (in order of increasing Rf values). Carotenes (1 spot) (yellow-orange) Pheophytin a (gray, may be nearly as intense as chlorophyll b) Pheophytin b (gray, may not be visible) Chlorophyll-a (blue-green, more intense than chlorophyll b) Chlorophyll-b (green) Xanthophylls (possibly 3 yellow spots) 6 CH242 Lab 1: Chromatography of Spinach (W15)
7 LAB 1: CHROMATOGRAPHY PRE LAB EXERCISES: NAME DATE 1. Rank the following solvents in order of increasing polarity (lease polar to most polar). Ethanol 50% Ethyl acetate/ 50% hexane 75% Ethyl acetate/ 25% hexane 70% acetone/ 30% hexane Ethyl acetate acetone hexane 30% acetone/ 70% hexane water Dichloromethane 2. Match the following terms with the phrase that best describes it: 1. Sorbent D. line marking the placement of a mixture on a chromatogram. 2. Eluent E. solvent used as a mobile phase 3. chlorophyll-b F. the final edge of the mobile phase after development of a chromatogram. 4. Eluent front G. adhesion of a substance to the surface of the stationary phase 5. Origin H. penetration of one substance into the inner structure of another. 6. Adsorption I. thin coating of porous material used as a stationary phase. 7. Absorption J. yellow-green 3. What safety precautions are necessary when using acetone and hexane (or ligroin)? A. Avoid flames as these solvents are flammable. B. Avoid inhaling the solvents as they are hazardous. C. Both A and B. 4. Why is a pencil used to mark the origin line and not a ball-point or ink pen? A. Pencils are more dependable since they never run out of ink. B. Ink could dissolve in the eluent and rise up chromatogram. C. Pencils are less likely to flake off the sorbent. D. Ink is harder to erase if you make a mistake in the labeling. E. More than one of these. 5. Point out two errors that would prevent an accurate R f determination in the TLC setup illustrated below: 6. Calculate the R f value for substances A and B on the chromatogram shown at the right. Show your calculations: A B & A&B 7. About 0.5 g of a sample mixture is to be separated on a silica chromatography column. How much silica should be used to pack the column? CH242 Lab 1: Chromatography of Spinach (W15) 7
8 8. Once the adsorbent is packed in the column, it is important that the level of the elution solvent not drop below the top of the adsorbent. Why? 9. What precautions must be taken when you introduce the mixture of compounds to be separated onto the adsorbent column? Why? 10. What effect will the following factors have on a chromatographic separation: a. Too strong an adsorbent b. Collection of large elution fractions c. Solvent level below the top of column adsorbent 8 CH242 Lab 1: Chromatography of Spinach (W15)
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