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 2: CHROMATOGRAPHY PART 2: 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.; Liquid Chromatography (introduction, preparation & development; Sources of Confusion LIQUID COLUMN CHROMATOGRAPHY: Introduction: 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.) Resources: Pavia; Introduction to Organic Laboratory Techniques CH242 Lab 2: Chromatography 2 (W14) 1

2 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. 2 CH242 Lab 2: Chromatography 2 (W14)

3 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 2: Chromatography 2 (W14) 3

4 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) 4 CH242 Lab 2: Chromatography 2 (W14)

5 LAB 2: CHROMATOGRAPHY 2 QUESTIONS: NAME DATE 1. About 0.5 g of a sample mixture is to be separated on an alumina chromatography column. How much alumina should be used to pack the column? 2. 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? 3. What precautions must be taken when you introduce the mixture of compounds to be separated onto the adsorbent column? Why? 4. 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 CH242 Lab 2: Chromatography 2 (W14) 5