WILLIAMSO5 AAecem, aliraatem EXPERIMENTSOrganic Experiments Seventh Edition Louis F. Fieser Late Professor Emeritus Harvard University Kenneth L. Williamson Mount Holyoke College D. C. HEATH AND COMPANY Lexington, Massachusetts TorontoSS CHAPTER 9 Thin-Layer Chromatography: Analysis of Analgesics and Isolation of Lycopene from Tomato Paste Prelab Exercise: Compare thin-layer chromatography (TLC) with column chroma- tography with regard to (1) quantity of material that can be separated, (2) the speed, (3) the solvent systems, and (4) the ability to separate compounds. ‘Thin-layer chromatography (TLC) is a sensitive, fast, simple, and inexpen- sive analytical technique that will be used repeatedly in carrying out organic experiments. It is a micro technique; as little as 10°° g of material can be detected, although the usual sample size is from 1 to 100 x 10~¢ g. TLC requires micrograms TLC involves spotting the sample to be analyzed near one end of a of material sheet of glass or plastic thatis coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end ina covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent and the stationary adsorbent phase. The more strongly a given component of the mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the TLC plate. Uses of Thin-layer Chromatography 1. To determine the number of components in a mixture. TLC affords a quick and easy method for analyzing such things as a crude reaction mixture, an extract from some plant substance, ora pa r. Know- ing the number and relative amounts of the components aids in planning further analytical and separation steps. 2. To determine the identity of two substances. If two substances spotted on the same TLC plate give spots in identical locations, they may be identical. If the spot positions are not the same the substances cannot be the same. It is possible for two closely related compounds that are not identical to have the same positions on a TLC plate. 3. To monitor the progress of a reaction. By sampling a reaction from time. totime itis possible to watch the reactants disappear and the products appear using TLC. Thus, the optimum time to halt the reaction can be determined, and the effect of changing such variables as temperature, concentrations, and solvents can be followed without the necessity of isolating the product. 4. To determine the effectiveness of a purification. The effectiveness of NZ118 Avoid the use of benzene, carbon tetrachloride, and chloroform. Benzene is a carcinogen, the others are suspect carcinogens. Organic Experiments distillation, crystallization, extraction, and other separation and purification methods can be monitored using TLC, with the caveat that a single spot does not guarantee a single substance. 5. To determine the appropriate conditions for a column chromatographic separation, Thin-layer chromatography is generally unsatisfactory for purifying and isolating macroscopic quantities of material; however, the adsorbents most commonly used for TLC—silica gel and alu- mina—are used for column chromatography, discussed in the next chapter. Column chromatography is used to separate and purify up to about a gram of a solid mixture. The correct adsorbent and solvent used to carry out the chromatography can be determined rapidly by TLC. 6. To monitor column chromatography. As column chromatography is cartied out the solvent is collected in a number of small flasks. Unless the desired compound is colored the various fractions must be ana- lyzed in some way to determine which ones have the desired compo- nents of the mixture. TLCis a fast and effective method for doing this. Adsorbents and Solvents The two most common coatings for thin-layer chromatography plates are alumina, Al,O,, and silica gel, SiO,. These are the same adsorbents most commonly used in column chromatography (Chapter 10) for the purification of macroscopic quantities of material. Of the two, alumina, when anhy- drous, is the more active, i.c., it will adsorb substances more strongly. Itis thus the adsorbent of choice when the separation involves relatively nonpolar substrates such as hydrocarbons, alkyl halides, ethers, alde- hydes, and ketones. To separate the more polar substrates such as alcohols, carboxylic acids, and amines, the less active adsorbent, silica gel. is used. In an extreme situation very polar substances on alumina do not migrate very far from the starting point (give low Rf values) and nonpolar com- pounds travel with the solvent front (give high Rf values) if chromato- graphed on silica gel. These extremes of behavior are markedly affected, however, by the solvents used to carry out the chromatography. A polar solvent will carry along with it polar substrates, and nonpolar solvents will do the same with nonpolar compounds—another example of the general- ization “like dissolves like.”” In Table | are listed common solvents used in chromatography, both thin-layer and column. Only the environmentally safe solvents are listed; the polarities of such solvents as benzene, carbon tetrachloride, or chloro- form can be matched by other less toxic solvents. In general these solvents are characterized by having low boiling points and low viscosities that allow them to migrate rapidly. They are listed in order of increasing polarity. A solvent more polar than methanol s seldom needed. Often just two solvents are used in varying proportions; the polarity of the mixture is a weighted average of the two. Ligroin~ether mixtures are often employed in this way.Chapter 9 Thin-Layer Chromatography: Analysis of Analgesics and Isolation of Lycopene 119 A very dilute solution (1%) of the test substance ix employed for TLC. TABLE 1 Chromatography Solvents Solvent Petroleum ether (pentanes) Ligroin (hexanes) Diethyl ether Dichloromethane Ethyl acetate Acetone 2-Propanol Ethanol Methanol Water Acetic acid Bp CC) 35-60, 35 7 82 B 65 100 ug. ‘The order in which solutes migrate on thin-layer chromatography is the sameas the order of solvent polarity. The largest Rf values are shown by the least polar solutes. In Table 2 the solutes are arranged in order of increasing polarity. TABLE 2 Order of Solute Migration ‘on Chromatography Solute Fastest Alkanes Alkyl halides Alkenes Dienes Aromatic hydrocarbons Aromatic halides Ethers Esters Apparatus and Procedure Solute Ketones Aldehydes: Amines Alcohols Phenols Carboxylic acids Sulfonic acids Slowest ‘A 1% solution of the substance to be examined is spotted onto the plate about | cm from the bottom end, and the plate is inserted into beaker or a 4-oz wide-mouth bottle containing 4 mL of an organic solvent. The bottle is lined with filter paper wet with solvent to saturate the atmosphere within the container. The top is put in place and the time noted. The solvent travels rapidly up in the thin layer by capillary action, and if the substance is a pure colored compound, one soon sees a spot traveling either along with the solvent front or, more usually, at some distance behind the solvent front.120 Colored compounds And colorless ones TLC tests for 1. Completeness of reaction 2. Purity of product 3. Side reactions FIG. 1 Thindayer chromatography plate, Organic Experiments One can remove the slide, quickly mark the front before the solvent evaporates, and calculate the Rf value. The Rf value is the ratio of the distance the spot travels from the point of origin to the distance the solvent travels (Fig. 1). If two colored compounds are present and an appropriate solvent is selected, two spots will appear. Fortunately the method is not limited to colored substances. Any organic compound capable of being eluted from alumina will form a spot, which soon becomes visible when the solvent is let evaporate and the plate let stand in a stoppered 4-0z bottle containing a few crystals of iodine. Iodine vapor is adsorbed by the organic compound to form a brown spot. A spot should be outlined at once with a pencil because it will soon disappear as the iodine sublimes away; brief return to the iodine chamber will tegenerate the spot. The order of elution and the elution power for solvents are the same as for column chromatography. The use of commercial TLC sheets such as Eastman silica gel with fluorescent indicator (No. 13181) is strongly recommended. These poly(ethylene terephthalate) sheets are coated with silica gel using polyac- rylic acid as a binder. A fluorescent indicator has been added to the silica gel so that when the sheet is observed under 254-nm ultraviolet light, spots that either quench or enhance fluorescence can be seen. Iodine can also be used to visualize spots. The coating on these sheets is only 100 microns thick, so very small spots must be applied. Unlike student-prepared plates, these coated sheets (cut to 1 x 3-in. size with scissors) give very consistent results. A light pencil mark 1 cm from the end will guide spotting. A supply of these little precut sheets makes it a simple matter to examine most of the reactions in this book for completeness of reaction, purity of product, and side reactions. fp} —— Salvent front ay = Q for Substance A en Substance B 9 9 o—} ‘Substance A. = ST Starting spot, <1 mm dia. the Depth of solventChapter 9 Thin-Layer Chromatography: Analysis of Analgesics and Isolation of Lycopene 121 FIG, 2 Micropipette. FIG. 3. Preparation of TLC plates by dipping, The two microscope slides are grasped at ‘one end, dipped into the silica gel siury, removed and separated, and dried and activated by heating. Spotting Test Solutions This is done with micropipettes made by drawing open-end mp capillaries in a microburner flame (Fig. 2). The bore should be of such a size that, when the pipette is dipped deep into ligroin, the liquid flows in to form a tiny thread which, when the pipette is withdrawn, does not flow out to form a drop. To spot a test solution, let a 2-3 cm column of solution flow into the pipette, hold this vertically over a coated plate, aim it at a point on the right side of the plate and about | cm from the bottom, and lower the pipette until the tip just touches the adsorbent and liquid flows onto the plate; withdraw when the spot is about | mm in diameter. Make a second 1-mm spot on the left side of the plate, letit dry, and make two more applications of the same size(1-mm) at the same place. Determine whether the large or the small spot gives the better results. Making TLC Plates The adsorbent recommended for making TLC plates is a preparation of finely divided silica gel with gypsum binder and fluorescent indicator (Aldrich 28855-1) or alumina containing plaster of paris as binder (Fluka’). A slurry of this material in water can be applied to microscope slides by simple techniques of dipping (A) or coating (B); for a small class, or for occasional preparation of a few slides by an individual, the more economi- cal coating method is recommended. (A) By Dipping. Place 15 g ofsilica gel or alumina formulated for TLC in a40 x 80-mm weighing bottle* and stir with a glass rod or with a magnetic stirrer while gradually pouring in 75 mL of distilled water, Stir until lumps are eliminated and a completely homogencous slurry results. Grasp a pair of clean slides’ at one end with the thumb and forefinger; dip them in the slurry of adsorbent (Fig. 3); withdraw with an even, unhurried stroke; and touch a corner of the slides to the mouth of the weighing bottle to allow excess fluid to return to the container. Then dry the working surface by. mounting the slides above a 70-watt hot plate, using as support two pairs of 1 X 7-cm strips of blotting paper (or a pair of applicator sticks), Drying takes about a minute and a half and is evident by inspection. Remove the plate with a forceps as soon as it is dry, for cooling takes longer. If you dip 1. Aluminumoxid Fluka for Dannschicht Chromatographie, Typ DS, Fluka AG, Buchs, 8. G. ‘Switzerland: Fluka Alumina is distributed by Tridorn Chemical, Inc., 255 Oser Av., Haup- pauge. N.Y. 11787 2. Kimble Exax, 15146, 3. The slides should be washed with water and a detergent, rinsed with tap water and then with distilled water, and placed on a clean towel to dry. Dry slides should be touched only on the sides, for a touch on the working surface may spoil a chromatogram.122 Jodine chamber Don't look into lantp Organic Experiments and dry 8-10 slides in succession, finished plates will be ready for use when. you are through. Alternatively, dry the slides in an oven at 110°C. This heating activates the alumina. Coated slides dried at room temperature do not yield spots as intense as those that have been heat-dried. Keep the storage bottle or adsorbent closed when not in use. Note that is is not a stable emulsion, but that the solid settles rapidly on standing. Stir thoroughly with a rod before each reuse, Plates ready for use, as wellas clean dry slides, are conveniently stored in a microscope slide box (25 slides). {B) By Coating. Place 2 g of silica gel or alumina and 10 mL of distilled water ina 25-mL Erlenmeyer flask, stopper the flask, and shake to produce anevenslurry. Keep the flask stoppered when not inuse. Place aclean, dry slide ona block of wood ora box, with the slide projecting about 1 cm onthe left-hand side (if you are right-handed) so that it can be grasped easily on the two sides. Swirl the flask to mix the slurry and draw a portion into a medicine dropper. Hold the dropper vertically and, starting at the right end of the slide, apply emulsion until the entire upper face is covered; make further applications to repair pin holes and eliminate bubbles. Grasp the left end of the slide with a forceps and even the emulsion layer by tilting the slide to the left to cause a flow, and then to the right; tilt again to the front and to the rear. Dry the slide on a hot plate as you did when preparing slides by procedure A. The adsorbent should be about 0.25 mm thick. Visualization of the Chromatogram If the substance being chromatographed is colored then it is possible to detect the components visually. Colorless substances can be detected by placing the dry TLC plate in a jar containing a few crystals of iodine. The iodine vapor will be preferentially adsorbed by the substances on the plate and they will appear as brown spots on a lighter-colored background. The plate is removed from the jar and the outline of the spots traced lightly in pencil because the iodine will soon evaporate. Plates that have been impregnated with a fluorescent indicator will show dark spots for the compounds under an ultraviolet light due to ‘quenching of the fluorescence by the substance on the plate. Again trace the spots lightly in pencil while the plate is under the uv light. Don't look directly into the light; it will damage the eyes. ‘A large number of specialized spray reagents have been developed that give specific colors for certain types of compounds, and there is a large amount of literature on the solvents and adsorbents (o use for the separation of given types of material.* “Thin-Layer Chromatography.” Springer Verlag, New York, 1969.Chapter 9 Thin-Layer Chromatography: Analysis of Analgesics and tsolation of Lycopene 125 Cleaning Up Solvents should be placed in the organic solvents container, and dry, used chromatographic plates can be discarded in the non- hazardous solid waste container. 2. Plant Pigments The botanist Michael Tswett discovered the technique of chromatography and applied it, as the name implies, to colored plant pigments. The leaves of plants contain, in addition to chlorophyll-a and -b, other pigments that are revealed in the fall when the leaf dies and the chlorophyll rapidly decom- poses. Among the most abundant of the other pigments are the carotenoids, which include the carotenes and their oxygenated homologs, the xantho- phylls. The bright orange B-carotene is the most important of these because it is transformed in the liver to Vitamin A, which is required for night vision. Lycopene (CioHse) MW 536.85 mp 173°C, AS 475om_ B-Carotene (Cols) mp 183°C, ez" 451 nm Chlorophyll-a126 HO’ Lycopene and B-carotene from tomato paste and strained carrots As an interesting variation, ry extraction of lycopene from commercial catsup Organic Experiments Se OEREEOOEOYYOODBYOD Lutein (a xanthophyiD) ‘¥-Dehydrolutein (a xanthophyll) Hs N=N NO CH, Butter Yellow (carcinogenic) Cows eat fresh, green grass that contains carotene, but they do not metabolize the carotene entirely, and so it ends up in their milk. Butter made from this milk is therefore yellow. In the winter the silage cows eat does not contain carotene because it readily undergoes air oxidation, and the butter made at that time is white. For some time anazo dye called Butter Yellow was added to winter butter to give it the accustomed color, but the dye was found to be a carcinogen. Now winter butter is colored with synthetic carotene, as is all margarine. Lycopene, the red pigment of the tomato, is a Cy-carotenoid made up of eight isoprene units. B-Carotene, the yellow pigment of the carrot, is an isomer of lycopene in which the double bonds at C,—C, and C}—C} are teplaced by bonds extending from C, to C,and from C; to C; to form rings. ‘The chromophore in each case is a system of eleven all-trans conjugated double bonds; the closing of the two rings renders A-carotene less highly pigmented than lycopene. Fresh tomato fruit is about 96% water, and R. Willstatter and H. R. Escher isolated from this source 20 mg of lycopene per kg of fruit, They then found a more convenient source in commercial tomato paste, from which seeds and skin have been eliminated and the water content reduced by evaporation in vacuum toa content of 26% solids, and isolated 150 mg of lycopene per kg of paste. The expected yield in the present experiment is 0.075 mg. ‘A jar of strained carrots sold as baby food serves as a convenient source of B-carotene. The German investigators isolated | g of B-carotene per kg of ‘‘dried’’ shredded carrots of unstated water content. ‘The following procedure calls for dehydration of tomato or carrot paste with ethanol and extraction with dichloromethane, an efficient solvent for lipids.Chapter 9 Thin-Layer Chromatography: Analysis of Analgesics and isolation of Lycopene 127 These hydrocarbons air oxidize with great rapidity Procedure Ina small mortar grind 2 g of green or brightly colored fall leaves (don’t use. ivy or waxy leaves) with 10 mL of ethanol, pour off the ethanol, which serves to break up and dehydrate the plant cells, and grind the leaves successively. three 1-mL portions of dichloromethane that are de- canted or withdrawn with a Pasteur pipette and placed in a test tube. The pigments of interest are extracted by the dichloromethane. Altern: place 0.5 gof carrot paste (baby food) or tomato paste in a test tube, sti shake the paste with 3 mL of ethanol until the paste has a somewhat dry or fluffy appearance, remove the ethanol, and extract the dehydrated paste with three I-mL portions of dichloromethane. Stir and shake the plant material with the solvent in order to extract as much of the pigments as possible. ‘These pigments are very sensitive to light-catalyzed photochemical air oxidation. Work quickly, keep containers stoppered where possible, and protect solutions from undue exposure to light. Fill the tube containing the dichloromethane extract from leaves or vegetable paste with a saturated sodium chloride solution and shake the mixture. Remove the aqueous layer and to the dichloromethane solution add anhydrous sodium sulfate until the drying agent no longer clumps together. Shake the mixture with the drying agent for about 5 min and then withdraw the solvent witha Pasteur pipette and place it ina test tube. Add to the solvent a few pieces of Drierite to complete the drying process. Gently stir the mixture for about 5 min, transfer the solvent toa test tube, wash off the drying agent with more solvent, and then evaporate the combined dichloromethane solutions under a stream of nitrogen while warming the tube in the hand or ina beaker of warm water. Carry out this evaporation in the hood. Immediately cork the tube filled with nitrogen and then add a drop or two of dichloromethane to dissolve the pigments for TLC analysis. Carry cout the analysis without delay by spotting the mixture on a TLC plate about 1 cm from the bottom and 8 mm from the edge. Make one spot concen- trated by repeatedly touching the plate, but ensure that the spot is as small as possible —less than 1.0 mm in diameter. The other spot can be of lower concentration. Develop the plate with 70:30 hexane: acetone. With other plates try cyclohexane and toluene as eluents and also hexane/ethanol ‘mixtures of various compositions. The container in which the chromatogra- phy is carried out should be lined with filter paper that is wet with the solvent so the atmosphere in the container will be saturated with solvent vapor. On completion of elution, mark the solvent front with a pencil and outline the colored spots. Examine the plate under the uv light. Areany new spots seen? Report colors and Rf values for all of your spots, and identity each as lycopene, carotene, chlorophyll, or xanthophyll.128 Make your own selections Except for tetraphenylihiophene, the structures for all of these molecules will be found in this book Organic Experiments Cleaning Up ‘The ethanol used for dehydration of the plant material can be flushed down the drain along with the saturated sodium chloride solution, Recovered and unused dichloromethane should be placed in the halogenated organic waste container. The solvents used for TLC should be placed in the organic solvents container. The drying agents, once free of solvents, can be placed in the nonhazardous solid waste container along with the used plant material and TLC plates. 3. Colorless Compounds You are now to apply the thin-layer technique to a group of colorless compounds. The spots may be visualized under an ultraviolet light if the plates have been coated with a fluorescent indicator, or chromatograms may be developed in a 4-07 bottle containing crystals of iodine. During development, spots appear rapidly, but remember that they also disappear rapidly. Therefore, outline each spot with a pencil immediately on with- drawal of the plate from the iodine chamber. Solvents suggested are as follows: Cyclohexane. Toluene (3 mL)-Dichloromethane (1 mL) Toluene 9:1 Toluene-Methanol (use 4mL) Compounds for trial are to be selected from the following List (all 1% solutions in toluene): . Anthracene* . Cholesterol . 2,7-Dimethyl-3,5-octadiyne-2,7-diol . Diphenylacetylene trans, trans-1 4-Diphenyl-t,3-butadiene* . butyl-2,5-dimethoxybenzene . trans-Stilbene . 1,2,3,4-Tetraphenylnaphthalene* 10. Tetraphenylthiophene IL. p-Terphenyl* 12. Triphenylmethanol 13. Triptycene “(Fluorescent under uv light.) tis upto you tomake selections and to plan your own experiments. Do as many as time permits. One plan would be to select a pair of compounds estimated to be separable and which have Rf values determinable with the same solvent. One can assume that a hydroxyl compound will travel less rapidly with a hydrocarbon solvent than a hydroxyl-free compound, and soChapter 9 Thin-Layer Chromatography: Analysis of Anaigesics and Isolation of Lycopene 129 Preliminary trials on used plates you will know what to expect if the solvent contains a hydroxylic compo- nent. An aliphatic solvent should carry along an aromatic compound with aliphatic substituents better than one without such groups, However, 2 on assumptions, you can do brief preliminary experi- ments on used plates on which previous spots are visible or outlined. If you spot.a pair of compounds on such a plate and let the solvent rise about 3. cm from the starting line before development, you may be able totellifa certain solvent is appropriate for a given sample. If so, run a complete chromato- gram on the two compounds ona fresh plate. If separation of the two seems. feasible, put two spots of one compound on a plate, let the solvent evaporate, and put spots of the second compound over the first ones. Runa chromatogram and see if you can detect two spots in either lane (with colorless compounds, it is advisable not to attempt a three-lane chromato- gram until you have acquired considerable practice and skill). Cleaning Up Solvents should be placed in the organic solvents con- tainer, and dry, used chromatographic plates can be discarded in the nonhazardous solid waste container. Discussion Tf you have investigated hydroxylated compounds, you doubtless have found that it is reasonably easy to separate a hydroxylated from a nonhy- droxylated compound, ora diol from a mono-ol. How, by a simple reaction followed by a thin-layer chromatogram, could you separate cholesterol from triphenylmethanol? Heating a sample of each with acetic anhydride- pyridine for 5 min on the steam bath, followed by chromatography, should do it, A first trial of a new reaction leaves questions about what has happened and how much, ifany, starting material is present, A comparative chromatogram of reaction mixture with starting material may tell the story. How crude is a crude reaction product? How many components are present? The thin-layer technique may give the answers to these questions and suggest how best to process the product. A preparative column chromatogram may afford alarge number of fractions of eluent (say 1 to 30). Some fractions probably contain nothing and should be discarded, while others should be combined for evaporation and workup. How can you identify the good and the useless fractions? Take a few used plates and put numbered circles on clean places of each; spot samples of each of the fractions; and, without any chromatography, develop the plates with iodine. Negative fractions for discard will be obvious and the pattern alone of positive fractions may allow you to infer which fractions can be combined. Thin-layer chromatograms of the first and last fractions of each suspected group would then show whether or not your inferences are correct.