enoid pigments (7) and should be collected in one ofthe test tubes as it comes offthe column. When all the chlorophyll has been removed from the column, the solvent is changed to acetone. The acetone can he added before all the petroleum ether reaches the top of the sodium bicarbonate. Depending on the leaf species, acetone usually elutes a pale green or yellowish band, most probably one of the phytochrome pigments (7). When the band(s) corresponding to the acetone solvent has (have) been collected, the solvent is again changed, this time to isopropyl alcohovwater, producing two or three bands composed of yellow and brown pigments. The final band often has a reddish brown hue and can be collected using the saturated aqueous sodium bicarbonate. These latter bands, eluted with isopropyl alcohovwater and bicarbonate solution, are the water soluble flavonoid pigments (7). Chromatography OffersAdvantages This method of chromatography has numerous advantages over more traditional methods, particularly for instructors with limited resources. The equipment and chemicals required are inexpensive and easy to obtain. Because virtually any kind of leaf can be used, it would be instructive to have different students in the same class analyze different leaves and compare results. Different leaves contain different pigments, and even a non-auantitative method such as this one c& often produce results that allow students to distinguish various types and levels of pigments. Examining yellow or red leaves, such as coleus, can be very interesting as students discover that these plants, although not green, still contain a significant amount of chlorophyll. Students also could look at the same leaves at different time intervals in the fall to oh-~ serve the changing composition of leaf pigments as the leaves change colors in the fall. ~
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Chromatography Unites Chemical and Biological Principles Chromatographing leaf pigments is a n instructive method of uniting chemical and biological principles. Chromatography uses chemical and physical properties to separate components in a mixture. The pigments described above have numerous different biological functions in the plant. Chlorophyll and carotenoid pigments are involved in photosynthesis. Phytcchrome pigments are responsible for regulating membrane and other metabolic activities in response to light signals from the environment. Flavonoids are found in highest concentrationin the flowersand fruits Colors and Rt Values of the Candy Shells and Dyes Sample Yellow candy Orange candy Red candy Green candy Brown candy Tan candy Tartrazine Food color
Rt valuesa
Colors after development Yellow Orange Red Blue, yellow Blue, yellow, orange?, red Blue, yellow, orange?, red Yellow Yellow
0.43 0.35 0.21 0.68, 0.41 0.67, 0.46, 0.37?, 0.16 0.67, 0.48, 0.30?, 0.18 0.54 0.58
'Except for the food mlor, which was measured from asingle run on chmmatography paper, f h values were determined from averaging 2 3 runs, one on filter and 1-2 an chromatography paper. Tailing made it hard todetermine exad spot pos~ions,but the color mmposition (with the exception of brawn and tan candies as explained in the text) was easy to see.
988
Journal of Chemical Education
of plants. Their predominant function there is presumably to act as an attractant for insects to distribute pollen and seeds; however, in leaves, flavonoids are thought to act as sunscreens, protecting plants against damaging ultraviolet radiation. The deep colors in leaves observed in autumn are due to carotenoids and flavonoids (7). The experiment can be performed at various levels including middle school and secondarv school. as well as college-level general chemistry lab. Literature Cited 1. Mewald, W; Radolph, D.;Sady, M . J. C h m Edue. 1985,62,530. 2. DiehlJones, S. M. J Chm.Edue. 1984,61,454. 3. Wollrab,A. J ChemEduc. 1975.52.8W. 4 . w s ~ s h , J. ~M r J c h m . E ~ U Cis%,aa. 294. 5. Pavia. D. L.; Lampman, G. M.; Kriz. G . S. Intmduefion fo O ~ n i e L o b o m f o r*k. y niqups, 3rd ed.; Saunders College Publahing, Philadelphia, PA, 1988;pp286291. 6. Reser, L. F; Williamson. K L. Oraonre &mriiifs. 6th ed.: D . C. Heath and CO.. . Lexington, MA, 1981; pp 328330. I. Gmdwin, T. W . Ckamlsfry and Bloehemlsfw ofplant Plgmmls, Vol. 1,md ed.;Aesdemie Press,Iandon, 1976.
Chromatography of M & M Candies Marjorie Kandel State University of New York at Stony Bmok Stony Brook, NY 11794 The popular M & M candies are the subject of an experiment using paper chromatography to detect the presence of F, D & C approved yellow #5 (tartrazine). This dye causes allergic reactions in some people and must therefore be named as an ingredient in products that contain it.! The .M & M package states that yrllowk5 IS one of the Lcolors added." However, is the dye present in all of the candies or only those of certain colors? The answer to this question is important to a person who is allergic to yellow#5 and wants to know which of the candies, if any, are safe to eat. The Experiment An 8- x 7-an strip is cut from Whatman #1 chromatography or filter paper. Along the longer side, 1 an from the edge, seven marks are made at 1cm intervals. Six of these are identified by code for the different-coloredcandies2and the seventh for the standard, either the compound tartrazine or a food color known to consist of yellow #5.3Atoothpick is dipped in water and rubbed along the shell of one of the candies to extract the dye. Care must be taken not to dissolve away so much of the shell that the chocolate is exposed. The toothpick is blotted lightly on a piece of paper towel and the extract applied in a quick motion to its mark on the paper. The above procedure is repeated for each of the different-colored candies. Compressed air, if available, may be passed over the wet spots to dry them faster. Each candy extract is reapplied to its position 3-4 times until the color is strong. The commercial food color solution, being more concentrated (about 4%), needs to be spotted only once; the same is true for a tartrazine solution of similar concentration. To be free-standing. the DaDer is rolled into a half evlin~"--der, attached at t h e & ~by'astrip of tape. Although a full cylinder is more stable. in a ~reliminarvtrial overla~of the edges caused the solvent Gont to rise unevenly &id the spots to run crooked. The chromatogram is developed in a beaker with no top. The developing solution is O.l'i sodium chloride as recom'The paper by Markow (1)gives i3, values forall F, D & C approved dyes in avariety of solvents. The informationcould serve as the basis fordetermining the presence of other dyes in other foods. An example of this type of experiment is found in Pavia, et al. (a.This text gives the structures of all F, D & C approved dyes. he colors are yellow, orange, red, green, brown, and tan. 'Durkee yellow is labelled as a minimum of 4% yellow #5.
mended by Markow (I).The paper is taken from the beaker when the solvent front is about 2 m from the top. Because the paper remains wet for a long time, the solvent continues forward for several minutes aRer the paper is removed from the developing solution. For this reason, it is important to draw the solvent line and outline the spots immediately. The entire experiment can be completed in less than onehalf hour. As noted by Markow, development time is very fast, about 5 min for these small strips of paper. The long step is the drying between and aRer spottings. Results Com~ositionsof the candy shells are shown in the table. The v&es for the tartrazcne and yellow food color agree well with Markow's reported Rt of 0.58. Those for the yellow component of the candy samples are considerably smaller. It was noticed that initially the solvent front was indented when passing the candy extract spots. Perhaps not fully wetted, these spots moved sluggishly a t first. A sample of tartrazine plus sucrose behaved as tartrazine alone; but the candy also contains corn syrup, corn starch, and gum acacia, which in the extracts may have contributed to the dragging effect. In the bmwn and tan candies, there is either an orange spot between the yellow and red, or an overlap of the two colors. This ambiguity could not be resolved by a longer run (solvent front = 7 em, as opposed to about 4 on the trials re~ortedin the table). To answer the question posed in the introduction to this experiment, all the candies contain yellow #5 except red and orange. Intuitively, green could be produced from blue + vellow. and this is borne out: however. the r e c i ~ for e makis not used, but rather a sining orange fmm red + gle dye. Intuition also leads to the hypothesis that tan and brown have similar compositions, and this is indeed the case - they contain the same pigments in different pmportions (with the question of orange unanswered). Acknowledgment This experiment is part of a n elementary course in scientific investigation, partial support for the development of which is beine ~ r o v i d e dbv t h e National Science Foundation's ~ n d k a d n a t ~e b u r s eand Curriculum Program through grant number USE-9055829. Literature Cited 1. Markow, P.G.J Chrm. Edue. 1988,65,89&900. 2. Pa&, D. !4 h p m a n . G. M.; Knl,G. S. Intmdudlon to O~ganichborafory%h niqus, 3rd ed.; Saunders:Philadelphia, 1988; pp 26?-280.
A Thin-Layer and Column Chromatography Experiment Adapted for Use in Secondary Schools A Quick, Safe, Colorful, Convenient Introduction to Two Basic Techniques Robert C. Reynolds and C. Allen O'Dell Southern Research Institute P.O. Box 55305 Birmingham. AL 35255 While a large variety of chromatography experiments is available in undergraduate texts and in the chemical literature (1-7), none of these fits the stringent time and safety requirements of a high schwl laboratory; in many cases, only 50 min are available for completion, and the students generally work in large open moms with poor ventilation. Furthermore, many of the published experiments require UV or chemical visualization in the thin-layer chromatog-
raphy (TLC) experiment, which increases time requirements and expense and also introduces health hazards. The Improved Experiments Thin Layer Chromatography Our experiment involves both a TLC portion and a column chromatography section. The TLC portion separates six highly colored dyes, which are used as standards, in addition to an unknown mixture that is prepared from some combination of the six knowns. The developing solvent is a 2-propanollacetic acid mixture (15:2). The thinlayer plates are commercially available and are composed of a layer of silica gel deposited on an aluminum support. These plates can be easily cut to size using a standard paper cutter or scissors. Column Chromatography The column chromatography portion of the experiment separates a mixture of three of the six dyes on an inexpensive silica gel column using acetonell-propanollwater as the eluting solvent (1:l:lvlvlv). Improvements These experiments have many advantages over others in the literature. They are discussed below. Safe Soluents The eluting solvents are composed of relatively nontoxic components that produce little odor and introduce minimal health or fire hazard. This is not the case with the common eluting mixtures composed of petroleum ether, diethyl ether, benzene, chloroform, or methylene chloride. lIEmes . Short The thin-layer unknown mixture can be identified within 10 min, and an excellent separation of the six components occurs within 20 to 30 min. The column experiment requires 1 6 1 8 min to elute the first component and 2 5 3 2 min to elute the second component. This experiment demonstrates both column chromatogr a ~ h vand thin-laver chmmatoera~hvwithin one laborato& beriod ifstudents work in i i i r &o n e can carry out thc TLC r x ~ r r i m r n while t the othcr runs thc column chromatography experiment. Safety of the Compounds and Procedure This ex~erimentuses six hiehlv colored dves that allow visual analysis of the experim&< Because the dyes are so hiehlv colored. the students handle extremelv small auantices"of each relatively nontoxic dye. One ndted exceition is rhodamine B, a suspected carcinogen. Also, the students are not exposed to potentially hazardous UV light or chemical sprays. In this regard, expense and laboratory preparation are minimized. Conuenience and Stability ofthe Dyes The students can retain the d a t e s for later analvsis. " . such as Rf determination because the spots remain visible for a t least one week when stored without soecial Drecantions. Furthermore, we found no degradation of a solution of the six dyes when tested by TLC in our labs eight months af'cer preparation. This allows the teacher to prepare unknowns and store them until needed. Availability and Cost of Materials Finally, all materials in this experiment are relatively inexpensive and readily obtainable by the high school teacher. With such a large sampling of separable dyes, the number of possible unknowns is greater than 50. This alVolume 69 Number 12 December 1992
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