edited by JAMESO. SCHRECK
filtrates ac residues
Univenfiy of Northern Colorado Greeley, CO 80639
Four Exciting Chromatography Activities Supermarket Column Chromatography of Leaf Pigments Doris R. Kimbrouah University of colorad; at Denver P 0. Box 173364 Denver, CO 30317
Examples of column chromatography of the variety of pigments found in plants are many and varied ( 1 4 ) .For example, most organic laboratory manuals contain an experiment in which chlorophyll and carotenoid pigments are isolated from spinach (51,or p-carotene and lycopene are isolated from carrots or tomatoes (6).In most cases the column support used is either silica or alumina gel. The rising cost of chemicals, coupled with the decreasing science budgets of many secondary and middle schools, precludes doing most of these procedures, even as a demonstration. This paper introduces a simple and effective method of column chromatography of leaf pigments that uses chemicals and equipment that can be purchased a t most grocery, hardware, and/or drugstores. Equipment and Chemicals Required Column
acetone (fingernail palish remover, nanscented and noncolored) * isopropyl alcohol/water,70130, vlv (mmmercialrubbing alcohol) saturated aqueous sodium bicarbonate solution (make a saturated solution of baking soda in water) Caution .Petroleum ether, acetone, and isopropyl alcohol are highly flammable. These organic solvents should he kept away from spark sources and should he disposed of properly in waste solvent cans. Test Tubes
Test tubes are needed to collect fractions. Any size will work. A 13 x 100 mm test tube works well. One can substitute any small container made of glass (baby food jars, vials, etc.). Leaves
Many kinds of leaves will work. Leaves from trees, bushes, or house plants (coleus and spider plants) work well. Miscellaneous
If a standard chromatography column is unavailable, a buret can be substituted. Simpler versions can be made using a plastic syringe barrel or a pasteur pipet. ~f either of the latter two are used, a wnstant flow from the column will occur and a solvent receptacle must be provided. Alternatively, the end can be fitted with a narrow piece of rubber or plastic tubing. The flow can then be stopped by folding the tubing and holding it or securing the folded portion with a paper clip, rubber band, or clothespin. Mortar and Pestle
end
a mortar and pestle to the leaves to the pigments. An adequate mortar and pestle can be obtained at most stores selling cooking equipment. One can spoon handle to crush substitute a bowl and use a jar the leaves in the solvent. Sodium Bicarbonate (Baking Soda)
This is the column support material. Commercial baking soda is adequate. The syringe barrel column requires less than 1tablespoon of baking soda, so one package can supply an entire class. The larger wlumns will necessitate a little more. The syringe can be supported using a rubber band attached to a ring stand. A buret or column will require a clamp. Solvents
The following solvents are required. Simple suhstitutions are shown in parentheses. petroleum ether or bexanes (mineral spirits),
Medicine droppers or pipets (2 or 31, cotton, small filter funnel or spatula (for pouring support slurry into the column), and a 'lamp or band and a ring stand (or to the column. Procedure The following procedure is for separating the pigments in leaves from a spider plant using a column made from a 10-mL plastic syringe barrel. The leaf extract is prepared by grinding up two small leaves in a mortar with a pestle in the presence of 1-2 mL of acetone. The liquid extract is drawn up in a pipet (dropper) to separate it from the leafpulp, Plug the mouth of the syringe barrel with a small piece ofcotton. ~h~ is packed to 2.5-3.0 in height by ~ o u r i n ae s l u m of sodium bicarbonate in oetroleum ether through a funnel. Alternatively, one can scoop up the slurry with a spoon or spatula and pour it into the column. The excess petroleum ether is allowed to run out the bottom of the column. When the level of the solvent reaches the top of the sodium bicarbonate, the leaf extract is added carefully with a pipet to the top of the column without disturbing the packing material. The extrad is allowed to run on to the top of the column. When the level of the leaf extrad reaches the top of the sodium bicarbonate, more petroleum ether is added through the clean funnel, to the top of the column. As the petroleum ether elutes, or runs out of the end of the column, a bright green hand separates from the top and moves down the column. This fraction is com~osedmedominantly of chlorophyll (both a and b) and'the &rot-
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Volume 69 Number 12 December 1992
987
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 C h m ~ E d u c1975.52.8W. 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.
