Objective Separations Techniques of separating mixtures will be illustrated using chromatographic methods. The natural pigments found in spinach leaves, β-carotene and chlorophyll, will be separated using column chromatography. The artificial colors used to make M & M candies will be analyzed utilizing paper chromatography. Background The purpose of all chromatography is to separate mixtures into individual components. The word chromatography, formed from the Greek words khroma, meaning color, and graphein, meaning to draw a graph or to write, was coined by the Russian botanist M.S. Tswett around 1906 to describe his process of separating mixtures of plant pigments. All forms of chromatography employ the same general principle: a mixture of substances in a moving phase passes over a selectively adsorbing surface, the stationary phase. Two different techniques, column chromatography and paper chromatography, are utilized in this experiment to separate components of spinach and M & M candies, respectively. Column chromatography operates on the principle that different substances are adsorbed on the surface of a solid adsorbent (such as silica) to an extent that depends on their polarity and other structural features. Because some compounds are more strongly adsorbed than others, they will be washed down a column of adsorbent at a slower rate and thus become separated from those less strongly adsorbed. The naturally colored components of spinach and other leaves can be separated using this technique. The dark green color of spinach is actually a combination of β-carotene and chlorophylls. β-carotene, a naturally occurring dye, is red in crystal form but yellow when dissolved in petroleum ether solvent. β-carotene is considered a vitamin A precursor in the body and is also used as a natural color additive in foods. Chlorophylls are organic molecules that play an important role in the photosynthetic cycle of green plants. Common uses for extracted chlorophylls include deodorants and leather dyes. In paper chromatography, the paper itself is the stationary phase. The cellulose structure of the paper allows for formation of hydrogen bonds to water molecules, so the stationary phase can be regarded as a layer of water, hydrogen-bonded to cellulose. The mobile phase containing the solute dissolved in the aqueous solvent passes through the paper fibers by capillary attraction. After the chromatogram has been developed and the solutes on the paper have been located, the movement of the solute on the paper is mathematically expressed by the R f value (called the retention factor), where R f distance traveled by the solute ( center of spot) = distance traveled by the solvent front Separations 1
The distances used in calculating R f values are measured as shown in the figure. Solvent front 5 9 12 A B 5 A: R f = = 0.42 12 9 B: R f = = 0.75 12 M & M candies will be analyzed using paper chromatography to detect the presence of FD&C approved yellow #5. 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 yellow #5 is one of the dyes 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. Materials needed mortar and pestle fresh spinach leaves (unfrozen) Whatman #1 chromatography or filter paper M & M candies (1 small package) 0.1% sodium chloride (NaCl) 80:20 petroleum ether/acetone wire and spatula petroleum ether plastic funnel acetone 100 ml beaker (1) sand 250 ml beaker (2) silica 50 ml beaker (4) scissors chromatography column (1) distilled water tape yellow food coloring (dropper bottle) buret clamp and ring stand toothpicks Cotton ruler Separations 2
Procedure Part I: Paper Chromatography of M&M candies 1. On a strip of Whatman #1 chromatography or filter paper, use a ruler to make seven marks (in pencil) in 1 cm intervals near the bottom of the longer side. Label six of the marks with the colors of the candies in your package of M & M s. Label the seventh mark the standard or control color, yellow #5. 2. Draw a finish line 2 cm from the top of the strip across of paper in pencil. 3. Dip a toothpick in a beaker of water and rub along the shell of one of the candies to extract the dye onto the tip of the toothpick. Be careful to not dissolve away so much of the shell that the chocolate becomes exposed. Lightly blot the toothpick above its mark on the chromatographic strip to create a small, single dot (about the size of a pinhead). Keep adding dye until half of the shell has been used and the color is concentrated on the single dot. Repeat this procedure for each of the other colored candies. 4. Using a toothpick, carefully add a very small drop of the commercial food coloring, yellow #5, to its mark on the strip. Because this solution s concentration is relatively high, one tiny drop or spot is sufficient. Allow all seven spots to air-dry before proceeding. 5. Roll your chromatographic strip into a half cylinder with the spots at the bottom and a place a piece of tape at the top to hold it in that shape. 6. In a dry 250-mL beaker, add 15 ml of 0.1% sodium chloride. Place your rolled strip into the beaker with spots at the bottom. Do not allow the strip to contact the sides of the beaker. 7. When the solvent front (wet line moving up the paper) reaches the finish line, remove the strip and quickly outline the different colors for each spot with a pencil. 8. Determine the various color dyes that compose of each candy shell and calculate R f values. 9. Record the color dye components that make up each candy shell. Disposal Procedure ** Discard the paper, M & M s, and toothpick(s) in the trash can. ** Discard the sodium chloride and water in an available sink. ** Take all glassware to an available sink and wash thoroughly. Place them back at your station upside down on a paper towel to drip dry. Separations 3
Part II: Column Chromatography of Spinach Leaves 1. In a mortar containing freshly thawed spinach leaves, add 40 ml of 80:20 mixture of petroleum ether/acetone in and a pinch of sand. Grind the spinach with a pestle until the added liquid is dark green. Decant the liquid (leave the solid behind) into a 100-mL beaker. 2. In 2 separate 50-mL beakers, add 30 ml of petroleum ether and 20 ml of acetone from the stock containers. 3. Secure the glass chromatography column to a ring stand using a buret clamp. Place a small wisp of cotton at the bottom of the column using a long piece of wire. On top of the cotton, carefully add 0.25 cm of sand. Tap the column several times afterward to help level and settle the sand. 4. On top of the thin layer of sand, use a ruler and slowly and carefully add about 5 cm of silica in small increments, tapping the column as you go to help settle and level the silica. CAUTION: DO NOT BREATHE THIS SOLID. After adding the silica, add another 0.25 cm of sand, tapping the column when finished. 5. Place a 250 ml beaker underneath the column and slowly add some of the petroleum ether to the top of the column. After the liquid reaches the bottom, the column should drip at about 1 drop per second. [If the dripping rate is unsatisfactory (3 drops per second is too fast and 1 drop every 5 seconds is too slow), stop and empty the solid material into the appropriate collection container and construct another column beginning at Step 3.] 6. Allow the chromatographic column to drip petroleum ether until the solvent level drops to just above the upper sand layer but no farther. Quickly and carefully add your dark green spinach to the column. 7. Allow the column to drip until the spinach extract level drops to just above the upper layer of sand. Slowly and continually add additional petroleum ether to keep the level of solvent above the upper sand level at all times. 8. After adding the additional petroleum either from Step 7, two colored bands will begin to separate from the original green spinach mixture. When the lower, yellow band reaches the bottom of the column, collect this fraction in a small 50 ml beaker. 9. After the yellow band has been collected and when the petroleum ether level drops down to the upper sand layer, begin adding acetone instead of additional petroleum ether, being care to make sure the solvent level stays above the upper sand layer. Once the acetone is added, the other color band (green) will begin to move down the column. When it reaches the bottom, collect it in an additional 50 ml beaker. Put the 250 ml beaker back under the column until you are ready for clean-up. Disposal Procedure ** Discard the remaining petroleum either, acetone and collection beaker fluids in the appropriately labeled containers located under the fume hood. ** Discard the solid column materials in the appropriate container under the fume hood. ** Take all glassware to an available sink and wash thoroughly. Place them back at your station upside down on a paper towel to drip dry. Mount the clean column back on the ring stand. Separations 4
Report Sheet Data Analysis Part I: Paper Chromatography of M & M Candies For each spot, describe the observable color(s) and then calculate the R f value for each color. (Begin at the top with component 1 being the fastest moving spot.) M & M Candy color Red Yellow Green Orange Blue Brown Yellow #5 Component 1 Color of spot by spot mm mm mm mm mm mm mm by solvent front mm mm mm mm mm mm mm R f value Component 2 Color of spot by spot mm mm mm mm mm mm mm by solvent front mm mm mm mm mm mm mm R f value Component 3 Color of spot by spot mm mm mm mm mm mm mm by solvent front mm mm mm mm mm mm mm R f value Describe the composition of the M & M candies. Do they consist of single components or a mixture of dyes? Red: Yellow: Green: Orange: Blue: Brown: Separations 5
Part II: Column Chromatography of Spinach Leaves Color Band # Color Materials ID 1 2 Post-lab questions: 1. Which M & M candies would be safe to eat for a person who has an allergy to yellow #5? 2. Why is a pencil used to mark the points of application rather than a ballpoint pen on the Chromatography paper? 3. What would happen if acetone were the first solvent used to elute the dyes in spinach leaves instead of petroleum ether? {Elution is the process of separating or exiting the column one after the other.} 4. The column chromatography experiment could be performed on any leaves. What results might you expect if this experiment was performed on tree leaves in the fall? Separations 6
Summary/Conclusions: Separations 7