Experiment #7 Chromatography

Experiment #7: Chromatography Abstract— Chromatography is an analytical technique commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed, and chromatography is thus a form of purification. Based on the ‘like dissolves like’ principle, the polarity of the mobile phase directly affects the distance each pigment will travel such that a polar mobile phase results in polar pigments moving up the strip the furthest and a non-polar mobile phase results in non-polar pigments moving up the furthest. Carotene changed from yellow to yellow-orange and moved 35.0 mm with an Rf factor of 0.34, xanthophyll stayed yellow and moved 30.0 mm with an Rf of 0.29, chlorophyll a changed from bright green to blue green and moved 15.0 mm with an Rf of 0.15, and chlorophyll b changed from yellow green to olive green and moved 25.0mm with an Rf of 0.24. Introduction— There are two phases in chromatography, one is mobile phase, which is the moving solvent, and the other is stationary phase, which is the chromatography paper. In chromatography a mixture of two or more solutes are placed on a stationary material over which a moving fluid is passed. The solutes (different pigments of color) will have a competing tendency to be attracted to the stationary phase and also to be dissolved in the mobile phase. The competition will cause some pigments to move relatively quickly and some to move through more slowly, the different travel speeds cause them to separate. If pigments have a higher affinity for the mobile phase rather than the stationary phase, it will travel further up the paper (Rf value close to 1). If pigments have a higher affinity for the stationary phase rather than mobile phase, it won’t travel very far up the paper, and will remain closer to the original line (Rf value close to 0). Experimental Materials and Equipment Spinach Leaves Chromatography Chamber Procedure— In this experiment, paper chromatography will be applied to Spinach leaves and the components of the mixture characterized. We placed 10 leaves of Spinach, 6.0 grams of sand, 2.0 grams of anhydrous magnesium and 2.0 mL of acetone in a mortar and grinded the mixture to a pulp. After adding petroleum ether and acetone to the chromatography chamber, we drew a ‘start’ line on the chromatography paper with a pencil. We added small dots of spinach pigment to the start line and then placed the paper into the chamber and quickly closed it. We gave the pigment ample time to move up the paper in different shades of color, which we then measured. The experiment controls three variables in paper chromatography: pigment, paper, and distance pigment moves. The latter is difficult to repeat precisely and to compare experiments the ratio called the representative fraction, Rf, is calculated. This value is invariably reported in manuscripts so that people who replicate the synthesis of a compound can verify that they too are getting the same Rf values for the same compounds. Rf is defined as the distance travelled by the individual pigment (Carotene 35.0 mm, Xanthophyll 30.0 mm, Chlorophyll a 15.0 mm, Chlorophyll b 25.0 mm) divided by the total distance travelled by the solvent (103.0 mm). Data 1) Data table Distance Travelled by Solvent: 103.0 mm Band Color Plant Pigment Distance (mm) Rf (use formula) 35.0 mm 0.34 Xanthophyll 30.0 mm 0.29 toChlorophyll a 15.0 mm 0.15 toChlorophyll b 25.0 mm 0.24 Yellow to Yellow-Carotene orange Yellow Bright Green Blue Green Yellow Green Olive Green Results Sample Calculation Rf = Distancetraveledby . pigment Distancetraveled by solvent = 35.0 mm ÷ 103.0 mm Rf Carotene = 0.34 Limitation on ResultsTriple Balance Beam ± 0.01 g Conclusion— The experiment outlined the Chromatography process used to separate a mixture of pigments into their individual components so that they can be distinctly analyzed. In a polar mobile phase, the high polarity pigments will travel the farthest. In a nonpolar mobile phase, the nonpolar pigments move furthest up the paper. Carotene was not only the most nonpolar as it traveled the furthest up the paper, but it was also fairly soluble as it experienced a color change from yellow to yellow-orange. Chlorophyll a was shown to be most polar as it traveled the least furthest up the paper. However, based on a molecular analysis, Chlorophyll b should be the most polar pigment tested in this experiment. This disparity can be attributed to inaccurate measurements of distances, human error in mixing of colors, and other colors may have been present. Questions 1. How many spots (pigments) did you identify? There were four different pigments indicated. 2. Which of your pigment molecules was the most nonpolar? Polar? Carotene was most nonpolar and Chlorophyll b was most polar. 3. Why should you not use ink on the coating to mark your pigment placement? Ink is a liquid or paste that contains pigments or dyes that may dissolve in the solvent. The ink is likely to move during the Chromatography process and interfere with the components, hindering the interpretation of results. Pencil is made of graphite, which cannot dissolve in solvent. 4. What other pigment mixtures might this technique be used for (HINT: black is a mixture of many colors)? Chromatography can be used to distinguish the different pigments that make up a particular substance. For instance, this method can be used to distinguish the different colors that make up black ink since black is a mixture of various colors. Chromatography takes advantage of the differences in molecular characteristics, specifically solubility in water and rate of absorption by the paper used in order to separate colors in a mixture. 5. The leaves of maple trees are green in the summer, but turn orange or red in the fall. Considering your results from the spinach leaf chromatography, why do you not see the orange and red maple leaf pigments in the summer? Why are they visible in the fall? During the summer, the deep green color of chlorophyll hides any other color from the leaves. In the fall, trees break down their nutrients from the leaves. In the fall chlorophyll production slows down because of a lack of light and water, which allows the carotenoid colors (red, orange, and brown) to come through.