Pigments in Paprika - Introduction to Organic Chemistry Lab | CHM 122L, Lab Reports of Organic Chemistry

Download Pigments in Paprika - Introduction to Organic Chemistry Lab | CHM 122L and more Lab Reports Organic Chemistry in PDF only on Docsity! Pigments in Paprika Chemistry 122L Objectives: To learn thin-layer chromatography (TLC) and column chromatography techniques. To separate paprika into the 3 major classes of compounds (listed below) by column chromatography. To use TLC to identify the 3 major bands to be isolated by column chromatography and to check the purity of the fractions after the column. Overview of the experiment: In this experiment you will extract paprika with diethyl ether. The major compounds that are extracted into the ether include 1) fatty acid esters of capsanthin, 2) fatty acid esters of capsorbin, and 3) β-carotene, but there are other “carotenoids” as well. These compounds are shown below. Because of the extended conjugation (alternating single and double bonds) present in these substances, they are colored. The esters of capsanthin and capsorbin are red and β-carotene is yellow. Capsanthin Capsorbin β-carotene Since these compounds have different polarities they can be separated by adsorption chromatography, and because they are colored it is easy to visually follow the progress of the separation. Procedure 1 CH3 CH3 CH3 CH3 O CH3 H3C H3C O CH3 CH3 CH3 OR O R O O CH3 CH3 CH3 CH3 CH3 CH3 CH3 H3C CH3 CH3 HO O O R O CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H3C H3C One student in a pair should begin extracting paprika for TLC. The other student should begin packing the chromatography column at the beginning of the lab period. Extraction of the Pigments. To a dry 50 mL Erlenmeyer flask add 15 mL of ether, 0.5 g of ground paprika, and 0.5-1.0 g of anhydrous magnesium sulfate. Add a clean, dry, magnetic stir bar, stopper the flask, and stir for 15 min. Gravity filter (Zubrick p. 106 and 117) the solution into a clean, dry 50 mL round bottomed flask and evaporate to near-dryness with a rotary evaporator. Do not apply much heat to the flask, for ether is very volatile. To the concentrated extract, add 1 mL of 15% diethyl ether, 85% heptane, stopper, and set aside in a dark place. Thin-layer Chromatography. (Zubrick Ch. 28)The adsorbent will be silica gel, the developing solvent is 15% diethyl ether, 85% heptane, and the developing chamber will be a clean, dry, 250 mL beaker containing a piece of filter paper and covered with aluminum foil (Zubrick Fig. 28.6, p. 227). Commercial silica gel sheets and micro capillary tubes for spotting are available in the lab. Using a micro capillary, spot the sheet with your paprika extract, making sure that the spot will be slightly above the developing solvent in the beaker. A 2 cm sheet is wide enough for two chromatograms, so you could make a second spot using more (or less) extract than before. Be sure to make a pencil mark to denote the starting position of your spots. Carefully lower the TLC plate into the developing beaker and cover the beaker with foil. When the solvent front climbs to within 1 cm of the top of the sheet, remove the sheet and quickly mark the solvent front with a pencil. After the sheet has dried, circle each spot with a pencil and make a note of the color. (Spots will fade.) Sketch a picture of the TLC plate into your lab notebook. Calculate and record the Rf value for the three major spots and indicate which compound is present in each spot. Column Chromatography. (Zubrick Ch. 29) We will use a chromatography column that has an internal diameter of about 22mm. It will be equipped with a short length of relatively inert tubing and a pinch clamp to control the flow of eluate. The adsorbent will be silica gel, and the solvent will be 15% diethyl ether, 85% heptane, changing to 50% ether, 50% heptane as elution progresses. Please do not to take more organic solvents than you actually need for the experiment! If a TA has not already done so, begin by placing a small plug of cotton in the narrow neck of the chromatography tube. This will keep the silica gel from running out the bottom of the column. The plug is best inserted from the bottom of the column, using a short length of wire to position the plug just below the wide portion of the tube. Be careful not to break the tube. If the plug doesn’t easily slip into place, you are using too much cotton! Mount the tube vertically using a ring stand and clamp, and close the bottom of the tube with a pinch clamp. Fill the tube about one-third full with 15% ether, 85% heptane, then add sand to form a uniform 5-10 mm layer at the bottom of the tube. If some sand sticks to the walls of the tube, wash it down with a little of the 15% ether solution. Next, introduce the silica gel. In the hood, carefully pour 40-45 mL of silica gel into a 50 or 100 mL clean, dry beaker. The exact amount is not important. Be careful not to breathe the dust, or spill the fine powder all over the hood! In another dry beaker place about 50 mL of the 15% ether, and make a slurry by carefully (slowly) introducing about half of the silica gel with stirring. Pour the silica gel into the solvent, not the other way around! Pour this suspension into the chromatography tube; rap the tube with a rubber stopper to ensure that the silica gel settles uniformly. Open the pinch clamp at the bottom of the tube to drain off some of the solvent into a clean, dry, Erlenmeyer flask. Use this solvent to make a slurry with the remaining silica gel as before, and pour this suspension into the column with tapping as before. The slurry will rapidly settle, 2