Chem 333L-021 Experiment 4 – Thin Layer Chromatography of Analgesics Objectives 1. Perform thin layer chromatography analysis on various standard analgesics. 2. Calculate Rf values. 3. Compare Rf value of unknown analgesic to those of standard analgesics for identification. Theory Thin layer chromatography (TLC) is a sensitive and simple analytical technique that is often used in organic experiments. Because it uses only 10-9 grams of material, it is considerably fast and inexpensive to perform. 1. Explanation of TLC TLC involves applying a given sample in solution form at one end of a sheet of plastic or glass that is coated with a thin layer of an adsorbent. The sheet is put on end in a covered jar that has a shallow layer of solvent. The solvent rises up the sheet by capillary action through the absorbent which causes differential partitioning to occur between the components of the mixture dissolved in the solvent and the stationary absorbent phase. Although many of the substances are colorless, exposure to a UV source or iodine chamber can allow for detection. The organic substances will absorb the iodine vapor and form a brown spot on the plate. Once all substances can be detected, Rf values can be calculated. a. Capillary Action The force that causes the solvent to rise through the adsorbent. b. Mobile Phase and Stationary Phase The solvent is the mobile phase. What a solvent will carry through the adsorbent depends on its polarity. The rule of “like dissolves like” determines whether a solvent will display polar or nonpolar substances on the sheet. The adsorbent is the stationary phase. Alumina and silica gel are often used as adsorbents for TLC plates. When anhydrous, alumina absorbs substances stronger than silica gel. This makes it ideal for more nonpolar substances. Inversely, silica gel is more ideal for polar substances. c. Absorption and Desorption The more component that is absorbed onto the stationary phase, the less time it spends in the mobile phase. This means it will migrate slowly up in the TLC plate. 2. Rf Values a. Solvent frontThe solvent front is the moving edge of the solvent up the plate. The farther a compound travels with the solvent front, the higher it is displayed up the plate and the higher its Rf will be. b. Calculation of Rf (response factor) Rf = (distance traveled by compound) / (distance traveled by solvent) 3. Utility of TLC a. Determination of number of compounds This method works well for analyzing crude reaction mixtures to determine the number of compounds present in the final product. b. Identification of substances (co-spots) Occasionally two substances will give identical Rf values using the same solvent locations. In these instances, it is possible that the two substances are identical but they could also be different substances who give similar, but not identical, values. c. Monitoring progress of reactions It is possible to monitor a chemical reaction by observing the disappearance of the starting materials and the appearance of the product. The ability to watch the products appear in real time allows for better control to stop a reaction at a certain at a desired stage. d. Monitoring purification processes Whether a substance has been recrystallized, distilled, extracted, or has been separated and purified by other methods, its purity can be evaluated using TLC. e. Determination of good solvent system for column chromatography Column chromatography is used to separate and purify a solid mixture. By using TLC, the correct solvent and adsorbent can be determined for column chromatography. TLC can also monitor its progress when the appropriate fractions are collected. Analgesics are substances that are used to relieve pain. In this experiment, analgesics will be run on TLC and compared to an unknown in order to identify it. In order to do this, the unknown will be run on TLC along with known standard compounds. The Rf values can then be compared to determine which matches the unknown. Depending on the compounds needing to be separated, the solvent mixture can be changed accordingly. Very polar molecules such as carboxylic acids and alcohols will need a more polar solvent. Good solvent mixtures usually contain both a nonpolar solvent and a polar solvent.Mechanism Procedure 1. Obtain a TLC plate from the desiccator on the side shelf. On the plate, make a light pencil line about 1 cm from one end of the plate. 2. Obtain a TLC jar. 3. Place the TLC solvent system (95% ethyl acetate/5% acetic acid) in the jar, ensuring that the solvent is not higher than 1 cm. 4. Spot one small circle of each of the three standards and one unknown onto the pencil line on the TLC plate. 5. Gently place the TLC plate in the jar, making sure that the pencil line is above the solvent. 6. Allow the solvent front to reach approximately 1 cm from the top of the plate. Remove the plate and draw a pencil line to indicate the solvent front. 7. Observe your plate under UV light for a few minutes and then circle the spots that are visible in the UV light. 8. Use a metric ruler to measure the distance from the center of each spot to the pencil line and record your values. 9. Measure and record the solvent front. 10. Using this information, calculate the Rf value for each of the standards. Compare the values with that of the unknown. 11. Determine unknown based on values. ApparatusReagent Table Name, Structure, MW (g/mol) Melting C Boiling C Density g/mL Properties Ethyl Acetate C4H8O2 88.10 -83.6 77.1 0.8945 Colorless, flammable liquid with a pleasant, fruity odor Acetic Acid CH3COOH 60.0524 16.6 117.9 1.0492 Corrosive material. Colorless liquid or solid with a strong vinegar-like odor Methanol CH3OH 32.042 -98 64.6 0.791 Colorless liquid with a characteristic pungent odor Aspirin C9H8O4 180.1598 135 140 1.35 Analgesic, anti-inflammatory and antipyretic. White crystals or white crystalline powder Acetaminophen C8H9NO2 169 - 1.293 Analgesic. Antipyretic. White crystalline powder. Caffiene C8H10N4O2 238 - 1.23 White powder or white needles. LIGHT SENSITIVE Ibuprofen 76 - -- White PowderDisposal • Dispose of TLC plate in proper container. • Wash excess standard solutions down the drain with excess water. • Solvents are to be placed in the pre-designated organic liquid waste container separate from other organic liquid waste. • Dispose of