Organic Chemistry Laboratory Experiment (TLC Analysis of Analgesics)

TLC Analysis of Analgesics Introduction TLC analysis, or thin-layer chromatography, refers to a laboratory technique used heavily in organic chemistry experiments to identify the specific components of a substance by determining the retention factor (Rf) of the particular compound being tested and comparing it to the known retention factor values of other compounds. In can also be applied in identifying the purity of a particular substance, comparing two substances with an unknown, or observing certain reactions. In the case of this experiment, the compounds being tested using the TLC method are common analgesic (pain-relieving) drugs containing active ingredients including aspirin, acetaminophen, caffeine, and ibuprofen. TLC plates are coated with a polar adsorbent (such as silica) attached to a solid support (such as plastic) so that organic compounds can interact with the polar characteristics of the plate. The analgesics are spotted onto the plates, which are then transferred to a development chamber like the one shown below: Figure: a thin layer chromatographic experiment set-up (2) However, thin-layer chromatography is not limited to identify and isolating these compounds. Their versatility also ranges from being able to separate amino acids as well as sugars. Choosing the right solvent is very important when constructing a TLC experiment. It has been noted over and over that molecules with similar structures, properties, and intermolecular forces react very well together, while non-similar molecules—take a nonpolar solvent and a polar molecule, for example—will not produce such dramatic results (1, Dale & Court). Experimental Preparation of Capillary Spotters and TLC plates: To begin setting up the experiment, four open-ended capillary spotters were made by heating two separate capillary tubes over the low-intensity flame of a Meeker microburner. Upon becoming soft and flexible in the middle, the capillaries were cleanly pulled apart, resulting in four spotters that corresponded with each of the four compounds used in this experiment: ibuprofen, caffeine, acetaminophen, and aspirin. Both of the TLC plates required were separated into three sections where the samples would later be tested. On the first TLC plate, the sections were separated as follows: A 1 (ibuprofen), B1 (acetaminophen), and C1 (co-spot). The second TLC plate contained the other two compounds in this same fashion: A2 (aspirin), B2 (caffeine), and C3 (co-spot). The co-spot on each plate is where both of the compounds will be added (e.g. C 1 will contain ibuprofen and acetaminophen). A line was lightly drawn at the base (about an inch above the bottom of the plate) to indicate where the compounds would be placed and set a standard for measuring R f values later. Next, the spots were made by dipping a capillary spotter into one of the samples, allowing capillary action to “pull” up the sample, and adding it to its respective positions on the TLC plates. This was repeated for all four compounds using different capillary spotters for each one to avoid contamination on the plates. TLC Plate Development and Visualization: The solvent used to interact with the polar compounds on the plate was made by mixing 15 mL of pentane with 4 mL of ethanol and 5 mL of ethyl acetate: Table 1: Information about the solvent being used for TLC development Mixture 5 Ethanol 4 mL Ethyl Acetate 5 mL Pentane 15 mL Dielectric Constant 6.50 The dielectric constant refers to how much a solvent “resists” the formation of an electric field —higher dielectric constants correlate to a more polar solvent. After mixing and corking this solvent, a beaker was lined with filter to paper to create the environment in which the reactions will occur (the developing chamber). A small amount of solvent was added to the chamber, where it was insured that the filter paper was completely saturated, and then the first TLC plate was added to the developing chamber. The beaker was covered in a watch glass and observed for 10-15 minutes before it was removed and analyzed. A line was lightly drawn at the point where the solvent reached during evaporation (called the solvent front). This process was repeated for the other TLC plate after development of the first one. Examining these TLC plates under a UV light indicated where the spots reached during the process, which were then circled for measurements to be taken later on. In order to determine the retention factors of each compound, the distance travelled by the spot was divided by the distance travelled by the developed solvent. Results Table 2: Measured Rf values for each compound Retention Factor Ibuprofen Caffeine Aspirin Acetaminophe N/A 0.44 0.89 n 0.786 Table 3: Class average of retention factors based on mixture used Solvent Dielectric Ibuprofen Caffeine Aspirin Acetaminophe Mixture 1 Mixture 2 Mixture 3 Mixture 4 Mixture 5 Constant 2.10 3.57 4.74 5.70 6.50 0.345 0.466 0.83 0.943 0.903 0.072 0.0625 0.35 0.241 0.307 0.191 0.375 0.84 0.811 0.768 n 0.089 0.1875 0.23 0.463 0.504 Following development and visualization of the TLC plates, they should look something like this: Discussion The solvent mixture used for this particular experiment was the most polar of the available solvents and proved to be very effective at separating each of the compounds, except for ibuprofen. The solvent was not effective in isolating ibuprofen because, of the four compounds tested, ibuprofen is the least polar molecule. As stated earlier, molecules with similar structure, intermolecular forces, and polarities tend to interact much more strongly than those that differ largely in their properties. Therefore, it stands to reason that a molecule with relatively low polarity (ibuprofen, in this case) would not be effected much by the highly polar solvent used to separate it. The difference in polarity can be seen by the effect that the solvent had on the more polar molecules aspirin, acetaminophen, and caffeine. They were much more capable of interacting with the plate as the vaporization process continued, allow them to move further up the coating. The visualization of the TLC plates was made simple by the UV light technique. Bromocresol green had no beneficial aspects in this case because the UV light displayed the spots equally well. However, not all compounds are as easily visible under UV rays and the bromocresol mixture may be necessary for other compounds under certain conditions. It is clear that a more effective solvent mixture for this experiment would be mixture 3 or 4 (listed in table 3). These solvent mixtures were not too polar and not too nonpolar, allowing them to react readily with all of the compounds and give proper data for each one. If a solvent is too polar then it will not react strongly with nonpolar compounds, and if the solvent is nonpolar then it will not react strongly with polar compounds. Mixture 1 proved to be relatively poor at separating the more polar compounds in this experiment (acetaminophen and caffeine). As the data shows, retention factor values for more polar substances are typically lower than those of less polar substances. Therefore, as polarity increases, R f values decrease. More polar compounds have a tendency to adhere to the TLC plate more, causing them to move slower up the plate (resulting in an ultimately lower retention factor). In addition, more polar solvents increase the speed of movement up the plates. That is why it is so crucial to use a solvent that will not react too strongly (too polar) or too weakly. Because of the solvents ability to move compounds further up the plate, more polar solvents typically cause a higher R f value to be calculated.