Lab 1- Thin Layer Chromotography of Analgesics PDF

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Thin Layer Chromatography of Analgesics CHEM 2821 January 27th, 2014

Abstract Unknown analgesic #1 (1) and unknown analgesic #2 (2) were analyzed using thin-layer chromatography (TLC). When the Rf values of the unknowns were compared to the experimental standards, 1 was determined to be anacin and 2 was inconclusively determined to be either empirin or vanquish, the strongest candidate being empirin. TLC test for 1 produced two spots with Rf values of 0.29 and 0.873. TLC test for 2 produced three spots with R f values of 0.273, 0.709, and 0.909. These values were compared to and coincided with the experimental R f values of the given standard analgesics of aspirin (0.923), ibuprofen (0.954), acetaminophen (0.615), caffeine (0.215), and phenacetin (0.677). In part two of the experiment, the optimal solvent ratio of hexanes to ethyl acetate was determined to be 3:1, which gave Rf values for an unknown mixture that ranged from 0.113 to 0.811.

Introduction Many mixtures containing two or more compounds can be analyzed and identified using a special technique known as TLC. TLC is the primary tool for speedy qualitative analysis of a compound. It is the method of separation when a mixture is between a stationary phase and a mobile phase. A stationary phase such as silicon dioxide SiO2 • xH2O (silica gel) is coated on a thin plate. The mobile phase is usually an organic solvent or a combination of organic solvents that move up the plate through capillary action. As the mobile phase moves up the plate, the mixture interacts with the solid phase. The components of the mixture move at different speeds due to differences in structure of each individual compound present. The greater the affinity a compound has for the mobile phase, the faster it moves, and the more distance it covers. The distance a compound travels is expressed by an R f value. The Rf value is the ratio of distance from the starting line to the compound location divided by the distance from the starting line to the solvent front. The R f values of different compounds can be compared to each other, similar values mean similar structures, which in turn mean similar compounds.

Figure 1. Thin-layer Chromatography analysis of Standards I-V. TLC developing solvent was a mixture of ethanol, ethyl acetate, and acetic acid.

Figure 2. Potential hydrogen bonding between Standards I and II and SiO 2.

Figure 3. Hydrogen bonding between Standards III and V and SiO2.

Figure 4. Lewis Acid- Lewis Base interaction between Standard IV and SiO2.

Results and Discussion Rf values and analysis of Standards I-V. The TLC data for Standards I-V: aspirin, ibuprofen, acetaminophen, caffeine, and phenacetin, respectively, are shown in Figure 1. Standards I and II obtained relatively high Rf values of 0.92 and 0.95, indicating that there was weak interaction with the silica gel and spontaneous bonding with the organic solvent. Although Standards I and II have alcohol groups that could potentially form hydrogen bonds with the silica gel as shown in Figure 2, the nonpolar sections of the molecules are larger and therefore make the two compounds of more nonpolar character. The nonpolar qualities of Standards I and II allow for the majority of time spent to be in the mobile phase, bonded to the organic solvent. Standards III and IV obtained midrange R f values of 0.62 and 0.68, showing that the two compounds share nonpolar characteristics, but also exhibit slight polar qualities. The N-H groups of the two compounds allow hydrogen bonding to occur with the silica gel as shown in Figure 3, but due to the nonpolar middle section of the compounds, the compounds also bind readily to the organic solvent. Standard IV obtained a relatively low R f value of 0.22, showing that there was strong bonding to the silica plate. Looking at the structure of the Standard IV, shown in Figure 4, a coordination Lewis acid-Lewis base complex can be formed with the silica gel, with Standard IV donating a pair of electrons to silicon. Standard IV can also form a dipole-dipole interaction with silica gel with the negative charge on the lone oxygen atoms and the positive charge on the carbon.

Figure 5. Thin-layer Chromatography analysis of 1, 2, and Standard VI. TLC developing solvent was a mixture of ethanol, ethyl acetate, and acetic acid.

Identifying the active ingredients in each unknown analgesic. The Rf values of 1 and 2 are shown in Figure 5. 1, with two spots on the TLC plate, was determined to be anacin containing both aspirin and caffeine. The Rf values of 0.87 and 0.29 are comparable to that of aspirin (0.92) and caffeine (0.22), two of the main active ingredients in anacin. 2, with three spots on the TLC plate, was determined to be either empirin or vanquish. Two R f values of 2, 0.26 and 0.91 indicated that the analgesic contained aspirin and caffeine, both active ingredients in empirin and vanquish. The identity of the third spot, with the Rf value of 0.71, could not be established without further evidence. This is because empirin has the active ingredient phenacetin, with an Rf value of 0.68 and vanquish with the active ingredient acetaminophen, with an Rf value of 0.62. The Rf value of the third spot is too close to both of the components of empirin and vanquish for any solid conclusion to be made about the identity of the unknown analgesic. The best inference for the identity of 2 would be empirin because of the closer proximity in the Rf values between the third spot and phenacetin than that with acetaminophen.

Figure 6. Thin-layer Chromatography analysis of the unknown mixture. TLC developing solvent was a mixture hexanes and ethyl acetate in the ratios of 1:1, 3:2, and 3:1.

Separation of an Unknown Mixture. Figure 6 shows the Rf values of the unknown mixture, in various ratios of solvent. A 3:1 mixture of hexanes: ethyl acetate was determined to be the best solvent for separating the components of the unknown mixture. A 3:1 ratio solvent resulted in R f values that ranged from 0.113 to 0.811. A 1:1 ratio solvent produced Rf values that ranged from 0.51 to 0.91. A 3:2 ratio produced Rf values that ranged from 0.38 to 0.95. Because the 3:1 ratio solvent was the most effective at separating the components, it suggests that the components of the unknown mixture have non polar qualities and require a less polar solvent such as hexanes to distribute the spots evenly across the TLC plate. On another note, five spots were supposed to appear but only four spots showed; one factor that might have caused this was that the TLC plate was not long enough and the last spot did not have room to show up. There was significant streaking between spots two and three, a potential location for the last spot. Conclusion TLC allowed for the identification of two unknown analgesics and the determination of the best solvent composition that best separates an unknown mixture. When compared to the R f values of given commercial standards of aspirin, ibuprofen, acetaminophen, caffeine, and phenacetin, 1 was determined to be anacin. 2 was determined to be either empirin or vanquish, the more likely being empirin. A 3:1 ratio of hexanes to ethyl acetate gave the best separation of components of the unknown mixture. This suggested that the components of the unknown mixture were mostly non-polar and bonded better to hexanes than ethyl acetate. Experimental Section Analysis/Identification of Commercial Analgesics. Small samples of aspirin, ibuprofen, acetaminophen, caffeine, and phenacetin were placed on one silica gel TLC plate. Samples of the two analgesic unknowns and Standard VI were placed on another silica TLC plate. The plates were developed in a mixture of ethanol, ethyl acetate and, acetic acid. The spots were detected under UV light and using an iodine chamber. Rf values were calculated by dividing the distance the samples traveled by the distance of the solvent line. Separation of an Unknown Mixture. Small samples of the unknown mixture were placed on three different silica TLC plates. The plates were developed using three different ratios of ethyl acetate to hexanes to obtain the best separation of the components of the mixture. The ratios used were 1:1, 3:2, and 3:1 hexanes to ethyl acetate. Rf values were calculated by dividing the distance the samples traveled by the distance of the solvent line....