Spectroscopy can be a helpful method used to deter-mine the structure of a compound and identifying the functional groups present (2). In this lab specifically, the spectroscopy techniques utilized was Proton Nuclear Magnetic Resonance (HNMR) and Infrared (IR). HNMR was used to analyze a liquid mixture of toluene and acetyl acetate and IR was used to analyze solid caffeine and liq-uid toluene. Another technique used in identifying com-pounds that was utilized in this lab was Thin-Layer Chromatography (TLC). The TLC technique was used to separate a solid mixture of caffeine and aspirin into the individual components and analyzed by calculating the retention index (Rf).
Experimental SectionThe TLC was performed using a mixture of caffeine and aspirin as the compounds to be separated and analyzed. A TLC plate was labeled with a depth solvent line approxi-mately 1 cm away from the bottom that was divided by 5 dashes evenly using pencil. The dashes were labeled cor-responding to the compound the dash would be dotted with.
From left to right in Figure 1, the dashes can be de-scribed as: A) aspirin, A+B A) mixture and aspirin, A+B) mixture, A+B B) mixture and caffeine, B) caffeine. Before the compounds were dotted onto the TLC plate, each needed to be dissolved methanol. The TLC plate was dot-ted with a capillary of each solution with the correspond-ing dash lines. In order to obtain an HNMR spectrum for a liquid sam-ple, a pipet is used to transfer an approximate 3-4 drops of the liquid, in this case a mixture of toluene and ethylacetate, to a vial. Using a different pipet, approxi-mately 1.0 mL of chloroform-d was added to the vial that contained the toluene and ethylacetate mixture. The sample is pulled repeatedly in and out of the pipet to en-sure the mixture of the sample and the solvent. The sam-ple is transferred an NMR tube using a pipet, capped, and inserted into the Varian 400mHg using the software Vnmrji version 4.2 revision A and Patches 4.2-LNX-DPR-WS to obtain the spectrum. For IR spectroscopy, the procedure does differ from the procedure expressed for HNMR. The solid sample chosen to be analyzed using this technique was caffeine. The caffeine and potassium bromide were grinded finely to-gether using a mortar and pestle. Using a spatula, a small amount of the grinded mixture was added until a thin layer of the solid mix covered the bottom of the KBr pellet when placed on top of the die and placed into a hand press in order to make the solid mixture a nearly trans-parent film. The KBr pellet was removed from the 3-piece assembly, placed on a sample holder, and inserted into the Thermofisher Scientific Nicolet i550 FT-IR using the software OMNIC 9.2.98 Driver version 9.2 to obtain the spectrum. On the other hand, the procedure for a liquid sample using IR as the technique is less complicated than that of a solid sample. Toluene was chosen as the liquid sample to be analyzed and a drop was placed on a salt plate using a pipet. Another salt plate was placed on top to spread the drop of toluene across the entire salt plate. The over-lapping salt plates were placed on sample holder, sealed with a cap, and inserted into the Thermofisher Scientific Nicolet i550 FT-IR using the software OMNIC 9.2.98 Driv-er version 9.2 to obtain the spectrum. Results The resulting TLC plate obtained is shown in Figure 1. The aspirin and caffeine mixture were separated into the individual compounds and can be identified by the loca-tions of the outlined circles. The aspirin (dash A) had a Rf value of 0.91 and the caffeine (dash B) had a Rf value of 0.73 (3). Displayed in Figure 2, the HNMR spectrum for the mix-ture of toluene and ethyl acetate is presented. The three different protons in ethyl acetate are represented by sig-nals at ґ: 2.5 (s, 3H, CH3 ), ґ: 4.14 (q, 2H, CH2), ґ: 1.3 (t, 3H, CH3). The signals at ґ: 2.1 (s, 3H, CH3 ), ґ: 7.2 (m, 5H) represent the different protons in toluene. The HNMR overall displays five different signals, each is labeled with the proton producing the signal. Discussion The resulting points displayed on the TLC plate indi-cate the separation of aspirin and caffeine by the two areas represented in the A+B A) mixture and aspirin, A+B) mixture, A+B B) mixture and caffeine shown Fig-ure 1. The two points result in one compound being car-ried for a longer distance by the mobile phase (solvent) than the other based of the Rf values. The Rf value for aspirin was 0.93 which is higher compared to the Rf val-ue for caffeine (approximately 0.22 higher). The higher Rf value of aspirin indicates that the aspirin is less polar than the caffeine, therefore less attracted to the polar stationary phase of the TLC plate and remain in the mo-bile phase longer as supported by having a greater Rf. The HNMR spectrum obtained for the mixture of toluene and ethyl acetate displays five signals that are produced by each different proton in each compound (Figure 2). In ethyl acetate, the point at (a) reported a singlet signal with a shift of 2.5 ppm due to its location near the carbonyl and had a relative integration that concluded 3H. The (b) signal displays a quartet multi-plicity and a relative integration that concluded it con-sisted of 2H. Due to the location of the 2H, the CH2 displayed a chemical shift of 4.14 ppm from being in the proximity of the oxygen. The (c) signals displays a triplet multiplicity and a relative integration that concluded 3H. The chemical shift of 1.3 corresponded to that of a CH3. For toluene, signals (d) and (e,f,g) are shown. The (d) signal displays a multiplicity of a singlet and the rel-ative integration determines 3H. The chemical shift at 2.1 ppm represents CH3 bonded to the aromatic. The (e,f,g) signal displays a multiplet multiplicity and a rela-tive integration that determined 5H. The chemical shift at 7.2 ppm is the chemical shift of the aromatic. The IR spectrum for caffeine is expected to display a peak for the C-N bond at 1250-1350 cm-1, stretched peak for the C=N at 1640-1690 cm-1, a stretch for the C=O at 1640-1810 cm-1, a peak at 1600-1680 cm-1 for the C=C, and a peak at 3000-3100 cm-1 for a sp2 H (1). The structure of caffeine was used to determine the ex-pected peaks based on the functional groups present in the structure and were accounted for in the spectrum obtained in Figure 3.The IR spectrum for toluene is expected to display a peak at 1600-1680 cm-1 for the C=C, a peak a peak at 3000-3100 cm-1 for a sp2 H, and a peak at 2850-3000 for the sp3 C-H (1). The structure of toluene was used to determine the expected peaks based on the functional groups present in the structure and were accounted for in the spectrum obtained in Figure 4. ConclusionThe techniques utilized in this lab demonstrated the usefulness of these methods in providing information about a compound. The TLC method is an easy tech-nique that reports speedy results of the types of compo-nents, number of components, purity of a compound and rate of a reaction (3). TLC does report vague results in that it separates the components based on polarity and does not provide any insight to the types of func-tional groups or arrangement. On the other hand, IR spectroscopy does provide types of functional groups present in a compound with each group having a corre-sponding shape of peak and the wavenumber it is transmitted at. IR does not provide the structure at which these functional groups are arranged as expressed in HNMR spectroscopy. The HNMR technique provides a framework for the structure of the compound based on each different proton displaying a signal that can be integrated to determine number hydrogens, the multi-plicity that represents the neighboring protons, and the chemical shift that provides a location based on the proximity of functional groups. It is through the com-bined use and analysis of these techniques the study of an unknown be determined most accurately.