Introduction: The goal of this experiment was to identify an unknown analgesic drug through the use of thin-layer chromatography. The drugs used were, Unknown #1, caffeine, aspirin, and acetaminophen. Thin-Layer Chromatography is a technique for identifying substances using a mobile phase (developing solvent) that draws up through a stationary phase (TLC plate) and marks spots of differing distances for differing substances. (1) The TLC plate used was a silica gel stationary phase with plastic backing.
It is very important to carefully handle the plate as it is very delicate and prone to flaking off, giving inaccurate readings. It is a very simple and effective method to convey this experiment but there are common errors needed to overt such as under, or over-spotting the sample, uneven plate cutting, or moving the developing chamber with the plate in it. (2) Results: TLC Plate after visualization Labels 1| Unknown#1| 2| Caffeine| 3| Aspirin| 4| Acetaminophen| Substance| Unknown| Caffeine| Aspirin| Acetaminophen| Rf Value| 1. 7| 0. 7| 2. 4| 1. 8| Calculating Retention Factor.
Molecular Weights Caffeine= C8H10N4O2 =(12. 01×8) + (1. 01×10) + (14. 01×4) + (16. 00×2) =194. 22g/mol Aspirin = C9H8O4 = (12. 01×9) + (1. 01×8) + (16. 00×4) =180. 89g/mol Acetaminophen = C8H9NO2 = (12. 01×8) + (1. 01×9) + (14. 01×1) + (16. 00×2) =151. 18g/mol The TLC plate showed the unknown to match with standard sample #4, which was determined to be acetaminophen. Also, comparing the Retention Factor values confirmed that the unknown most closely matched acetaminophen. Referring to Table 15. 1 of Lehman’s Experiment 15, acetaminophen is found in both Excedrin and Tylenol.
(1) Discussion: Filled a beaker with about 5mL ethyl acetate/acetic acid (200:1) to use as a developing chamber. Covered and set under fume hood for about 10 minutes to allow the gas to fill the chamber while prepared unknown drug and TLC plate. The developing solvent is considered a hazardous substance therefore, you should avoid contact and do not breath its vapours. It is also considered a pollutant and should be disposed of appropriately. (1) Compound| Molecular Weight (g/mol)| Caffeine| 194. 22| Aspirin| 180. 89| Acetaminophen| 151. 18|
Dissolved the unknown drug in 2. 5mL 1:1 ethanol/dichloromethane. This chemical is also hazardous and caution should be exercised when handling it. (1) The drug dissolved completely, unlike some of the other unknowns, meaning that it has a low molecular weight. Calculating the weights for the standard substances, we can assume that our unknown is not caffeine. The TLC plate was then marked with a line about 1 cm from the bottom, carefully, with a dull pencil so as to not scratch the surface and cause a non-uniform solvent movement, giving us inaccurate results.
The plate was spotted with the unknown and standard samples along the pencil line equally spaced out to avoid confusion and lessen an incidence of contamination. For this, we used separate capillaries for each substance to avoid contamination. The plate was then put into the developing chamber and allowed to soak up the mobile phase (liquid solvent). This took about 5-10 minutes. Afterwards, it was taken out and left to dry before observing it under a short wavelength (254 nm) ultraviolet light. Under the light, the spots were able to be seen and could be marked.
The eyes are highly susceptible to ultraviolet light damage; therefore it is of upmost importance to wear appropriate safety glasses and to not look directly at the light source. (1) Some of the spots were more streaked than the others. This is probably due to over concentrating the initial spot. (2) This could have given inexact results, as I included the highest spot for my calculations. Using a ruler, the distance that each substance traveled was recorded from the pencil line near the bottom, to the center point of each spot.
Due to the use of a standard ruler, we were only able to get within 1 millimetre of accuracy. The caffeine spot travelled the least, meaning, it was the most polar substance, compared to the aspirin, which was the least (2) We can confirm this by referring to the structures of the molecules. The caffeine molecule has more electronegative atoms pulling the electron density away from its carbons, making it the most polar of the group. While aspirin’s charges are more spread out and due to the position of its non-polar benzene ring, makes it the least polar of the three.
Acetaminophen still has polar bonds, but its benzene ring evens out the charge of the molecule putting it in between caffeine and aspirin in terms of polarity, and, in turn, retention factor. Conclusion: Thin-Layer Chromatography functions on the principle that a compound will have its own attractive forces to the stationary phase differentiated by their polarities, allowing the identification and comparison of substances. In this experiment, the unknown turned out to be acetaminophen which has a polarity that is in between the aspirin and caffeine, and therefore, had the middle Rf value.
References: 1) Lehman, John W. “Operational Organic Chemistry: A Problem-Solving Approach to the Laboratory Course” 4th Edition, Pearson-Prentice Hall (2008) 2) http://chemwiki. ucdavis. edu/VV_Lab_Techniques/Thin_Layer_Chromatography. UC Davis ChemWiki by University of California (2010) 3) http://pubchem. ncbi. nlm. nih. gov/. National Center for Biotechnology Information, U. S. National Library of Medicine (2009) i) http://pubchem. ncbi. nlm. nih. gov/summary/summary. cgi? cid=2519&loc=ec_rcs ii) http://pubchem. ncbi. nlm. nih. gov/summary/summary. cgi? cid=2244&loc=ec_rcs.