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Thin Layer Chromatography (TLC) 10/5/2023 Asael Garcia Abisai Garcia Chem 2023 1L0 FL23 Sarita Sitaula

Purpose Thin Layer Chromatography, also known as TLC, is a type of chromatography technique that separates compounds based on their polarity. In this lab TLC will be preformed and it will be analyzed using the Retardation Factor (Rf). By analyzing the Rf of a component with a specific solvent, an unknown solute can be determined. Background TLC is a sensitive, fast, simple, and inexpensive analytical technique that you will constantly use during organic experiments. It is the separation of two or more compounds or ions caused by their molecular interactions with two different phases. These phases are the stationary phase, polar (SiO 2 or Al 2 O 3 ), and mobile phase where an organic solvent or two more miscible solvents begin to move and separate. These phases can take the form of solid and liquid, liquid and liquid, gas and a solid, or a gas and a liquid. TLC is a micro technique meaning that little as 10 -9 g of material can be detected. The uses of TLC are 1.to determine the number of components in a mixture, 2. Determine the identity of two substances, 3. To monitor the progress of a reaction, 4. Determine effectiveness of purification, 5. Determine the appropriate conditions for a column chromatographic separation, and 6, Monitor column chromatography. Table of Physical Constants Substance Mol Formula Mol Weight m.p (C) b.p. (C) Density Aspirin C 9 H 8 O 4 180.158 g/mol 136 140 1.4 g/cm 3 Acetaminophen C 8 H 9 NO 2 151.163 g/mol 168 420 1.26 g/cm 3 Ibuprofen C 13 H 18 O 2 206.28 g/mol 75-77 157 1.03 g/cm 3 Caffeine C 8 H 10 N 4 O 2 194.19 g/mol 235 178 1.23 g/cm 3 Acetic Acid CH 3 COOH 60.05 g/mol 16 117 1.049 g/mL Ethyl Acetate C 4 H 8 O 2 88.11 g/mol -83 77 0.9006 g/mL Hazards Aspirin is toxic if swallowed and causes eye, skin, and respiratory tract irritation. Acetaminophen causes skin and eye irritation. Ingestion can cause nausea, vomiting, and diarrhea. Ibuprofen causes skin and eye irritation. Caffeine dust may irritate the eye or the skin if in contact. Ingestion can lead to gastrointestinal irritation with nausea, vomiting, and diarrhea. Acetic acid is a flammable liquid and vapor which can cause sever skin burns and eye damage. Must be kept away from open flames. Ethyl Acetate can irritate the skin, eyes, nose, and throat. Lab gloves and safety glasses must always be worn covering all exposed skin. Procedure 1. Before proceeding, practice the TLC spotting technique.

2. Draw a light pencil line about 1 cm from the end of a chromatographic plate. 3. On this line, make a 5 light dots equidistant with a pencil and name dots As, Ac, Ib, Ca, UK. On the line spot aspirin (As), acetaminophen (Ac), ibuprofen (Ib), and caffeine (Ca), which are available as reference standards. Use a separate capillary for each standard (or rinse the capillary carefully before reusing). 4. Make each spot as small as possible, preferably less than 0.5 mm in diameter. 5. Examine the plate under UV light to see that enough of each compound has been applied; if not, add more. 6. The unknown(uK) spot will be placed in the middle and four known will work as standards. 7. To the developing jar or beaker (see Fig. 8.9 or Fig. 8.10 on page 173 Laboratory text book), add 4 mL of the mobile phase, a mixture of 95% ethyl acetate and 5% acetic acid. Insert the spotted TLC plates with tweezers. 8. After the solvent has risen nearly to the top of the plate, remove the plate from the developing chamber, mark the solvent front with a pencil, and allow the solvent to dry. 9. Examine the plate under UV light to see the components as dark spots against a bright green- blue background. 10. Outline the spots with pencil 11. Calculate the Rf values for the spots and identify the components in the unknown. Observations • First it was a clean white sheet with 5 dots equally spaced that can only be seen under the UV light. • After placed in solvent and developed, traces of the dots moved up across the paper. The solvent can be seen creeping up across the chromatography paper.

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• The unknown had developed three dots when the other substances only developed 1. These dots matched with As, Ac, and Ca. • In Ac there was a small trail left behind it. Clean Up Solvents should be placed in the organic solvents waste container; used dry chromatographic plates can be discarded in the nonhazardous solid waste container. Clean used containers with acetone and disconnect any lab equipment used during experiment. Return all equipment to their designated areas in the laboratory. Data Rf = Distance traveled by compound / Distance traveled by solvent • Rf of As = 6.5cm/7.5cm = 0.867 • Rf of Ac = 5.2cm/7.5cm = 0.693 • Rf of Ib = 6.8cm/7.5cm = 0.907 • Rf of Ca = 2.3cm/7.5cm = 0.307 • Rf of Unknown = 6.5cm/7.5cm = 0.8667/ 5.2cm/7.5cm = 0.693/2.3cm/7.5cm = 0.307 Unknown contained traces of Acetaminophen, Aspirin, and Caffeine. Unknown is Excedrin. Discussion Our Chromatography paper was damaged prior to doing the experiment. On the right side of the paper can be seen the damage causing Ibuprofen to not appear fully in a dot when the experiment was conducted. However, it was separated correctly and did travel up across the paper. A trail can also be seen left behind by Acetaminophen. The reason for this was because the loaded sample was too heavy. It couldn’t carry itself across the paper leaving a trail behind. Conclusion Thin Layer Chromatography is a type of chromatography technique that separates compounds based on their polarity. In this lab the Retardation Factor was analyzed for each of the 5 different substances and was found to be: Rf of As = 0.867, Rf of Ac = 0.693, Rf of Ib = 0.907, Rf of Ca = 0.307, Rf of Unknown = 0.867 and 0.693 and 0.307. Unknown contained traces of Acetaminophen, Aspirin, and Caffeine and is identified as Excedrin.

References Acetaminophen | C8H9NO2 | CID 1983 . (n.d.). PubChem. Retrieved October 12, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Acetaminophen Acetaminophen SAFETY DATA SHEET Section 2. Hazards Identification . (2017, June 14). Cayman Chemical. Retrieved October 12, 2023, from https://cdn.caymanchem.com/cdn/msds/22629m.pdf Acetic Acid | CH3COOH | CID 176 . (n.d.). PubChem. Retrieved October 12, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Acetic-Acid Aspirin | C9H8O4 | CID 2244 . (n.d.). PubChem. Retrieved October 12, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Aspirin Chapter 8: Thin-Layer Chromatography: Analyzing Analgesics and Isolating Lycopene from Tomato Paste. (2016). In Macroscale and Microscale Organic Experiments (pp. 165-170). Cengage Learning. Ethyl acetate . (2019, March 11). American Chemical Society. Retrieved October 12, 2023, from https://www.acs.org/molecule-of-the-week/archive/e/ethyl-acetate.html Ibuprofen | C13H18O2 | CID 3672 . (n.d.). PubChem. Retrieved October 12, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Ibuprofen ICSC 0405 - CAFFEINE . (n.d.). Inchem.org. Retrieved October 12, 2023, from https://www.inchem.org/documents/icsc/icsc/eics0405.htm

Post Lab 2.What error is introduced into the determination of an Rf value if the top is left off the developing chamber? Solvent will evaporate from the developing chamber if the top is left off. You need to follow the solvent as it moves up the TLC plate until it reaches the finish point. You must stop the procedure when it reaches this stage. If the top is left open, the solvent will evaporate, and you won't be able to track it precisely any longer. As a result of the solvent continuing to evaporate, its level will appear lower than it is. As a result, the chemical location you are working on will rise above its proper level. 3.What problem will ensue if the level of the developing liquid is higher than the applied spot in a TLC analysis? The issue that would arise in a TLC analysis is that the examined chemical would not adequately go up the TLC plate if the amount of the developing liquid was higher than the applied spot. Instead, it will be swept away, leaving you with no useful information. 5.In carrying out an analysis of a mixture, what do you expect to see when the TLC plate has been allowed to remain in the developing chamber too long, so that the solvent front has reached the top of the plate? The solvent will eventually reach the top of the TLC plate if you let the plate develop in the chamber for too long. The chemicals will finally all reach the top of the plate since it will continue to develop and migrate up. Additionally, they will eventually merge into a single blob of spots at the top. Additionally, they have the potential to spread out, making it impossible for you to employ the results because they are no longer optimal. 7.What will be the result of applying too much compound to a TLC plate? You would expect precise and specified outcomes when conducting this experiment. A TLC plate's spots get overly large and start to overlap when too much compound is applied to them.This will produce inaccurate findings, making it difficult to distinguish between the purity of the sample and the separations. 8.Why is it necessary to run TLC in a closed container and to have the interior vapor saturated with the solvent? First off, it's crucial to have a closed container because doing otherwise will result in the solvent evaporating during the course of the experiment. The chemicals won't properly separate if it evaporates. The dipped section of the TLC plate won't remain constant over the course of the experiment if the lid is left open. Additionally, keeping the container closed will maintain an undamaged environment for the process to proceed as intended. 10.A TLC plate showed two spots with Rf values of 0.25 and 0.26. The plate was removed from the developing chamber, the residual solvent was allowed to evaporate from the plate, and then the plate was returned to the developing chamber. What would you expect to see after the second development was complete? We may anticipate that the distance between the two sites will be higher than previously after the second development is finished following the first one. Their compounds will be able to move up with the solvent thanks to the second development. You will see afterwards that the space

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between the dots is wider than before, with the solvent moving the chemicals up once more. enabling the viewer to see results at a higher resolution. 12.Using a ruler to measure distances, calculate the Rf value for substance B in Figure 8.10. You must measure the distances and enter them into this formula to determine the Rf value. Rf = distance spot travels / distance solvent travels. This will be 4.1mm/5.7mm= Rf of 0.72