Chem143 Lab 1 Martin Case Corinne Sadlowski Active Ingredients in Analgesics by TLC http en wikipedia org wiki Thin layer chromatography Write up for this lab to be completed on 9 22 and 9 24 Reagents and Equipment Analgesic tablets 1 each Advil Anacin Bayer Excedrin Tylenol TLC standards few mg each Ibuprofen Acetaminophen Aspirin Caffeine TLC eluents 25 mL each Hexane Ethyl acetate Chloroform Methanol Ethanol 2 mL per tablet 10 mL total TLC plates silica 5 x 3 cm 10 plates Disposable pipettes Test tubes Glass rod 100 mL beaker Filter paper Foil Capillary tubes Hand held UV lamp Iodine Chamber Purpose The purpose of this lab is to introduce to you the technique of thin layer chromatography TLC TLC is an extremely effective method for separating a mixture of different components The benefits of this technique are that it is relatively fast 10min per run is inexpensive is non destructive the sample can be recovered unchanged requires very little sample gives reproducible results you can compare your experimental sample with a known sample with a fair degree of confidence Because of these benefits organic chemists use TLC on a daily basis for Monitoring a reaction The disappearance of starting material and appearance of product on the TLC plate can be used as a method to check the progress of a reaction 1 Chem143 Lab 1 Martin Case Corinne Sadlowski Determining the purity of a compound TLC allows you to separate and visualize the components of a mixture Identifying a compound If a spot on a TLC plate lines up precisely with a known compound it is a good indication but not assured that the mixture contains the known compound In this lab you will use TLC to determine the ingredients of the over the counter analgesics by comparing their organic soluble components with the following standards COOH O HN O COOH N N O N N O OH Ibuprofen acetaminophen O aspirin caffeine Experimental Procedure 1 Extraction of organic molecules Place each analgesic tablet Tylenol Excedrin Bayer Advil Anacin in a separate test tube and add 2 mL of ethanol to each Crush each pill with a glass rod to expose the organic soluble analgesic to the solvent 2 Preparation of TLC plates Use a pencil to draw a baseline 1 cm from the bottom of a 5 x 3 cm silica gel TLC plate Mark the line 4 times at roughly equal intervals so that you know where to spot your samples Don t mark too close to the edge of the plate Make a note of which spots will correspond to which sample 3 Identification of effective eluent We have to find the best solvent system eluent experimentally A good eluent will move all the spots away from the baseline and separate the compounds across the plate between the baseline and the top of the plate the solvent front We start with an apolar solvent system and repeat the TLC on a new plate with increasingly polar solvents as follows 50 ethyl acetate in hexane 100 ethyl acetate 100 chloroform 10 methanol in chloroform If we need to go less polar we can decrease the ethyl acetate increase the hexane If we need to go more polar we can add for example acetic acid to our eluent a Line the inside of a 100 mL beaker with a piece of filter paper pour 5 10ml of the least polar eluent into the bottom of the beaker cover with tin foil and leave to sit while the TLC plate is spotted The filter paper allows the eluent to saturate the air within the beaker which makes the TLC run more quickly and makes the results more consistent b Use a glass capillary to spot the samples onto the appropriate marks on the TLC plate The spots should be less than 5 mm across Clean the spotter between each sample c Run the TLC by placing it carefully in the beaker making sure the level of the solvent is below the sample position line When the solvent front is near the top of the plate remove the plate form the beaker and mark the position of the solvent front d Visualize the TLC plate under a UV lamp and circle the spots with a pencil and make note of any differences in how the spots appear Now place the plate in an iodine chamber for several minutes and mark any other spots that may appear Sketch the TLC 2 Chem143 Lab 1 Martin Case Corinne Sadlowski plate and the results in your notebook and calculate the Rf retention factor for each of the compounds Rf distance compound traveled distance solvent front traveled e If the spots are clustered close to the baseline increase the polarity of the eluent and repeat the process If the spots are clustered close to the solvent front decrease the polarity of the eluent and repeat the process from step 2 You may have to try 3 or 4 eluents before you settle on a good eluent composition 4 TLC of standards Prepare a TLC plate as described above and spot the standards on the plate Run the plate using your optimized eluent visualize by UV and Iodine and calculate the Rf s for the compounds Sketch the TLC plate and the results in your notebook 5 Confirmation of analgesic identities Make a best guess as to which compounds are contained in each analgesic and confirm this by co spotting the samples this is done by spotting the standard in lanes 1 and 2 Spot the unknown in lane 3 and also on top of the standard in lane 2 ie Standard Standard Unknown Unknown Run these plates visualize by UV and iodine sketch the TLC plate and the results in your notebook Write up In your write up state what compounds you believe are present in each of the analgesics How confident are you that your assignment is correct Comment on the purity of the formulations Do you see spots with iodine that are not visible under UV If so what do you think these substances might be If any of your analyses showed caffeine comment on why this substance is included in an analgesic formulation If you use any on line or library resources list them at the end of your report Waste disposal The eluent waste is flammable Discard of in the waste container labeled ethyl acetate hexane chloroform methanol trace Advil Anacin Bayer Excedrin and Tylenol TLC plates may be contaminated with ethyl acetate and acetic acid Discard as dry chemical waste in the solid waste container Do not mix solid and liquid wastes Pipettes and capillary tubes should be placed in the broken glass box at the front of the laboratory Safety Your pre lab should include a comprehensive safety assessment of all the solvents and chemicals used in this lab Additionally do not shine the UV lamp into unprotected eyes Safety glasses will filter out harmful UVA 3