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2. Looking at the structures of the 4 different drugs below, what do they all have in common, and why do you think they absorb UV light? он aspirin caffeine Но paracetamol ibuprofen 3. What are the stationary and mobile phases of this chromatographic technique? 4. Why is it important to use a pencil, and not pen, for marking TLC plates? 5. Which compounds stain immediately with the permanganate dip? Why do vou think this is?

2. Looking at the structures of the 4 different drugs below, what do they all have in common, and why do you think they absorb UV light? он aspirin caffeine Но paracetamol ibuprofen 3. What are the stationary and mobile phases of this chromatographic technique? 4. Why is it important to use a pencil, and not pen, for marking TLC plates? 5. Which compounds stain immediately with the permanganate dip? Why do vou think this is?

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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Chromatography of painkiller drugs Then dip a micro-pipette into a solution of your sample (just take the liquid from your premade solutions, leave the solid to settle to the bottom), and make a tight spot on the plate by lightly touching the plate on the baseline at a marked point. Make sure that you number/label these so that you know which lane is which drug. Now check the plate before you develop it under the UV TLC lamp, to make sure the sample is strong enough to see - you should be able to see a dark spot in each lane along the baseline. Make sure that the solvent has evaporated from the spots before you develop the plate. You can reuse the same micropipette, as long as you draw up some ethanol and spot it onto some paper towel, between samples. AIM To analyse the components of painkiller tablets by comparison to known standards YOU WILL NEED • Fluorescent silica gel TLC plates • 100 mL glass beaker and watch glass • 6 glass vials or small beakers • Filter paper • Ethanol • Ethyl acetate • Micro-pipettes • Caffeine tablet Paracetamol tablet Aspirin tablet • Ibuprofen tablet • Mixed painkiller tablets X and Y • UV TLC lamp (short (254 nm) wave) Permanganate dip and tweezers • Spatula • Paper towels Caffeine, paracetamol, aspirin, caffeine, and ibuprofen all absorb UV light, and can be identified on the fluorescent silica plates under short wave (254 nm) UV radiation; When viewed under short wave UV the zinc sulfide in the silica plates fluoresces green, except where an eluted substance quenches this fluorescence – these stand out as dark spots on the plate. If the sample is too weak, allow the spot to dry and reapply. This can be done several times, but allow the spot to dry each time to keep the spot size small. Devoloping the TLC plate: PROCEDURE Place the loaded TLC plate into the tank carefully, making sure the baseline is at the bottom, the back of the plate leans against the tank wall at a slight angle, and the baseline is above the level of the eluent. Do not move the tank during the plate development. Place the lid (watch glass) on the tank and allow the eluent to rise up the plate, until it is about 1 cm from the top. Carefully remove the plate, mark the solvent front with a pencil, and allow the plate to dry (preferentially in a fumehood). Making your solutions: Crush up 1 aspirin tablet with a spatula, and add 2-3 mL of ethanol, in a vial or small beaker, and label the beaker 'ethanolic aspirin solution'. Repeat this with a caffeine, paracetamol and ibuprofen tablet, to make up your 4 known standard solutions - if the drug is in a capsule, you can open the capsule and tip the powder into the ethanol. Stir the solutions for several minutes to try to dissolve as much as possible (you can shake them if they are in vials with lids). Look at the TLC plate under 254 nm UV light, and use a pencil to circle the dark spots that you can see on the plate. Measure the Rf (retention factor) of each of the standards, and draw a copy of your TLC. Dip your plate into a permanganate dip using tweezers, and put it onto a paper towel to dry, aluminium side down against the towel. Avoid getting the permanganate solution on your hands. Once the plate has dried, draw what you observe, and then determine by comparison which drugs are in X and Y. Finally, take a tablet/powder of 2 unknown mixtures of painkillers, and repeat the procedure above to make a solution labelled 'ethanolic solution X', and 'ethanolic solution Y. Making the TLC tank: Now you need to make yourself a TLC tank, with either a 100 mL beaker and a watch glass, or a wide jam jar with a screw-top lid. Alternatively foil can be used to make a lid. Line the tank with filter paper (this helps to saturate the air in the tank with solvent), and pour a small amount (approximately 10 mL) of ethyl acetate into the tank - if you are doing this outside of a fumehood, do not leave the tank without a lid on. To measure the retention factor, Rf: this is the relationship between the distance moved by a given TLC spot up the plate, as a fraction of the total distance travelled by the eluent. This ratio is always constant, regardiess how long the TLC plate is. solvent front Allow the ethyl acetate to soak up into the filter paper (you can swirl the tank to do this), and either add or remove some ethyl acetate so that the solvent level is roughly 0.5 cm high. R, bla Loading the TLC plate: baseline To load your TLC plate, first draw a line on the plate in pencil (not ink) 1.0 cm from the bottom of the plate; this marks the starting point. Mark 6 dashes along this line, in pencil, as markers for your samples: aspirin, paracetamol, caffeine, ibuprofen, X and Y. Be careful to mark the silica gently. without scoring it. To measure distance 'b', measure from the baseline to the middle of the TLC spot.
Transcribed Image Text:Chromatography of painkiller drugs Then dip a micro-pipette into a solution of your sample (just take the liquid from your premade solutions, leave the solid to settle to the bottom), and make a tight spot on the plate by lightly touching the plate on the baseline at a marked point. Make sure that you number/label these so that you know which lane is which drug. Now check the plate before you develop it under the UV TLC lamp, to make sure the sample is strong enough to see - you should be able to see a dark spot in each lane along the baseline. Make sure that the solvent has evaporated from the spots before you develop the plate. You can reuse the same micropipette, as long as you draw up some ethanol and spot it onto some paper towel, between samples. AIM To analyse the components of painkiller tablets by comparison to known standards YOU WILL NEED • Fluorescent silica gel TLC plates • 100 mL glass beaker and watch glass • 6 glass vials or small beakers • Filter paper • Ethanol • Ethyl acetate • Micro-pipettes • Caffeine tablet Paracetamol tablet Aspirin tablet • Ibuprofen tablet • Mixed painkiller tablets X and Y • UV TLC lamp (short (254 nm) wave) Permanganate dip and tweezers • Spatula • Paper towels Caffeine, paracetamol, aspirin, caffeine, and ibuprofen all absorb UV light, and can be identified on the fluorescent silica plates under short wave (254 nm) UV radiation; When viewed under short wave UV the zinc sulfide in the silica plates fluoresces green, except where an eluted substance quenches this fluorescence – these stand out as dark spots on the plate. If the sample is too weak, allow the spot to dry and reapply. This can be done several times, but allow the spot to dry each time to keep the spot size small. Devoloping the TLC plate: PROCEDURE Place the loaded TLC plate into the tank carefully, making sure the baseline is at the bottom, the back of the plate leans against the tank wall at a slight angle, and the baseline is above the level of the eluent. Do not move the tank during the plate development. Place the lid (watch glass) on the tank and allow the eluent to rise up the plate, until it is about 1 cm from the top. Carefully remove the plate, mark the solvent front with a pencil, and allow the plate to dry (preferentially in a fumehood). Making your solutions: Crush up 1 aspirin tablet with a spatula, and add 2-3 mL of ethanol, in a vial or small beaker, and label the beaker 'ethanolic aspirin solution'. Repeat this with a caffeine, paracetamol and ibuprofen tablet, to make up your 4 known standard solutions - if the drug is in a capsule, you can open the capsule and tip the powder into the ethanol. Stir the solutions for several minutes to try to dissolve as much as possible (you can shake them if they are in vials with lids). Look at the TLC plate under 254 nm UV light, and use a pencil to circle the dark spots that you can see on the plate. Measure the Rf (retention factor) of each of the standards, and draw a copy of your TLC. Dip your plate into a permanganate dip using tweezers, and put it onto a paper towel to dry, aluminium side down against the towel. Avoid getting the permanganate solution on your hands. Once the plate has dried, draw what you observe, and then determine by comparison which drugs are in X and Y. Finally, take a tablet/powder of 2 unknown mixtures of painkillers, and repeat the procedure above to make a solution labelled 'ethanolic solution X', and 'ethanolic solution Y. Making the TLC tank: Now you need to make yourself a TLC tank, with either a 100 mL beaker and a watch glass, or a wide jam jar with a screw-top lid. Alternatively foil can be used to make a lid. Line the tank with filter paper (this helps to saturate the air in the tank with solvent), and pour a small amount (approximately 10 mL) of ethyl acetate into the tank - if you are doing this outside of a fumehood, do not leave the tank without a lid on. To measure the retention factor, Rf: this is the relationship between the distance moved by a given TLC spot up the plate, as a fraction of the total distance travelled by the eluent. This ratio is always constant, regardiess how long the TLC plate is. solvent front Allow the ethyl acetate to soak up into the filter paper (you can swirl the tank to do this), and either add or remove some ethyl acetate so that the solvent level is roughly 0.5 cm high. R, bla Loading the TLC plate: baseline To load your TLC plate, first draw a line on the plate in pencil (not ink) 1.0 cm from the bottom of the plate; this marks the starting point. Mark 6 dashes along this line, in pencil, as markers for your samples: aspirin, paracetamol, caffeine, ibuprofen, X and Y. Be careful to mark the silica gently. without scoring it. To measure distance 'b', measure from the baseline to the middle of the TLC spot.
2. Looking at the structures of the 4 different drugs below, what do they all have in common, and why do you think they absorb UV light? он aspirin caffeine OH но paracetamol ibuprofen 3. What are the stationary and mobile phases of this chromatographic technique? 4. Why is it important to use a pencil, and not pen, for marking TLC plates? 5. Which compounds stain immediately with the permanganate dip? Why do you think this is?
Transcribed Image Text:2. Looking at the structures of the 4 different drugs below, what do they all have in common, and why do you think they absorb UV light? он aspirin caffeine OH но paracetamol ibuprofen 3. What are the stationary and mobile phases of this chromatographic technique? 4. Why is it important to use a pencil, and not pen, for marking TLC plates? 5. Which compounds stain immediately with the permanganate dip? Why do you think this is?
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