Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY Caution: Do not ingest any materials from this lab. Ethyl acetate and ethanol are used in the solvent mixture and both are flammable. Purpose: In this lab you will separate the components of different pain medications using thin‐layer chromatography, and use this information to determine what drugs are in pills that contain more than one drug. Use your textbook if you need a refresher on polarity. Background: We sometimes refer to pain‐pills in terms of their brand name, rather than their generic name, which is also different than their chemical one. We sometimes ask for Tylenol or Advil to mean “pain drug”, when in actuality, these drugs contain two different pain‐killers, acetaminophen and ibuprofen, respectively, and these work in different ways in the body. Some people refer to any pain killer as simply aspirin, while aspirin itself is a generic name for Bayer. This is most likely due to aspirin being the only pain drug available for many years. In this lab you will be examining a number of pain killers and determining which are components in common brand name drugs using a technique called thin‐layer chromatography, or TLC. You will need to be familiar with some terms prior to the lab:
Polarity—electronegative atoms (oxygen, nitrogen, chlorine, and fluorine) when bound to a non‐ electronegative atom (carbon and hydrogen usually) create a polar bond, due to the electronegative atom “stealing” electrons from the other. Polarity can be in terms of “yes” and “no”, but also in terms of magnitude. Water, for example, is more polar than ammonia, but both are polar. Methane is not polar because it has no electronegative atoms. Oxygen gas (O2) isn’t polar because there are no non‐electronegative atoms bound to it. Solvent—a solvent is a liquid that you can dissolve a chemical in. If the chemical is not soluble in the liquid, it can’t be a solvent. Water, for example, is a good solvent for both sugar and salt, because they dissolve completely in it. This follows the “like dissolves like” rule, regarding polarity and solubility. Solute—a chemical that is being dissolved in a solvent is the solute. In the water‐sugar example, sugar is the solute. Solution—a solute dissolved in a solvent creates a solution. Most often this is a liquid with other chemicals dissolved in it. In the water‐sugar example, the water with the dissolved sugar is the solution. Thin layer chromatography (TLC)—a method chemists use to separate one chemical from another using their differences in polarity. There are several components to TLC: o Stationary phase—In order to separate chemicals, we have to have a platform on which to separate them. In our case, the platform is a thin sheet of the polar chemical silica attached to a plastic support. The silica does not move in the process of chromatography, therefore, we call it stationary. o Mobile phase—A mobile phase is the solvent that carries the chemicals through the stationary phase. A small spot of chemicals won’t move on silica alone, but if we add a solvent, it can draw the chemicals up through the paper using capillary action.
Revision S10 Page 1 of 7
Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY o o
o
Point of origin—the spot where you put your chemicals on the stationary phase. Developing—after putting chemicals on the point of origin, the chromatogram can be “developed”, that is, the mobile phase can be pulled through the stationary phase to separate the chemicals. Retention factor (Rf)—The distance our chemicals move during chromatography is typically less than the distance the mobile phase moves. We can measure the difference in distance by using the Rf value. This is the distance a chemical moves on our silica (stationary phase) during our separation divided by the distance the mobile phase moves. If I use water as a mobile phase, sugar as my solute, and silica as my stationary phase, my Rf value will always be the same in this system. Changing the mobile phase to alcohol will change the Rf for the sugar, because sugar has a different affinity for alcohol than it does water. The following formula is used to determine Rf:
TLC works off of affinities. If a chemical has a higher affinity for the silica plate and a low affinity for the solvent (which would happen if the compound is quite polar) it will stick to the plate (it wouldn’t migrate far). If the chemical has a low affinity for the plate and a high affinity for the solvent (which would happen if it is quite non‐polar), it will fly up the plate with the solvent, because it “hates” being in contact with the polar silica, but is “happy” being in contact with the solvent. Analgesic—a pain killing drug. This could be aspirin. This could be morphine. All pain killers are considered analgesics. Antipyretic—a drug that reduces a fever. H Anti‐inflammatory—a drug that reduces swelling. CH 3 N Tylenol™‐‐generic name is acetaminophen, chemical name is N‐ O HO (4‐hydroxyphenyl) acetamide. Acetaminophen is both an analgesic and an antipyretic. OH Bayer™‐‐generic name is aspirin, chemical name is 2‐acetoxybenzoic O acid. Aspirin acts as an analgesic, antipyretic, and an anti‐ O CH 3 inflammatory, which makes it a very good overall pain reliever for many ailments. Aspirin also has the ability to prevent blood from clotting, which is why it is so widely used in people prone to heart O disease. Advil™‐‐generic name is ibuprofen, chemical name is CH3 H2 C 2‐(4‐isobutylphenyl)propanoic acid. Works mostly as HC OH C O
CH3 H C
Revision S10 Page 2 of 7 CH3
Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY an anti‐inflammatory drug, but acts as an analgesic when pain is associated with swelling.
Caffeine—not sold as is, but a component of pills. Not an analgesic, but a stimulant and diuretic (makes you urinate). Can help a body absorb other drugs more quickly; it is commonly found with analgesics to provide faster pain relief.
Equipment: Beaker silica plates aspirin soln watch glass Solvent* Acetaminophen soln ibuprofen soln caffeine soln filter paper UV lamp Unknown drugs soln ruler capillaries (*Solvent is 25:1:1 ethyl acetate: ethanol: acetic acid) Procedure: 1. You will start this experiment by determining the chromatographic properties of the drugs in their pure form. Obtain a TLC plate and very gently draw a line about 1 cm from the bottom of the plate using a dull pencil on the side that has the silica. If you push too hard, you will scratch off the silica. DON’T use ink! On the line, and 4 small lines as shown in Figure 1, and label the lines T (for acetaminophen, Tylenol), A (for aspirin), I (for ibuprofen), and C (for caffeine). 2. Touch a clean capillary tube to the acetaminophen solution to draw up about 1‐2 cm of solution. Being careful to not disturb the silica, touch the capillary to the silica on the T line. You may have to jiggle it slightly to get the solution to come out onto the silica. YOU WANT TO GET AS SMALL OF A SPOT AS POSSIBLE! When a small amount of solution comes out, remove the capillary and let the spot dry. Continue until all of the solution in the capillary is gone. Repeat the process using clean capillaries each time for the remaining 3 solutions.
Revision S10 Page 3 of 7
Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY 3. Add the developing solvent to a depth of about 0.5 cm to the beaker, and add a piece of filter paper to the side of the beaker, swirling the solvent to get the filter moist. When all 4 spots on the TLC have dried, place the, spots down, in the beaker. You will want to lean it slightly to get better results (see Figure 2). Put the watch glass on top of the beaker to seal in the solvent. 4. Allow the solvent to reach within a half cm to the top of the plate and pull it out of the beaker. Immediately mark with a line the spot where the solvent stopped. This is called the solvent front and you will need it for measurement. Lay the TLC plate with silica side up until it dries. 5. When the plate is dry, you can visualize the spots. The chemicals are present in very small quantities, and are not colored, however, they absorb UV light. The silica contains an indicator dye that glows green under UV light. This means when you look at your plate under UV light it will glow green except where your spots moved to. Look at your plate under the UV light, being careful around the UV lamp. You don’t want to get a sunburn, or do damage to your eyes by looking at it (both of which are possible). Where you see spots, lightly circle with a pencil. Set aside your plate for measurements at the end of the experiment. 6. Mark a new TLC plate for 3 new samples. Take the three mixed pain relievers and spot each of them onto the plate. Develop as you did the previous examples. Visualize under UV light, and mark what spots you see. You will see more than one spot per sample. 7. Fill in the data sheet and answer the remaining questions.
Revision S10 Page 4 of 7
Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY DATA SHEET and POST LAB QUESTIONS Name:__________________________________ Partner: ________________________________ 1. Sketch your TLC plates below: Knowns: Unknowns: 2. Measure the distance between your two drawn lines in mm. This is the distance your mobile phase traveled. a. Knowns: ____________mm b. Unknowns: ____________mm 3. Measure the distance the spots moved in mm, measuring from the bottom line (where you initially spotted them) to the middle of where they ended up. Use this value to determine the Rf values for each. a. Knowns: i. Acetaminophen (T) ________________mm Rf=____________ ii. Aspirin (A) ________________mm Rf=____________ iii. Ibuprofen (I) ________________mm Rf=____________ iv. Caffeine (C) ________________mm Rf=____________ b. Unknowns: i. Excedrin Migraine ________________mm Rf=____________ ________________mm Rf=____________ ________________mm Rf=____________
Revision S10 Page 5 of 7
Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY ii. Excedrin Tension ________________mm ________________mm iii. Excedrin Back and Body ________________mm ________________mm
Rf=____________ Rf=____________
Rf=____________ Rf=____________
4. Comparing your Rf values, which drugs are in your unknowns? a. Excedrin Migrane: b. Excedrin Tension c. Excedrin Back and Body 5. How did your anticipated polarities compare to what you actually saw in this experiment? ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 6. If two components have an identical Rf value, does this mean they have to have the same structure? Explain. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 7. Where would the spots have appeared, relative to what you found, if you used a more polar solvent? Explain. ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________
Revision S10 Page 6 of 7
Austin Peay State University Department of Chemistry
CHEM 1021
ANALYSIS OF DRUGS IN PAIN‐RELIEVERS BY THIN‐LAYER CHROMATOGRAPHY PRE‐LAB ASSIGNMENT Name: ____________________________________ Date: _____________________________________ 1. If you try and chromatograph a very polar compound using TLC, would you expect the compound to move a short distance or a far distance? Why? 2. I want you to form a hypothesis regarding the separation of the chemicals. Draw a plate below that outlines what you think the chromatograph will look like for the 4 single compounds. Look at the provided structures and determine what you think is the relative polarity of the compounds, and place them on the plate accordingly. Explain why you picked the order of chemicals that you did regarding polarity. 3. You have a chromatograph that has two spots on it, spot 1 moved 15 mm from the origin and spot 2 moved 35 mm from the origin. The solvent moved a total of 57 mm. What are the Rf values for the two chemicals? Which is more polar?
Revision S10 Page 7 of 7
