Lab: Analysis of Analgesics

Purpose: To analyze over the counter analgesics using thin-layer chromatography. Background: Thin-Layer Chromatography Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. We'll look at the reasons for this further down the page. Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of solvents. The compounds are spotted on a pencil line (1 cm from the bottom of the plate) and are placed in a chamber with a solvent. As the solvent slowly travels up the plate, the different components of the dye mixture or the drug compound will travel at different rates and the mixture is separated into different colored spots. Measuring Rf values Measurements are often taken from the plate in order to help identify the compounds present. These measurements are the distance travelled by the solvent (marked when the plate is removed from the beaker), and the distance travelled by individual spots. These measurements are then taken The Rf value for each dye is then worked out using the formula: For example, if the red component travelled 1.7 cm from the base line while the solvent had travelled

5.0 cm, then the Rf value for the red dye is: (see calculation to the right) If you could repeat this experiment under exactly the same conditions (same solvent), then the Rf values for each dye or compound would always be the same. For example, the Rf value for the red dye would always be 0.34. What if the substances you are interested in are colorless? As referenced in the background, the stationary phase on a thin layer plate often has a substance added to it which will fluoresce when exposed to UV light. That means that if you shine UV light on it, it will glow. That glow is masked at the position where the spots are on the final chromatogram - even if those spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow apart from where the spots are. The spots show up as darker patches. While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again. How does thin layer chromatography work? Silica gel is a form of silicon dioxide (silica) and is the stationary phase. The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups. The surface of the silica gel is very polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it as well as van der Waals dispersion forces and dipole-dipole attractions. What separates the compounds as a chromatogram develops? As the solvent begins to soak up the plate, it first dissolves the compounds in the spot that you have put on the base line. The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards. How fast the compounds get carried up the plate depends on two things:

How soluble the compound is in the solvent. This will depend on how much attraction there is between the molecules of the compound and those of the solvent. How much the compound sticks to the stationary phase - the silica get, for example. This will depend on how much attraction there is between the molecules of the compound and the silica gel.

Suppose the original spot contained two compounds - one of which can form hydrogen bonds, and one of which can only take part in weaker van der Waals interactions. The one which can hydrogen bond will stick to the surface of the silica gel more firmly than the other one. We say that one is adsorbed more strongly than the other. Adsorption is the name given to one substance forming some sort of bonds to the surface of another one. Adsorption isn't permanent - there is a constant movement of a molecule between being adsorbed onto the silica gel surface and going back into solution in the solvent. Obviously the compound can only travel up the plate during the time that it is dissolved in the solvent. While it is adsorbed

on the silica gel, it is temporarily stopped - the solvent is moving on without it. That means that the more strongly a compound is adsorbed, the less distance it can travel up the plate. In the example we started with, the compound which can hydrogen bond will adsorb more strongly than the one dependent on van der Waals interactions, and so won't travel so far up the plate. Introduction: Answer the following questions in paragraph form. Remember to cite all of your sources. What do the following terms mean (or what do they do to a body): analgesic agent, antipyretic agent anti-inflammatory agent? Look up the following over the counter medications and find out what active ingredients each contains. Table I: Composition of Some Over-the Counter Analgesics (mg/tablet) Product Alka-Seltzer Ibuprofen Bayer Aspirin Excedrin Excedrin (E.S.) Tylenol Tylenol (E.S.) Provide the structure of aspirin, acetaminophen, and caffeine. List the factors that influence the Rf of a given compound. Explain why you will use a UV light to see the analgesics on the TLC plate? Why must ink be avoided in marking TLC plates? Two compounds have identical Rf values under identical conditions. Does that mean that they have identical structures? Explain. Look up the MSDS for the chemicals being used in this lab and make a list of safety precautions for YOUR handling of them in lab. Active Ingredient Aspirin Acetaminophen Caffeine Other

Materials: In each bin (need to be returned to the bin): o Ruler o Pencil o TLC plates o 3 pint sized jars with lids o 4 -10mL beakers o Unknown sample o Capillary tubes (put in the glass disposal immediately after use!) o Ethyl acetate o Ethanol o Hexane o Acetone

Shared materials (use and return to the same location): o Chromatography solvent o Ibuprofen o Tylenol o Excedrin o Aspirin o UV light In the fume hood: o Waste beakers for solvents

Procedure: 1. Pulverize a tablet of Excedrin, Tylenol, Aspirin and Ibuprofen and an unknown using a clean mortar and pestle. Use a little acetone and a paper towel (good luck finding them!) to clean the mortar and pestle between uses. You may share some of your pulverized tablet with another group to minimize waste. 2. Transfer a small pea sized amount of Excedrin to a 10mL beaker. 3. Add 2 mL of acetone to each test tube and stir with a clean stirring rod. Do not be concerned. All of the solid will not dissolve.

4. Obtain 2 TLC plates and draw a very light line with a pencil approximately 1.5 cm from the
bottom of the plate. Make 3 evenly spaced lines along the pencil line to mark where you will be spotting the samples. It should look like:

5. Place the end of a new capillary tube in the solution (you will see the liquid rise in the tube) 6. Lightly press the end of the capillary tube on the first intersection on the TLC plate quickly. 7. Allow the spot to dry completely. 8. Repeat steps 6-7 two more time in the SAME place on the plate. 9. Repeat steps 3-7 for Tylenol, Aspirin and Ibuprofen and an unknown sample. (DO NOT
CONTAMINATE SAMPLES use a new capillary tube each time). Discard the capillary tube into the glass recycle immediately after use for each sample! 10. Make a total of 3 SETS of identical plates (repeat steps 1-9) 11. Place enough solvent (see below) to coat the bottom of the jar. The solvent CANNOT be above the pencil line. a. Mason Jar 1 ethyl acetate b. Mason Jar 2 ethanol c. Mason Jar 3 hexane 12. Place each set of plates in the beaker with the pencil line down.

a. SMART IDEA: You do not want the two silica sides to face each other. If one falls over and the two silica sides touch you will have contamination between plates. 13. Screw the lid on the mason jars. 14. Allow the plates to develop until the solvent is close to the top (1 cm). 15. Take the TLC plate out of the solvent and MARK the solvent line with a pencil. 16. Allow the plate to dry. 17. Take your plate to the UV light (CAUTION: DO NOT STARE AT THE UV LIGHT! It should remain parallel with the table surface at all times), turn on the light and shine over the plate. 18. Using a pencil, circle the dark spots on the plate. 19. Calculate and record the Rf values for the spots. 20. Repeat steps 1-10 in the chromatography solvent and then try a mix of solvents. Results: Draw the TLC plates and record which sample you spotted in each row (OR Take a picture and tape it in your lab notebook!) Using a table format, record Rf values for all 5 samples (Knowing the composition for each analgesic, identify the components. Indicate any colors observed)

Analysis: 1. In one paragraph describe how compounds separate. (You may want to explain mobile and stationary phase) 2. Speculate as to the identity of the insoluble solid in step 3. 3. Relate the polarity of the solvent with the quality of the compound separation. Which solvent is the best? Which is the worst? Why? 4. The course of a chemical reaction (A + B C) was followed by TLC analysis at different times after the reaction was initiated. The TLC plates of the reaction mixture at time zero, and after 15 and 30 minutes of reaction, run under identical conditions, are shown below.

The Rf values of pure sample of A and B (under identical conditions) are 0.2 and 0.8, respectively. (a) Calculate the Rf value for all spots. (b) Identify the spots and explain the TLC results. (c) What compound was the limiting reagent in the preparation of C? answer.

Briefly justify your

5. Often drugs are analyzed using TLC on silica gel with three different solvents: Solvent 1: chloroform/acetone/diethylamine (5:4:1) Solvent 2: chloroform/diethylamine (9:1) Solvent 3: cyclohexane/chloroform/diethylamine (5:4:1) The Rf for different drugs are given in the table below.2-3 Alkaloid morphine quinine codeine cocaine strychnine aconitine reserpine Solvent 1 0.10 0.19 0.38 0.73 0.53 0.68 0.72 Solvent 2 0.08 0.26 0.53 0.90 0.76 0.90 0.80 Solvent 3 0.00 0.07 0.16 0.65 0.28 0.35 0.20

You are running a forensic laboratory. Which solvent would you chose to analyze: (a) A sample seized by the police in a drug smuggling case and suspected to contain morphine, cocaine, and aconitine? (b) A mixture of cocaine and reserpine? (c) A poison containing strychnine and reserpine? 6. What are some sources of error? Be specific! 7. What is the identity of the unknown? Use evidence as support. Chemical Disposal

The ethyl acetate layers must be put in an organic waste container in the fume hood. The hexane must be put in a separate organic waste container in the fume hood. The ethanol can be washed down the drain. Any solid waste should be discarded in the trash. References: This lab has been modified and adapted from: http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html (Thin Layer Chromatography Notes) www.newberry.edu/academics/documents/AnalgesicslabPart1.docx http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html o Adapted from:

Palleros, D. R. Experimental Organic Chemistry John Wiley & Sons, Inc., New York, 2000. Randerath, K. Thin Layer Chromatography Verlag Chemie, Weinheim, 1965. Waldi, D.; Schnackerz, K.; Munter, F. J. Chromatogr. 6, 61, 1961.