CHEMICAL SEPARATION EXPERIMENT 2 INTRODUCTION The term analysis in chemistry usually refer to the quantitative and qualitative determination of the components of a sample. Qualitative refering to the identity of each component, quantitative to the amount. Prior to both determinations, a separation of the components usually has to be done. There are several methods of separation, one of the most widely used is called chromatography. A typical chromatographic system would consist in a tube filled with a fine powder (like silica gel), through which the sample to separate is carried by a solvent. In chromatographic terminology, the tube is called a column, the powder stationary phase, the progression of the sample components elution, and the solvent mobile phase or vector phase. Separation occurs because the components elute through the column at different rate, depending on their affinity for the stationary phase versus their affinity for the mobile phase. The stronger the affinity for the stationary phase, the slower the progression of the component. The affinity depends on the physical and chemical properties of the individual components. THEORY Thin layer chromatography (TLC) is one type of chromatography where the stationary phase is a thin layer of adsorbent particles deposited on a solid plate. In this experiment, the plate is made of polyester coated with a 0.250 mm thick layer of silica gel A small amount of the sample to separate is spotted (applied) on a line drawn near the bottom of the plate. The plate is then placed standing in a developping chamber (Figure 1), containing a smal amount of the mobile phase, to wet only a few mm of the plate, below the line. The solvent is drawn up by capillary action. A good separation is achieved when each component interacts differently with the mobile or stationary phase, creating individual bands (spots) on the plate. Figure 1 Developing chamber (beaker covered with aluminum foil), containing a TLC plate and a small amount of solvent. 1
The retention time or retention factor (Rf) is used to characterize and compare components of various samples. Rf = distance from origin to spot distance from origin to solvent front The first of two goals in this lab is to determine the content of an unknown solution, which contains one or more of the molecules shown in Figure 2. Those are currently sold as over-the-counter medicines or energy drink. The second goal is to determine the composition of an unknown mixture of food dyes. The unknown may contain one or more of the molecules shown in Figure 3. Acetylsalicylic acid Acetaminophen Ibuprofen Caffeine Pseudoephedrine hydrochloride Figure 2 Candidate over-the-counter medicine compounds. 2
Erythrosine B (red) Brilliant blue Fast green Sunset yellow Tartrazine (yellow) Figure 3 Candidate commonly used food dyes. Intermolecular forces are the forces of attraction that exist between molecules. They are responsible for what we ve called so far affinity. Polarity is frequently the key factor in chromatographic resolution (separation). The affinity of a compound with the mobile phase vs stationary phase, depends on its polarity. Generally: polar compounds have strong affinities for polar compounds, nonpolar compounds have strong affinities for nonpolar compounds, polar compounds have weak affinities for nonpolar compounds. A simple (and very limited) way to put it would be: alike molecules have stronger affinities with one another than unlike ones. During elution, as the solvent progresses along the TLC plate, molecules of analytes (in this experiment, analytes will be drugs or dyes) are carried by the solvent. Along the way, they are constantly in contact with the highly polar silica (stationary phase). If the analyte is a highly polar compound, the forces of attraction (affinity) may be so strong that the analyte will not move (elute) at all. To make it move, one would have to use a more polar solvent. A polar solvent, having stronger affinity with the analyte, would therefore compete with the stationary phase. In the extreme situation where the affinity for the solvent is much stronger than for the stationary phase, the analyte would travel at the same rate as the solvent front the stationary phase not being able to retain the analyte at all. Finding a solvent or mixture of solvents that serves as an effective mobile phase is the most 3
difficult part of TLC. Often, several different combinations of solvents are tested before one is found that will separate the compounds of interest successfully. Obviously the task gets more complex with the increasing number of compounds to separate. This is where the TLC method find its limit of use. You will use TLC to separate food dyes, using a 5:1 mixture of isopropanol and ammonia for mobile phase. You will also use TLC to identify the active ingredient in an over-the-counter drug mixture, using a 4:2:1 mixture of hexane, ethyl acetate and ethanol as mobile phase. The identity of the compounds in both unknown samples will be verified by comparing their TLC Rf values, with those of authentic samples (standards). The different drugs shown in table 2 will not be visible after the plate has been developed. Although, the spots will become visible when viewed under short-wave ultraviolet light. APPARATUS AND CHEMICALS Apparatus 2 400 ml beakers 3 small tests tubes 1 50mL beaker 1 test tube holder 2 125 ml erlenmeyer 1 diamond sand cutting rod 2 TLC plates (5cm x 10cm) 1 spatula 4 long pasteur pipets 1 dropper bulb 2 short pasteur pipets 1 large petri dish 1 Bunsen burner aluminum foil 1 ignitor cotton 1 plastic funnel + tubing (mini fumehood) Kimwipes 1 thermometer transparent tape Chemicals Ethanol Isopropanol/ammonia 5:1 solution Erythrosine B red solution Brilliant blue solution Fast green solution Sunset yellow solution Tartrazine solution Hexane/ethyl acetate/ethanol 10:5:1 solution Tylenol (acetaminophen) Aspirin (acetylsalicylic acid) Advil (ibuprofen) Red Bull (caffeine) Sudafed (pseudoephedrine hydrochloride) 4
PROCEDURE Preparation of capillaries You will first use the flame of a Bunsen burner to soften a long pasteur pipet. When it is soft enough, take it away from the flame and immediately stretch it about one meter long. You will see the pipet transform into an extra long glass tube of capillary size. Break it into short pieces of about 5cm. Use the sanding disc to make clear and straight cuts. Keep only the capillaries with an outside diameter smaller than 0.9 mm. Prepare 15 of those capillaries per two students, keep them in a 50 ml beaker. Whenever a flame is used in the lab, everyone should pay special attention to all of their actions. To minimize the risk for accidents, burners will be used only in the first 30 minutes of the lab period. Plan your procedure accordingly. Separation of dyes Developing chamber. Prepare one for two students. Label a 400 ml beaker as 5:1 isopropanol/ammonia. Transfer 10 ml of that developing solvent (from a buret or solvent dispenser) into the beaker and immediately cover with a piece of aluminum foil. Verify that the solvent covers the bottom of the beaker to a depth of about 0.5 cm. The level of the liquid must be below the line when the plate is placed in the beaker. Preparation of the TLC plate for dyes. Prepare one for two students. Always wear gloves to manipulate undeveloped TLC plates. On one of the TLC plate, draw a line in pencil, not pen, 1 cm from the bottom along the short (5 cm) side of the plate. Below the line, going from left to right, write U1 E S B F T U2. Evenly space the labels, using the full width of the plate. Be careful not to disturb (contaminate) the silica gel as you write on the plate. Light pencil line 1cm U1 E S B F T U2 Mark names in pencil Figure 4 TLC plate before spotting 5
Spotting. Use the capillary tubes (one per dye) to spot each of the 5 dyes and the two unknowns, along the line drawn on the plate. Since two students are sharing the same TLC plate, U1 will be for the unknown of student #1 and U2 for the unknown of student #2. Make sure to record who is U1 and who s U2. To spot the plate, immerse the end of the capillary into the Erythrosine B red solution until some of it is drawn into the tube. Then, quickly and lightly touch the capillary to the surface of the plate (over the A label) so the spot is very small (diameter 2 mm). This is very important, to obtain a good resolution. The spot will be highly colored since the food dye solution is quite concentrated. Repeat the procedure, to respectively spot the Sunset yellow, Brilliant blue, Fast green, Tartrazine, and the Unknown solutions. Developing TLC. Place the plate in the beaker, cover it with the foil and allow the solvent front to move up the plate until it reaches approximately 6.5 cm from the drawn line (one hour maximum). Do not move or shock the beaker while the chromatogram is developing! Remove the plate and mark the solvent front with a pencil. Allow the plate to dry for 10-15 minutes under a mini-fumehood. Then circle any visible spots with a pencil. Measure the distance from the origin to the center of each spot and the distance from the origin to the solvent front. Report the values and colors of the spots in table 2. Separation of drugs Developing chamber. Prepare one for two students. Label the second 400 ml beaker as 10:5:1 hexane/ethyl acetate/ethanol. Transfer 10 ml of that developing solvent (from a buret or solvent dispenser) into the beaker and immediately cover with a piece of aluminum foil. Verify that the solvent covers the bottom of the beaker to a depth of about 0.5 cm. Preparation of the unknown drug solution. Prepare a hot water bath, using a hot plate and a large Petri dish half filled with tap water. Transfer approximately 50mL of ethanol into a 125mL Erlenmeyer. Cap it loosely with a piece of aluminum foil, and place it in the hot bath. Monitor the temperature by inserting a thermometer directly in the ethanol, make sure it doesn t go over 50 C. Push a small wad of cotton through the top of a short pasteur pipet so that is forms a plug at the point where the pipet narrows. Obtain a sample of unknown from your demonstrator. Put it directly into the plugged pipet. Fill the pipet with hot ethanol (50 C) to dissolve some of the drugs mixture. Then use the bulb to force the solution out, through the cotton filter, into a small test tube. Cap the tube and label it. 6
Preparation of the TLC plate for drugs. Prepare one for two students. On a second TLC plate, draw a line in pencil, 1 cm from the bottom along the short (5 cm) side of the plate. Below the line, going from left to right, write U1 AS AD TY RB PSE U2. Evenly space the labels, using the full width of the plate. Be careful not to disturb the silica gel as you write on the plate. Spotting. Use capillary tubes (one per drug) to spot each of the 5 solutions and the two unknowns, along the line drawn on the plate. Drug solutions were prepared from commercial tablets, using the same procedure you followed to prepare your solution of unknown. Because the drug solutions are more diluted than the dye solutions, it will be necessary to touch the plate several times on the same spot. Always allow the spot to dry in between applications. This operation will be completed much faster if your preparation is still warm. Developing TLC. Place the plate in the beaker, cover it with the foil and allow the solvent to rise to within 1 cm of the top of the plate. In this case, the solvent will travel up the silica gel plate much more quickly and could reach the top within a few minutes. Remove the plate and mark the solvent front with a pencil. Allow the plate to dry for 10 minutes under a mini-fumehood. Then observe it under short-wave ultraviolet light. With a pencil, circle any dark spots. Measure the distance from the origin to the center of each spot and the distance from the origin to the solvent front. Report values in table 3. Wastes The eluting solvents and drug extracts go into the Organic Waste Container. Capillaries go in the large beaker next to the liquid waste containers. DATA ANALYSIS Attach one TLC plate in the appropriate space of table 1 (use large bands of the provided transparent tape). Reproduce (sketch) the other TLC plate in the proper space of Table 1. Calculate the Rf of each spot on the developped TLC, report values in table 4 and 5. Identify the components present in the unknowns, by matching the Rfs of the unknowns with the standards. 7
Data sheet, hand in before leaving the laboratory Chemical Separation Experiment 2 - Data Name First Last McGill ID number Demonstrator Section Date Day Time Table 1 TLC of the food dyes and drug solutions TLC of the food dyes TLC of the drugs Attach TLC here Attach TLC here 8
Data sheet, hand in before leaving the laboratory Table 2 Elution of food dyes Spot # Dye Elution distance (cm) Spot color 1 2 3 4 5 Unknown code # Solvent front (cm): Table 3 Elution of drug solutions Spot # Drug compound Elution distance (cm) 1 2 3 4 5 Unknown code # Solvent front (cm): 9
Lab report, hand in within 24 hours Chemical Separation Experiment 2 - Lab Report Name First Last McGill ID number Demonstrator Section Date Day Time Table 4 Resolution of the dyes Spot # Dye name Rf Spot color 1 2 3 4 5 6 Unknown Code # : 10
Lab report, hand in within 24 hours Table 5 - Resolution of the drugs Spot # Drug name Rf 1 2 3 4 5 Unknown Code # : QUESTIONS 1. Explain how your observations in table 4 and table 5 led to the identification of the components of both unknowns. 11
Lab report, hand in within 24 hours 2. Discuss the relationship between the polarity of molecules and the Rf values. 3. Which one of the two mobile phases is the more polar? Support your answer. 12
Questions and problems 1. What are the stationary phase and the mobile phases involved in this experiment? 2. What is the purpose of the small wad of cotton in the pipette? 3. Why is a pencil (not a pen) used to mark the position of the spots? 4. Why is an Rf value rather than the distance the spot moved used to help identify a substance by TLC? Answers 1) Stationary phase : silica gel, mobile phases : 5:1 isopropanol:ammonia for dyes, 4:2:1 hexane:ethyl acetate:ethanol for drugs. 2) It acts as a filter, to remove the undissolved granules of drug. 3) Inks are dyes, they could elute and interfere with the compounds of interest. 4) Rf values are independent of the distance traveled by the solvent, it allows to compare values from different chromatograms, even when the solvent front positions (elution times) are different. 13