Isolation of Caffeine from Tea and Identification of an unknown analgesic. Two weeks Extraction, Distillation, Melting point, FTIR, and TLC Numerous purification methods are available to the organic chemist, including recrystallization, extraction, distillation, column chromatography, and sublimation. Each technique has advantages and disadvantages depending on the reagents involved and the desired results. Extraction, one of the most useful separation (purification) techniques in organic chemistry, is based on the simple concept that "like dissolves like." Typically, extraction describes the removal of a desired compound from a solid or a liquid by a solvent. For example, in a coffee or tea pot, caffeine and other compounds are extracted from the grounds or leaves with hot water, this constitutes a solid/liquid extraction. In this experiment you will use both melting point determination and thin layer chromatography (TLC) to determine the purity of your caffeine and to identify an unknown compound. Caffeine is classified as an alkaloid . Alkaloids are naturally-occurring molecules that contain a basic nitrogen. They have some level of structural complexity (a very subjective term) that normally involves rings or a number of stereocenters..
Compounds found in over-the-counter analgesics. analgesics.
Other alkaloids like caffeine.
Like both cocaine and morphine, caffeine can be isolated from plants. A major hurdle when trying to isolate a single compound from plant material is that the plant contains a large number of other compounds. Effective removal or extraction of the desired compound must exploit the different properties of each component. Properties to examine include polarity and acidity/basicity. In the extraction of caffeine from tea, we will need to consider both polarity and acidity. Our extraction begins by extracting regular tea bags with hot, basic water. This process of ‘steeping’ removes all the water soluble components of tea. In addition to caffeine, we will remove tannins and small amounts of chlorophyll. Tannins are acidic and will be deprotonated under the basic conditions. The conjugate bases of tannins are anions. Tannins can also decompose in base. The decomposition
products are also anions. So, at this point, our water solution contains caffeine, tannins in the form of anions, and small amounts of chlorophyll. Since caffeine is soluble in organic solvents and the tannin anions are not, we can extract the caffeine into an organic solvent (dichloromethane) and leave the tannin anions behind in the basic aqueous layer. Evaporating the organic solvent will isolate caffeine. Caffeine is a colorless solid, but our caffeine may have a greenish hue. This is due to small amounts of chlorophyll that will also be extracted into the organic solvent. The key step in this entire procedure is the extraction. Extractions can have many forms, and the most common is the liquid-liquid extraction. The goal of this experiment is to extract caffeine, a nitrogencontaining natural product, from tea leaves (about 2-3 % caffeine by weight) using solid/liquid and liquid/liquid extractions. A solid/liquid extraction will be used to extract caffeine from tea leaves. In the liquid-liquid extraction, a solution of dichloromethane (CH 2Cl2) will be used as the liquid phase of the extraction. Ideally, any other compounds present in the original solution will remain in the aqueous layer, and pure caffeine will be isolated from the organic layer. However, other compounds may also be soluble in the organic (CH 2Cl2) layer resulting in an emulsion and/or recovery of impure caffeine after extraction. Often extractions are followed by additional purification methods, such as distillation (for liquids) and recrystallization (for solids). In all experiments, TLC and/or NMR data are used after an extraction to determine product identity and purity. For liquid-liquid extraction to work, the liquids cannot be soluble in one another. Our two liquids are water (aqueous layer) and dichloromethane (organic layer). When these two liquids are vigorously shaken together, compounds that are dissolved in each layer will have the opportunity to pass into the other liquid. If a compound is more soluble in water than dichloromethane, it will be primarily dissolved in the water layer at the end of the extraction. After the liquids have been mixed, they will begin to separate. This is analogous to Italian salad dressing that has been shaken – the seemingly homogeneous mixture, called an emulsion, slowly begins to separate back into its water and oil layers. Once the separation of water and dichloromethane is complete, the two layers can be physically separated. The components in one layer will then be separated from the components in the other layer. Generally, extractions are performed multiple times to ensure that the compound of interest has been completely removed.
Extraction of caffeine into dichloromethane from water The success of an extraction involving a natural product is often expressed according to the percent recovery. The percent recovery is essentially the percentage of the natural product recovered from the initial plant or animal mass involved in the extraction. The percent recovery can be calculated as follows percent re cov ery
=
grams of caffeine re cov ered x 100 grams of tea leaves
Depending on whether or not the natural product has been further purified after the extraction, the percent recovery is called the "purified percent recovery" or the "crude percent recovery." To determine whether an extraction is efficient, the percent recovery is compared with the known composition of the natural product in the animal or plant mass. In addition, the efficiency of two extractions can be compared; typically, the extraction with the higher percent recovery (especially if it is a purified percent recovery) is considered the more successful (efficient) extraction. Experimental Place tea from four tea bags into a 400-mL Erlenmeyer flask. You will need to know the weight of the tea leaves used in the experiment. Record the brand of tea used. Add 4g of calcium carbonate and 100mL of water. Boil the solution for 15 minutes on a hotplate stirring every minute or so. Let the o mixture cool to around 50 C and then filter using vacuum filtration using Whatman no. 54 paper. Using two portions will help. Watch for a clogged filter and replace if necessary. Cool the filtrate to room temperature (add a few ice chips if necessary). Perform an extraction on the filtrate using 15mL of dichloromethane. See technique 8. Shaking the flask vigorously may cause an emulsion to form. (Why is this undesirable?) Be sure to properly vent your funnel. Once the layers have separated, drain the organic layer into a 50mL Erlenmeyer flask. If an emulsion layer exists, include the emulsion layer in the Erlenmeyer flask. . (Which layer, top or bottom, is the CH2Cl2 layer? What physical properties do you need to determine this?) Note that small amounts of water (dark liquid) that are accidentally removed will be taken care of later in the procedure. Cork the flask to prevent evaporation. Perform another extraction with a fresh 15mL of dichloromethane. Collect in the same 50mL Erlenmeyer flask. Wash the organic layer: Pour the tea solution out the TOP of the separatory funnel into a beaker and rinse with water. Pour the combined dichloromethane solution into the funnel and wash with 20mL of water. Drain the dichloromethane into a clean 50mL Erlenmeyer flask. Remove the aqueous layer from the separatory funnel. Repeat the wash process with an additional 15 mL of water. Dry the organic layer with anhydrous sodium sulfate or magnesium sulfate . (How much should be added? Which is best to use? How will you know when your solution is dry? See technique 8.7) Some of the drying agent will clump up, and the rest should remain as a fine powder. If there is no fine powder, add a little more drying agent and swirl again. Keep adding more until the drying agent does not completely clump up. Decant the organic layer into a round-bottomed flask (What size?). Eventually, you will need to calculate the weight of the crude, dry caffeine in the flask after the solvent is removed. (To get an accurate weight, what information do you need about your round-bottomed flask before you decant the CH2Cl2?) Do not fill the flask more than halfway. Avoid getting any drying agent into the roundbottomed flask--consider what glassware, pipets, or other techniques might be helpful in this. Rinse the Erlenmeyer with 5 mL of fresh CH 2Cl2 and add it to the round bottom. Setup a simple distillation to remove most of the solvent. Use a sand bath. DO NOT distill the roundbottom flask to complete dryness. Remove from the heat when any solid film appears. Allow the flask to air dry before taking a crude weight or preparing your melting point sample. Compare to the know melting point for caffeine. Take a TLC of your caffeine sample using acetone as the developing solvent (See technique 15). The caffeine standard solution will be in your hood. From these data, you can identify whether caffeine is in your sample and whether it is pure.
Melting Point
An organic compound’s melting point is one of several physical properties by which it can be identified. A physical property is intrinsic to a compound when it is pure. Since melting points are relatively easy and inexpensive to determine, they are handy identification tools to the organic chemist. If you want to use the melting point to identify a solid compound which you have isolated in the lab, you will need to compare the experimentally determined melting point with that of the true compound. Melting points are listed in various sources of scientific data and should be recorded in your “table of physical properties” in your notebook. If you look up the melting point of a compound in more than one source, you may find that the reported values differ slightly. The melting point ranges listed in company catalogues (Acros, Baker, SigmaAldrich, etc.) are the melting points of the compounds as they are sold. If the compounds are sold slightly impure, the melting point range will reflect this fact. The melting point listed in the CRC, Merck Index, or on the MSDS is the melting point of the pure compound. While theoretically these should be constant from source to source, in reality the reported melting points sometimes vary. Therefore: Always record the source of the physical data which you write in your lab report. An impurity consisting of 5% total mass in a sample will lower the melting point from that of the pure compound, and it will increase the melting point range. There is not a "formula" to predict how much the melting point will be lowered, however, the greater the amount of the impurity, the greater the melting point depression. For example, a pure compound with a melting point of 110-111° could show a value of 103-107° if a 5% impurity were present. If a compound changes color upon melting, it is likely that it has undergone a chemical reaction, or decomposed. If the sample has decomposed, it is no longer the same compound and will have a different melting point. Therefore, simply cooling and re-melting the sample will not give you the melting point (or in this case the decomposition point) of the compound you originally intended to analyze. To be safe, start with a fresh sample of the compound and heat at a slower rate. If a compound "disappears" on melting, it is likely that it has sublimed , or went directly from the solid to the gas phase. The melting point of such a compound needs to be taken in a sealed capillary tube. How to use the Mel-Temp
Pack the capillary tube by pressing the open end gently into a sample of the compound to be analyzed. Crystals will stick in the open end of the tube. Tap the bottom (closed-end) of the capillary on a hard surface so that the crystals pack down into the bottom of the tube. The solid should fill the tube to a depth of 2-3 mm. When the crystals are packed into the bottom of the tube, place the tube into the slot behind the eyepiece on the Mel-Temp. Make sure the unit is plugged in, set to zero, and then turn it on.
The rate of temperature increase in the vicinity of the melting o point must be small, about 2 C per min. This insures that the temperature of the hot plate, thermometer, and sample will be in thermal equilibrium. Increase the temperature rapidly at first and then slowly as the melting point is approached in the following manner:
Thermometer
1. Set the power level to 5. 2. When the temperature is about 15 degrees below the anticipated melting point, change the setting to that indicated on the chart below.
For example, if the aniticpated melting point is 150 degrees C, when the thermometer reads "135" set the the control control
Observe the crystals with your eye about 4" from the lens to prevent accidentally touching the hot apparatus. Record the temperatures at which melting begins and at which the last crystal disappears, this is your melting point range. When you do not know the melting point of a compound, first take a crude melting point by heating rapidly. Then cool the apparatus to 20 degrees below the crude melting point, and proceed to take a more careful melting point on a second sample of the compound. When finished, turn off both the unit and the thermometer and place the used melting point tube in the used melting point capillary tube beaker in the hood .
Thin Layer Chromatography: Sample Prep: Obtain a vial of your unknown pain reliever. Place 0.01 g of your unknown compound (about half of what you receive in the vial—you do not need to weigh it) in 1 mL of ethanol. Crush an Excedrin® pain reliever tablet in a mortar and pestle until it is a fine powder, add 10 mL of ethanol and mix well. Use a disposable pipet to transfer to a storage bottle provided by your instructor. Dissolve a small amount (0.01g) of your caffeine in 1mL of ethanol in a sample bottle (these amounts can be estimated). Samples of other pain reliever standards will be provided Developing Solution: You should choose one mixture while your partner chooses the other mixture; one mixture is ethyl acetate–hexanes–acetic acid 80:20:1 and the other is ethyl acetate–hexanes–acetic acid 50:50:1. Caffeine TLC: Obtain a 5 x 10 cm silica gel TLC plate. Do not touch the faces of the plate as fingerprints will mark the plate, and interfere with the chromatography. Using a pencil draw a faint line 1.5 cm from the bottom of the plate. Mark four faint x’s on this line 1 cm from the edge and each other. Below the line label pure caffeine (PCA), sample caffeine (SCA), Excedrin® (EX), and Aspirin (AS). and spot using a microcap to spot the samples. Fill the microcap by dipping it into the liquid until the microcap fills nearly to the top. Touch the microcap to the TLC plate on the pencil line above each label and allow the liquid to flow onto the plate. Rinse the microcap with ethanol between each spotting.
For the “developing jar”, use a beaker covered with a watch glass. Partially line the beaker with filter paper and fill it with one of the eluting solvent mixtures to a depth of 0.5–1.0 cm. Place the plate in the developing jar to which you have already added your assigned developing solution. Allow the solvent to come within ~1 cm of the top of the plate then remove the plate from the jar and mark the solvent line. Allow the plate to dry in the hood and then place the plate under the UV lamp. Carefully circle all the spots you observe. Make notations as to which spots are more intense. Determine the Rf’s of the spots for each sample. Pain Reliever TLC: Prepare your TLC plate by drawing a light pencil line 1.5 cm from the bottom of the plate. Below the line write (in pencil) from left to right acetaminophen (AC), ibuprofen (IBU), then choose from Excedrin® (EX), Aspirin(AS), and Caffeine (CA), the one that you think would best help you identify your unknown. The last spot is for your unknown pain reliever.
AC IBU XXX
UNK
Use a microcap to spot the samples. Fill the microcap by dipping it into the liquid until the microcap fills nearly to the top. Touch the microcap to the TLC plate on the pencil line above each label and allow the liquid to flow onto the plate. Rinse the microcap with ethanol between each spotting. When all of the ethanol spotting solvent has evaporated, the TLC plate is ready to be “developed.” Place the TLC plate in the developing beaker, cover the beaker with a watch glass, and develop until the solvent reaches 1 cm from the top of the plate. Remove the plate from the beaker, mark the solvent front, air dry, and visualize the spots by placing the plate under an ultraviolet lamp. Carefully circle all the spots you observe. Make notations as to which spots are more intense.
Determine the melting point of your unknown solid using the Mel-Temp Melting Point Apparatus. Each member of the pair must take a melting point of the unknown compound. Be sure to write down the number of the unknown in your lab notebook. When you have completed the TLC and melting points of the unknown compound, find which other pair in your lab section has the same unknown. And record their unknown number and names in your lab book. Wastes
Organic Waste:The eluting solvents and tablet extracts go into the Organic Waste Beaker in the hood. Used disposable glassware:Used microcaps and melting point capillaries go in the glass waste receptacle. Used TLC plates: TLC plates go in the small trash receptacle labeled “used TLC plates” (in the hood). Used filter paper: Place in the solid organic waste beaker in the hood.
Discussion Items: - Comment on the purity of your caffeine. Consider the color, odor, MP, and TLC results. - Identify your unknown pain reliever based on the TLC results, the MP results and the ingredients listed on the bottle. Defend/explain your choice. Questions: 1. You think that you have isolated ibuprofen in the lab. Since you don't totally trust your own laboratory techniques, you want to prove to yourself that you have ibuprofen before you ingest it. Using only melting point techniques, explain how you can prove that you actually have ibuprofen. (Assume the stockroom is able to supply you with any compound you need.)
2. The CRC lists the melting point for a compound as 182–183°C. You observe a melting point for this same compound isolated in your experiment as 177–181°C.What can you conclude about the compound isolated in your experiment? 3. Why does the presence of an impurity lower the melting point of a compound? 4. What would happen if your solvent level is above the level of the initial spots when you place the TLC plate in the developing jar? 5. What could happen if you used ink to draw in your base line and letters on the TLC plate? 6. Consider the following silica gel TLC plate of compounds A, B, and C developed in hexanes:
a) Which compound, A, B, or C, is the most polar? b) What would you expect to happen to the R f values if you used acetone instead of hexanes as the eluting solvent? c) How would the R f values change if eluted with hexanes using an alumina TLC plate?
7. You are trying to determine a TLC solvent system which will separate the compounds X, Y, and Z. You ran the compounds on a TLC plate using hexanes/ethyl acetate 95:5 as the eluting solvent and obtained the chromatogram below. How could you change the solvent system to give better separation of these three compounds?
8. After a rather lengthy organic chemistry synthesis procedure, a student ran the product of the reaction on a TLC plate and obtained the result below. What might he/she have done wrong, if anything?
adapted from http://www.wooster.edu/chemistry/organic/211_Experiments/LabIII-Caffeine.html http://www.chm.davidson.edu/erstevens/S04_201_LB8_tea.pdf http://orgchem.colorado.edu/index.html
