Microsoft word - $asq1097195_file000002_14541668.doc

Supplemental information
Detection of Salicylic Acid in Willow Bark: An Addition to a Classic Series
of Experiments in the Introductory Organic Chemistry Laboratory
*Author to whom correspondence should be addressed ([email protected]) Student handout
Detection of Salicylic Acid in Willow Bark
The use of willow bark to treat pain and fever dates back thousands of years to the time of Hippocrates (~400 BC), who advised patients to chew the bark of a willow tree to relieve their symptoms. Native peoples around the world also used extracts or teas made from local species of willow tree for the same purposes for thousands of years. By the early 19th century, the science of chemistry had evolved to the point that chemists began to question and search for the chemical in willow bark that was responsible for its curative properties. In 1828 Johann Buchner, a professor of pharmacy in Munich, isolated a yellow crystalline compound from the bark of a willow tree which he called salicin (after the Latin name for willow, salicaceae) and which was responsible for the therapeutic properties of the willow tree. The extraction procedure was later improved by French chemist Henri Leroux, and in 1838 Raffaele Piria (an Italian chemist) split salicin into a sugar (glucose) and an aromatic molecule. It was subsequently found that the active component was the aromatic molecule, which was given the name salicylic acid. This molecule, however, caused significant irritation to the stomach and was thus modified to give acetylsalicylic acid (Aspirin®), which was first sold in 1899 and has since become the most widely used drug in the world. Willow bark, however, contains very little salicylic acid as the bulk of it exists in the form of derivatives (such as salicin) whereby the phenol or carboxylic acid functionalities of the molecule have reacted with other molecules to form esters. Esters can be cleaved by the action of acid, which in this case will yield free salicylic acid, and thus by simply heating willow bark in acid, salicylic acid can be extracted from the bark! You will analyze your willow bark extract for the presence of salicylic acid using the technique of thin layer chromatography. The compounds will be visualized in two ways: UV light, and by dipping the plate in a 1% ethanolic solution of ferric chloride. You should expect to see several compounds under UV light as the willow bark extract is impure; however, ferric chloride reacts selectively with phenols (one of which is present on salicylic acid) and so only some of the compounds in the willow bark extract should be apparent on the TLC using this method. Hazards:
Students employ 1.0 M hydrochloric acid in this experiment. Hydrochloric acid is corrosive and all spills should be treated with sodium bicarbonate. Gloves should be worn to limit the risk of skin contact when working with hydrochloric acid. Areas of accidental skin contact should be washed for at least 15 minutes with cool water; eyes should be flushed at an approved eyewash station for at least 15 minutes, and in both cases prompt medical attention should be sought. The organic solvents used in this experiment (ethyl acetate in the extraction and toluene, acetic acid, methanol, and diethyl ether in the TLC eluent) and all are potentially harmful. All work involving the TLC eluent should be done in a fumehood. In case of inhalation of any solvent, remove to fresh air and seek medical attention. Areas of skin or eye contact should be treated as described above for hydrochloric acid. Students allergic to aspirin should take additional safety precautions to limit their exposure to willow bark and the extract. Required Techniques:
Experimental Procedures:
We will have samples of willow twigs from at least one of five different species of willow (Salix fragilis, Salix amygdaloides, Salix tameline, Salix bebbiana, and Salix pentandra) available for students. All five species contain sufficient quantities of salicylic acid for this experiment. Peel the bark from the willow branches, making sure to collect both the hard outer bark and softer, green, inner bark. Do not worry if the bark is dirty or otherwise imperfect. Using scissors, cut
the bark strands into pieces ~1 cm long and place ~10-15 g of these bark pieces into a 100 mL round-
bottom flask. Add 50 mL of 1.0 M HCl to the flask and set up a reflux apparatus (CAUTION:
Hydrochloric acid is corrosive. Immediately report all spills to the instructor
). Reflux the
mixture for 30 minutes. The solution should change to a very deep red color.
After 30 minutes, cool the flask to room temperature (CAUTION: It is very important that the
flask and its contents be at room temperature or cooler before proceeding) and filter the solution
into a 125 mL separatory funnel using Whatman No. 4 filter paper. (NOTE: The filtrate may be
cloudy. Do not worry about this). Rinse the flask and bark with 10 mL of water and transfer this water
to the separatory funnel as well. The salicylic acid and other organic molecules are extracted from the
acid solution using ethyl acetate. Add ~10 mL of ethyl acetate to the separatory funnel, shake
(remember to vent!), allow the layers to separate, and then drain off the lower aqueous layer into a
clean 125 mL Erlenmeyer flask. Pour the organic layer out of the top of the separatory funnel into a
clean and dry 50 mL Erlenmeyer flask. Pour the aqueous layer back into the separatory funnel and
repeat this procedure once more using 10 mL of fresh ethyl acetate, combining the ethyl acetate
extract with the previous one.
Dry the organic solution by adding the appropriate amount of anhydrous magnesium sulfate to the flask and filter into a clean and dry 50 mL Erlenmeyer flask using Whatman No. 2 filter paper. Decolorize the solution with charcoal. Filter the solution using Whatman No. 2 filter paper into a 40 mL sample vial. The filtrate should be colorless to pale yellow in color; if it is amber, you will need to repeat the decolorizing process. Allow the solution to cool to room temperature. The presence of salicylic acid will be detected by thin layer chromatography (TLC) using a salicylic acid standard. Obtain a 2 x 5 cm piece of fluorescent silica gel TLC plate, remembering not to touch the surface of the plate with your bare fingers! Using a pencil, lightly mark the plate about 0.5 cm from the bottom edge. Using a capillary, spot the salicylic acid standard and your solution of willow bark extract on the pencil line and approximately 0.5 cm from each other. A suitable solvent system to develop your TLC is provided in the fumehood and consists of a mixture of toluene, diethyl ether, glacial acetic acid and methanol (120:60:18:5, by volume)
(CAUTION: Do not allow the TLC solvent mixture to come in contact with your skin). Place ~5
mL of this solvent in a developing jar (a 4-oz glass bottle). The bottom of the jar should be completely
covered, but you do not want enough solvent that it will submerge the spots on the TLC plate. Keep
the developing jar and the (unpleasant smelling) solvent in the fumehood at all times
. To
develop your TLC, place the TLC plate in the developing jar, making sure that your spots are ABOVE
the level of the solvent. Allow the developing solvent to rise almost (but not completely) to the top of
the plate (~2 minutes) and then remove the plate from the jar using tweezers. Mark the solvent front
immediately with a pencil. Do not remove the TLC plate from the fumehood until the solvent on it
has evaporated
. .
When the TLC is complete, visualize the spots under the UV lamp and sketch what you see in your lab notebook; indicate the location, size and color of all spots. After this, dip the plate in a jar of 1% ferric chloride solution, then remove and sketch the appearance of the plate again. Note any differences. (Ferric chloride reacts with phenols to yield a rust-colored compound). For each spot record the distance from the origin, the Rf value, and the color. Report questions:
1. Why does it not matter whether you use 10 g or 15 g of bark, or any amount in between? 2. Salicylic acid is much more soluble in methanol than ethyl acetate. Why can’t you use methanol in the extraction instead of ethyl acetate? 3. Why was it important to use a pencil to mark the TLC plate rather than a pen or marker? 4. Why is it important that the sample spots on the TLC plate are above the solvent line? 5. How many different compounds in your willow bark extract were detected by TLC? How many of 6. Did your willow bark extract, in fact, contain salicylic acid? Explain how you know. Instructor notes
This experiment exposes students to the common techniques of extraction, refluxing, and thin layer chromatography and is designed to be completed in a three hour laboratory session. It is of appropriate difficulty to be completed in the first semester of organic chemistry. However, we advise that students should have previous experience with at least one of these key techniques to avoid “information overload”. Ideas for incorporating this laboratory into existing experimental sequences are discussed in the main article. Required equipment per pair of students:

5-7 willow branches, ~4-8 mm in diameter and ~30 cm in length (10-15 g of bark is required) One condenser with correctly sized joint to fit 100 mL round bottom flask One 125 mL separatory funnel and stopper Ring stand or clamp to hold separatory funnel Three pieces of fluted filter paper (Whatman No. 2 (2 pieces) and Whatman No. 4) 40 mL sample vial (must hold 20 mL solvent) One 4x5 cm silica gel TLC plate with fluorescent indicator
Required chemicals and solutions per pair of students:

Anhydrous magnesium sulfate (or sodium sulfate) Salicylic acid solution (as a standard for thin layer chromatography) ~10 mL of TLC eluent (composition described below) 1% ethanolic solution of ferric chloride (FeCl3)
CAS registry numbers:

Hydrochloric acid
Ethyl acetate
Magnesium sulfate
Salicylic acid
Diethyl ether ]
Acetic acid [
Methanol ]
Ferric chloride
Solution preparation:

To prepare 1.00 L of 1.0 M hydrochloric acid:
Add 83 mL of concentrated hydrochloric acid to 500 mL of water, then add sufficient water to make 1.00 L of solution. To prepare a salicylic acid standard solution: Add ~5 mg to ~10 mL of dichloromethane in a vial and shake to dissolve Mix 120 mL toluene, 60 mL diethyl ether, 18 mL glacial acetic acid, and 5 mL of methanol. To prepare 100 mL of 1% ethanolic ferric chloride (FeCl3): Dissolve 0.80 g of FeCl3 in 100 mL of 95% ethanol.
Safety and hazards

There are no unusual safety hazards present in this experiment that do not exist in most introductory organic chemistry laboratories. Standard safety practices, including the use of a lab coat and safety goggles, should be strictly enforced. Additional considerations are listed below: If students wish to use a utility knife to remove the bark from the willow branches, it is useful to remind them that the knives are sharp. Hydrochloric acid is corrosive. Treatment for exposure to the skin or eyes typically involves washing the affected area with cool water for 15 minutes or using an approved eyewash station; however, sound judgement should be exercised in the event of any spill. The extraction is performed using ethyl acetate, which boils at 78 °C. The reflux flask and acid solution must therefore be cooled well below this temperature prior to coming in contact with ethyl acetate. As students often shortcut with the assumption that it is “cool enough”, they should be made aware of this fact. The solvent system used for developing the TLC is noxious and should be kept in the fumehood. Students should be reminded that the TLC plate, after being removed from the developing jar, is still coated in these noxious solvents and should not be removed from the fumehood until dry.
Additional useful notes:

Extraction of the acidic willow bark solution with dichloromethane is also suitable; issues of toxicity notwithstanding. In this case the decolorization step is unnecessary as the brightly colored compounds are not extracted into the dichloromethane, but the dichloromethane extract must often be concentrated before running the TLC. Drying the ethyl acetate extract prior to TLC analysis is not necessary given the highly polar nature of the TLC developing solution. We, however, prefer to include this short step to give students practice with the drying procedure. Sodium sulfate works, for all practical purposes, as well as magnesium sulfate to dry the extract. In the event a student does not detect salicylic acid in the extract, the extract may be concentrated by boiling on a hotplate. In our experience, however, if students do not use excessive quantities of ethyl acetate (>>20 mL), salicylic acid has nearly always been readily detected by TLC. Branches can be stored in a plastic bag at –20 °C in a freezer for at least one year without issue. No special precautions or preservation techniques are required. The retention factor (Rf) for salicylic acid is 0.56 using the described TLC solvent system. As with all experiments involving natural sources, we strongly recommend testing this experiment on the local source of willow prior to incorporation into the laboratory curriculum. We have obtained clear, consistent results with the five species of willow listed in this paper, but have found that one species present in Calgary, Alberta, Salix interior, gives inconsistent results.
Answers to report questions

1. Why does it not matter whether you use 10 g or 15 g of bark, or any amount in between?
This experiment qualitatively detects salicylic acid using the reasonably sensitive technique of thin layer chromatography. We are not concerned with the absolute amount of salicylic acid in the bark, and thus it does not matter how much you use as long as sufficient bark is used so that enough salicylic acid is extracted to detect. More than 15 g, however, may not fit in the reflux flask! 2. Salicylic acid is much more soluble in methanol than ethyl acetate. Why can’t you use methanol in the extraction instead of ethyl acetate? An extraction requires two immiscible solvents; methanol is miscible with water and thus no layers would be obtained. In other words, you would not be able to extract or separate anything! 3. Why was it important to use a pencil to mark the TLC plate rather than a pen or marker? Thin layer chromatography separates organic compounds based on their affinities for the silica on the TLC plate and the solvent used in the developing system. Since the ink in pens consists of several organic compounds, if you marked your TLC plate with pen the ink would be separated into its individual components, streaking up the TLC plate and likely obscuring those spots due to organic compounds in the willow bark extract. 4. Why is it important that the sample spots on the TLC plate are above the solvent line? If the sample was spotted below the solvent line, it would simply dissolve in the solvent, essentially leaving a clean, unspotted TLC plate. 5. How many different compounds in your willow bark extract were detected by TLC? How many of This answer to this question may vary from student to student, but most students will have only one phenol present. If two are present, the major component has always been salicylic acid. They are able to distinguish phenols from other organic compounds as only phenols will be stained by the 1% ferric chloride stain. 6. Did your willow bark extract, in fact, contain salicylic acid? Explain how you know. A sample of pure salicylic acid was spotted on the TLC plate next to the spot from the willow bark extract. Once developed, a significant spot in the willow bark extract lane had exactly the same Rf value as salicylic acid. Both spots also exhibited identical behaviour under ultraviolet light (both should fluoresce a vivid blue color) and both were readily stained by exposure to 1% ethanolic ferric chloride. Taken together, this is strong evidence that the willow bark extract did indeed contain salicylic acid.


Microsoft word - saratov hiv audit form engl3.0.doc

SARATOV / ENGELS – BEMIDJI AIHA PARTNERSHIP ASSESSMENT OF HIV/AIDS Care - Adults HIV/AIDS Care and Outcomes Chart Audit for Quality Assurance and Quality Improvement Contents Preface and Quick-Start Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fact sheet: the north carolina aids drug assistance/hiv medications program (adap)

ADAP FACT SHEET The North Carolina AIDS Drug Assistance Program (ADAP) Communicable Disease Branch, Division of Public Health, NC DHHS What is the North Carolina AIDS Drug Assistance Program (ADAP)? The North Carolina AIDS Drug Assistance Program (ADAP) uses a combination of state and federal funds to provide low-income residents of North Carolina with assistance in obtaining ess

Copyright © 2010-2014 Medical Articles