Extraction of paracetamol (alvedon) from 4-aminophenol

I recently made a visit to Stockholm University to extract paracetamol, the chemical name for alvedon. Followed is a summary of the lab.

Aim

The aim of the lab is to extract one of the following products from an unknown substance and then investigate which chemical has been produced.

Method

• Take 1.1g of substance A and mix it with 3 ml of distilled water in a 50 ml boiling flask equipped with a magnetic stirrer.
• Add acetic anhydride and agitate the bottle.
• Then put a condenser on the flask and then but it in an oil bath with a temperature of 70-80°C until the solution becomes clear (this might take up to 30min).
• Let it cool gradually, first in air, then water and finally in ice.
• As the substance crystallizes filter it out using a Büchner funnel and water suction.
• Rinse the flask used with cold distilled water and pour it into the Büchner funnel to get all the crystals out to ensure the optimal yield.
• Put the crystals to an Erlenmeyer flask to prepare for recrystallization while heating distilled water in another.
• Add the water to the crystals just until the entire product is dissolved.
• Let it cool until crystals appear and then filter them off and wash with small amounts of cold distilled water and weight the product.
• Dissolve a small amount of the product produced, Ibuprofen, Acetylic acid, Paracetamol in approximately 1ml of methanol.
• Using a small capillary take small amounts of each sample and release it on a THC-plate. Between each sample be sure to clean the capillary with the use of acetone.
• Pour in a 1:1 mixture of pentane ethylacetate (about 3ml) into a TLC-chamber.
• Then place the THC-plate inside the TLC-chamber and be sure to check that the samples are not in direct contact with the liquid.
• After the THC-plate has dried, look at the samples under an UV-light and make observations. 

Discussion

It was concluded that the product created was paracetamol when a thin layer chromatography test was performed and observed under ultraviolet light (which is discussed further down the page). An unknown substance was treated with acetic anhydrate and resulted with paracetamol. A compound that behaves in this matter is 4-aminophenol and is widespread in the industrial production of this drug. Upon these observations was the build up of “Chemical A” decided.  

When 4-aminophenol is t treated with acetic anhydrate it produces paracetamol. This procedure is the last stage of an industrial production of the drug called “Alvedon”. 

Followed is a balanced equation of the reaction taken place. 

C₆H₇NO + C₄H₆O₃ --> C₈H₉NO₂ + C₂H₄O₂ (acetic acid)

4-aminophenol + acetic anhydrate à paracetamol + acetic acid

Mr 4-aminophenol - 109.13 g/mol

       acetic anhydrate - 102.09 g/mol

       paracetamol - 151.17 g/mol

       acetic acid - 60.05 g/mol       

As the experiment started, approximately 1.09 grams of 4-aminophenol was measured out to use in the experiment. This is (1.09/109.13) 9.99 x 10^-3 moles of acetic anhydrate which should result in the same amount of moles of paracetamol, as these are created in a 1:1 ratio. The theoretical yield is (9.99 x 10^-3 x 151.17) about 1.51g of paracetamol which is the amount that should be received. The actual yield at the end was 1.05g which is ≈ 70 % (69.54 %) (1.05/1.51 x 100) of the theoretical amount.

Recrystallization was performed to purify the compound received. The crystals of compound A were filtered out using filter paper. Doing a recrystalization purifies the substance but also decreases the yield as material is lost due to the finite solubility of paracetamol. This together with the product lost on the filter paper makes up for the primary loss of the substance, decreasing the yield.     

In practice it is impossible to receive the total theoretical yield (100%) as some molecules or atoms will be lost in the process. 4-aminophenol or acetic anhydrate could have been used in excess in the reaction. Having substances in beakers, boiling flasks, test tubes etc. will always result in loss of particles as they don’t get rinsed out of the container. In the reaction performed between 4-aminophenol and acetic anhydrate all molecules did not necessarily had to react with each other, leading to a lower percentage yield received. The main cause to loss of the product was in the filtration as visible amounts to the eye were left on the filter paper, drastically lowering the yield. The total amount of paracetamol could yet not have been crystallized when filtration was performed, once again leading to a loss of particles. The recrystallization performed also lowered the yield quite significantly.    

The product was analyzed using Thin-layer chromatography (TLC). The sample molecules of each drug were dissolved in a small amount of methanol and placed on the bottom of a TLC-plate. This was then placed in a TLC-chamber with a solvent at the bottom. The solvent rose up the plate by capillary action and because of different analytes they rose at different rates ending up at different positions. 

The THC-plate was exposed to ultraviolet light to see how high each sample had traveled and what color it emitted.  The effect of UV-light on fluorescence objects is more colesly discussed in the following post; 

http://www.blogger.com/blogger.g?blogID=997544598088907329#editor/target=post;postID=6376829843095828029

The conclusion was drawn that the product received was paracetamol as it emits an identical color, which in terms mean that they must have the same electronic configuration and hence be the same molecule.

The process of paracetamol acting as a pain reducer is not completely understood in modern times. It is believed that it’s vital function is to reduce the production of cyclooxygenase (COX), which causes easing of pain and reduce inflammatory symptoms. Cyclooxygenases are enzymes that produce prostanoids, which in terms cause pro-inflammatory symptoms. Currently three isoenzymes have been discovered; COX-1, COX-2 and COX-3. A research group in Lund, Sweden discovered in November 2011 that paracetamol effect the TRPA1-receptors, which are located in the spinal cord. They cause an inhibition of neural signals on the surface layer of the dorsal horn. The spinal cord is a part of the Central nervous system (CNS) and is the pathway of signals from the Peripheral nervous system (PNS) to the brain.  

Paracetamol metabolized into AM404, a molecule that mainly stops the uptake of anandime by the neurons. The effect of the substance being taken up is that it activates TRPV1, the major pain receptor of the body. AM404 also affects the sodium channels by restricting the movement of sodium ions in and out of the cell. Both these factors have tested to have a pain reducing effect, which leads to the conclusion that these factors are involved in the mechanism of paracetamol.      

Sodium channels are integral membrane proteins found in the plasma membrane which control the movement of sodium ions (Na⁺) in and out of the cell. The pain reducing factor comes from the blockage of the movement of sodium ions in neurons. Sodium ions are necessary in order to carry an electrical impulse along neurons. When a stimuli, (such as touching something on your fingertip) exceeded a certain threshold a brain wave is generated. This electrical impulse consist of the sodium-potassium pump, which are sodium ions (K⁺) and potassium ions (Na⁺) moving in and out on a membrane. As the sodium channels are not letting through sodium ions the signal is solaced, reducing the sensation of pain.        

Optical isomeres and amino acids

In terms of chemistry isomers are compounds that have an identical molecular formula but a different structural formula, which means that its atoms are differently geometrically arranged. There are several forms of isomers and one that will be looked more closely into is stereoisomerism.

If you look at your nose in the mirror, the reflection will be identical to your real nose. If you instead would look at you palm in the mirror it would look exactly as your left hand. The reflection is not superposed, which is the reason you cannot fit you left hand into your right glove. An optical isomer (stereo isomer) is a mirror reflection of a compound. All compounds are not able to form optical isomers as the mirror image will be identical to the original object. This is just like the example with the nose, achiral objects. For stereoisomerism to occur, the compound needs to bee chiral. This happens when there is a fixed center with three different groups; atoms or molecules boned to it. These kinds of isomers have identical chemical properties and perform identical chemical reactions. They can only be distinguished in the direction they bend light. 

In biological terms optical isomers can have significantly different behaviors. There was an incident in the late 1950s with a drug called Thalidomide and was used by pregnant women against morning sickness. It resulted in children being born with various deformations which were caused by the optical isomer from of the compound. These are the devastating effects stereoisomerism can have.

All life is based on amino acids, more precise on the optical isomer L-amino acids. Amino acids are chiral which means that there are optical isomers; L-amino acids and D-amino acids. If a pathogen was artificially created that was based on the D-amino acid, all organism on earth would not be able to defend themselves against this microorganism. White blood cells of animals would have no effect against them as they were designed to fight pathogens of the same amino acid build up. If someone would create an organism like this, they would have the power to destroy all life on earth.

Why do grains of pollen move continously in a random motion when placed in still water?

This phenomena can be explained by Brownian motion, which is the random drifting of particles in a liquid.  The kinetic theory states that all matter is made out of particles which are in constant motion in proportion to their energy and attraction between other particles. A particle can consist of atoms, molecules or ions.   

In water or other liquids the attraction between the particles is relatively low, which means that the water molecules can move freely. The grains of pollen move due to the water particles bumping into them causing movement. This is a random physical occurrence, which means that the water molecules can hit the grains of pollens from different directions at any time. 

The observation of grains of pollen moving in a random motion on water was made in 1827 by a botanist called Robert Brown. 

Below is a demonstration of Brownian motion. The red particles represent grains of pollen (out of proportion) being moved around by the blue water molecules.

Why do some objects glow under ultraviolet light?

Materials that glow under ultraviolet light, also called black light are called to be fluorescent. In normal light emission, photons are released from electrons at the same frequency as they were absorbed. In fluorescence materials this is not the case as they emit light at different wavelengths than absorbed. In normal color emission, electrons absorb energy from photons that is required to move up energy levels. As the electrons fall to their ground state they release the energy in the form of photons at the exact same frequency. Ultraviolet light cannot be seen by human eyes as the frequency is outside the visual light spectrum. When fluorescence materials are exposed to this black light they usually reemit photons with a lower frequency that is within the visual light spectrum. The difference in energy is emitted as heat.

Excitation: S₀ + hv_ex --> S₁

Emission: S₁ --> S₀ + hv_em + heat

(Where S₀ is the ground state and S₁ the first energy level)

These materials usually have rigid structures and delocalized electrons. Examples of materials are called white paper, vitamins, body fluids chlorophyll.  

Why are some objects seen as colorless?

In atoms and molecules the electrons surround the nucleus in energy levels. When an electron is not exited it usually occupies the lowest energy level available. When energy is given to an electron it moves up a different amount of energy levels depending on how much energy it gains. The distance between the energy levels decreases further away from the nucleus until the electron completely has escaped the nucleuses attraction. When this occurs, the atom or molecule has been ionized.   

When photons of light hit atoms or molecules, the electrons absorb this energy. In order for electrons to move to the next energy level the light need to be of a minimum frequency. This is as the electrons cannot rest in between energy levels. The amount of energy needed for electrons to move to the next energy levels is called a “quanta”. When electrons fall back to their ground state the same amount of energy is released in form of photons. It takes different frequencies to raise electrons of different atoms and molecules to different energy levels. This is what gives different colors.  

The visible light we see is only a small part of a much larger electromagnetic spectrum. The human eye only interprets a narrow range of frequencies. Photons that travel either below or above these frequencies are seen as colorless. Examples of theses are ultraviolet rays, x-rays and radio waves. Some materials have electrons that require a quantum of energy that is outside the frequencies of visible light. This means that they do not give off any photons and make perfect translucent glass.