PRAC 2 Iodination - CHEM1200 Sem 2 2021 PDF

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Summary

Practical 1 of CHEM1200, contains the introduction, procedure and results section....

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2-1

Experiment 2

Iodinate, iodinate In this experiment, you will react 4′-hydroxyacetophenone with hypoiodous acid, HOI. An electrophilic aromatic substitution reaction results, with similarities to a reaction carried out in your thyroid gland. The identity and purity of the iodinated product will be checked by melting point range determination and thin layer chromatography. This is an example of “green” chemistry, as the waste from the reaction can be safely poured down the sink at the end of the experiment. The assessment of this practical task focuses on your gain in manipulative skills.

Practical Skills – By the end of the lab you will be able to: • • • •

Complete a synthetic procedure; Be proficient in the use of filtration apparatus; Set up, run and interpret a TLC; Understand and apply stoichiometric relationships between reagents to calculate the yield of a reaction.

LabSkills links:

Useful Equations:

Vacuum filtration technique simulation and video; TLC video, set up and measurement simulations; Melting point analysis technique practice and video; Mole calculations to determine yield guide.

n = m/M; Yield calculations

Concepts: Synthesis of an organic product; Identification of the product using TLC and melting point; Isolation of the product from a mixture; Yield calculations. If you want to find out more in the textbook: This experiment will complement the Organic Chemistry lecture series, strengthening your understanding from lectures. In particular, they relate to lectures on electrophilic aromatic substitution reactions. Electrophilic aromatic substitution reactions are covered on pp. 1012 – 1018, Blackman, Chemistry 4th ed. Student Receipt Experiment 2 – Iodinate, iodinate I certify that this student has completed the experimental section of this practical to a satisfactory standard.

Student signature Demonstrator Signature

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2-2 INTRODUCTION Iodine is an essential element. Since 2009, bakers in Australia have been required to use iodised salt, to help avoid deficiencies in the population. 1 Around 2 billion people worldwide are estimated to be iodine-deficient, which can result in developmental issues in children. It is used in the body to make two hormones involved in the regulation of metabolism, in the thyroid gland. The structures of these hormones are shown below.

A key step in the synthesis of the hormones involves iodination (replacing a H atom with iodine) of certain tyrosine residues in a protein called thyroglobulin. Tyrosine is an amino acid that has a phenol (4-hydroxybenzene) side-chain. This is an example of a biological electrophilic aromatic substitution reaction, similar to those that you have been studying in lectures, such as chlorination, bromination, nitration and sulfonation.

There are three steps involved in an electrophilic aromatic substitution reaction. The first step is generation of the electrophile, followed by reaction of the aromatic molecule with the electrophile in the second step, to generate a resonance-stabilised carbocation intermediate. The third step is loss of a proton from the carbocation intermediate to regain aromaticity and generate the substituted final product.

1

http://www.foodstandards.gov.au/consumer/nutrition/iodinefort/Pages/default.aspx

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2-3 Below is an explanation of the specifics of the iodination mechanism. Just as in other electrophilic aromatic substitution reactions, the first step in the iodination mechanism, or reaction pathway, is the formation of the electrophile, a species which can accept an electron pair from a nucleophile. The synthesis of these hormones takes place in the thyroid gland, which has a special transporter protein which causes sodium iodide to be concentrated in the thyroid tissue. However, iodide ions are not electrophiles and there must be an oxidation reaction to convert iodide ions into the reactive electrophile. The reaction of hydrogen peroxide and iodide ions is catalysed by an enzyme called thyroid peroxidase to give the electrophile. This is thought to be hypoiodous acid, HO-I, which has a partial positive charge on the I atom.

A reaction then occurs between the aromatic ring of a tyrosine residue and the electrophile that has been formed. Typically, this step in an electrophilic aromatic substitution reaction would result in the formation of a resonance-stabilised carbocation intermediate. However, the carbocation structure shown is not the lowest energy Lewis structure that could be drawn – can you use electron-pushing arrows to draw a lower energy canonical structure? (The answer is below if you are unsure, but have a go first.)

The canonical structure on the right is the more stable, because there is no separation of charge. The third step is deprotonation of the intermediate, which is added by the water molecule which was formed as a leaving group earlier in the reaction. This regenerates aromaticity in the final substitution product. The iodinated tyrosine residue then undergoes a second round of iodination by the same mechanism. There are then further enzyme-catalysed steps to generate the T3 and T4 hormones.

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2-4 Details of the Experiment To make the workup of the product easier, you will not be using tyrosine as your starting material, but 4′-hydroxyacetophenone, which has the structure shown. This is dissolved in ethanol, as the compound only has limited aqueous solubility. The electrophilic equivalent to I+ is hypoiodous acid, HOI, generated by reaction of sodium iodide with sodium hypochlorite (NaOCl) in the form of a commercial bleach solution. The reaction to generate this is quite exothermic, so the reaction is cooled on ice. This also helps to prevent a second iodine being added to the aromatic ring. Hypoiodous acid disproportionates readily to form iodate (IO3–) and iodide ions, with further reaction making iodine. After reaction is complete, the iodine and excess bleach are removed by reaction with thiosulfate anions. Dilute hydrochloric acid is added to acidify the reaction solution and precipitate the iodinated product. This is collected by filtration, before being tested for purity. Draw the product for the iodination of 4′-hydroxyacetophenone. Use the mechanism on the previous page as a guide:

HOI

How do you know what you have made is pure? The iodinated product you will make is potentially impure. If the reaction has not gone to completion, then there could be starting material still present. The organic product could be contaminated with inorganic salts or be incompletely dried, with solvent molecules still present. There is also potentially a second product that could form. How do you tell if your product is pure? Thin-layer chromatography (TLC) is a powerful tool for determining whether two compounds are identical (Refer to the LabSkills simulations on Blackboard for a complete description of thin layer chromatography). Each compound possesses a characteristic retention time and should give a single spot if pure. Additional spots, with different retention times, indicate the presence of impurities. In this experiment, your iodinated product is compared by normal-phase TLC with the starting material, and a pure sample of the product, to see whether it is sufficiently pure. Measuring the melting point range of a substance is a good way to test for purity. A pure substance usually has a sharp melting point range (narrow temperature range during which it changes from a solid to a liquid). Any impurities in the substance cause a lowering and broadening of this characteristic temperature range. The closer to the literature melting point range, the purer the product.

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2-5 Gas-chromatography-Mass Spectroscopy (GC-MS) is a highly useful chemical analytical tool. You won’t have opportunity to run GC-MS on your own sample, because of time constraints, but you will be able to interpret GC-MS traces that have been run for you. GC-MS is a very useful technique, because it provides information not only on the number of compounds present in a mixture, but also tells the molar masses of each of those compounds. To a skilled chemist, the particular way that a molecular ion breaks down into smaller fragments can also give useful information about the structure of the molecule and can also be compared to databases to “fingerprint” the compound. In GC, the sample to be analysed is initially vaporised into a gas by heating and carried by a flow of nitrogen or helium gas through a very fine, long column (a capillary column) containing a stationary phase that consists of silica with a 5% hydrophobic coating. The gas that carries the sample through the column is called the mobile phase. As the sample passes through the column, the various components travel at different rates, depending on how strongly they interact with the stationary phase. Ideally, all of the components are separated from one another by the time they reach the column exit. As the components of the mixture elute from the end of the capillary column we need some way of detecting them. The instrument you will use detects molecules using a mass spectrometer (MS). The volatile molecules leave the capillary column of the GC and enter the mass spectrometer where they are ionized and then detected. Thus, a trace is recorded that provides a peak corresponding to each individual component at the time that it emerged from the column. The area under these peaks (or the size of the peak) is proportional to the amount of the component that was ionised. So, the GC trace allows us to quantify how much of each component is present in the mixture. The MS measurement tells us the molecular weight of the compound in each peak. The time required for a sample component to elute from the column is called its Retention Time (Rt) and is usually measured at the top of the peak. This retention time is constant for a particular compound if the conditions of the GC are not changed, and so the retention time is a property that can be used to confirm the identity of a component of a mixture. What if I find my product is impure? What would a chemist do? Recrystallising a solid will increase its purity, but there won’t be time in this experiment for you to do this. Recrystallisation is a technique which is based on the fact that different compounds will have different solubilities in a given solvent and that the solubility of a product changes with temperature. When added to a mixture of hot methanol and water, the iodinated product and any impurities will dissolve, but on cooling the impurities will remain in solution while the pure product will precipitate out of solution. The side-product is much less soluble and can be removed by filtering the solution while still hot. Once the filtrate is cooled, the desired product will precipitate and the product can be filtered and dried, before being tested for its purity.

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2-6

Experimental Procedure AIMS To synthesize an iodinated product from the reaction of acetovanillone, sodium iodide and sodium hypochlorite. To assess the purity of the product using melting point range and reverse-phase TLC. SAFETY NOTES The reaction of sodium iodide and sodium hypochlorite generates iodine, a corrosive substance. You should protect your hands from exposure by wearing disposable gloves and any area of skin contact should be rinsed with large amounts of water. If you have chemicals on your gloves you should change them as soon as possible to avoid accidental contamination.

PROCEDURE Part A – THE REACTION To give a mono-iodinated product, it is important to add the bleach solution slowly and maintain the temperature of the reaction below 10 ºC. 1. At the balance, weigh out approximately 1.0 g of 4′′ -hydroxyacetophenone and record the accurate weight of your sample on your results sheet (i.e. you don’t need exactly 1.0 g but you need to know exactly how much you have!). Weigh out 1.10 g of sodium iodide. 2. Add the 4′-hydroxyacetophenone into a 100 mL Erlenmeyer flask, add 10 mL of ethanol and swirl the flask to dissolve the solid. 3. Add the sodium iodide to the flask, along with a magnetic stirrer bar.

Erlenmeyer Flask

4. Cool the flask in an ice-water bath on top of a stirrer hot plate. Make sure that the heating is not turned on! 5. While the flask is cooling to below 10 ˚C, make 10 mL of an approximate 5.75% (by mass) NaOCl bleach solution. You will be provided with approximately 12.5% bleach solution. You may assume that the densities of the two solutions are 1 g mL-1, as the precise amount is not critical. 6. Add all the 5.75% bleach solution dropwise to the ice-cooled solution over 10 minutes (roughly a 3-second interval between drops), keeping the temperature below 10 ˚C. Note any colour changes observed. Do not add the bleach solution too fast. After the addition is complete, take the flask out of the ice bath and stir the reaction for a further 10 minutes. During this time, complete Part B before returning to Step 7. START PART B NOW while waiting for the reaction to complete (see page 2-14). 7. Make 10 mL of a 10% by mass solution of sodium thiosulfate. Add this to your reaction flask and note any colour changes. 8. Acidify your reaction solution with 1 mol L-1 aq. HCl solution. A precipitate should form after the addition of the acid. Add enough acid to precipitate all the solid. If this does not happen, consult with your tutor.

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2-7 9. Collect the precipitate by vacuum filtration, using a Büchner flask, funnel and filter paper. Make sure that the flask is clamped around the neck, as they are very easy to knock over. Have the flask sitting on the bench, not suspended in the air. Wash the solid with a few mL of ice-cold water. Instructions on how to perform a vacuum filtration are in the Lab Skills links on Blackboard.

Filter paper covers all the holes but does not come up the sides. Büchner Funnel (Top View)

10 mg is equivalent to an amount about the size of a matchstick head, or some solid on the end of a spatula

10. Dry your solid product by sucking air through the sample for a few minutes. Transfer the solid to a weighed watch glass and then weigh the watch glass plus crystals to determine the mass of your crude iodinated product and record the mass. You will need to keep ~10 mg (small amount on the end of a spatula – no need to weigh material) of your crude product for TLC (see next page). 11. Place your product into a warm oven to help dry it and remeasure the weight after 10 minutes. You should note that as water is driven from your product that the weight decreases. Record the mass of your dry product in your results sheet. Place your product back into the oven for further periods of 10 minutes until no more water is removed. You can use this time to complete the TLC task (see next page). 12. Transfer your product to a weighed sample bag. Label the sample with your and your partner’s name, the date, the melting point range and the name and weight of the product. Submit this sample to your demonstrator at the end of the session. 13. You will need to use ~10 mg (small amount on the end of a spatula – no need to weigh material) of product for melting point analysis (see next page). 14. Determine the melting point range for the product as described on the next page. 15. Calculate the yield of the reaction.

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2-8 Thin Layer Chromatography 1.

Collect an aluminium backed TLC plate from the trolley.

2.

On the side coated with white silica, gently draw a very faint pencil line all the way across, approximately 1 cm from the bottom. Then faintly mark three small dots, equidistant across the line and write underneath each dot the numbers 1 – 3.

3.

Dissolve the following 3 solids in acetone using 1.5 mL Eppendorf tubes: i. ii. iii.

4′-hydroxyacetophenone; the pure iodinated product; your product.

Take ~10 mg for each of the compounds. (This is the equivalent to a match stick head, N.B. do not weigh 10 mg – these balances are not accurate enough for milligram quantities.)

Eppendorf Tube

4.

Load as follows on to the TLC plates: spot 1 – 4′-hydroxyacetophenone, spot 2 – the pure iodinated product, spot 3 – your product. Smaller spots work better than large ones.

5.

The following 2 steps must be completed in the fumehood. Take a TLC chamber and pour a small amount of the dichloromethane/methanol solvent mixture into the bottom just so that there is less than 0.5 cm of solution in the bottom (A dispenser is set up with the volume of solution required). Using your tweezers, carefully lower your TLC plate into the beaker, spot end down so that it stands up against the side of the beaker and make sure that your spots are not submerged under the solvent. You should immediately see the solvent begin to slowly move up the TLC plate.

When spotting a TLC sample with a blunt needle, it is a good idea to draw some sample into the needle and blot the sample twice onto a piece of tissue before applying it to the TLC slide. This ensures that your sample will not be contaminated with the previous sample.

6.

Put the watch glass over the top of the beaker. Keep a careful eye on the plate and when the solvent level is about 1 cm from the top, carefully remove it with your tweezers and immediately mark the solvent level lightly with a pencil.

7.

Carry your plate to the UV lamp box and look at it under the lamp. Put a pencil circle around any spots you see. Note that the silica will be very soft, so just lightly circle the spots. Measure the Rf value for each of your spots. This is explained in the Lab Skills links on Blackboard.

Melting Point Range Determination Determine the melting point range of your product using the melting point apparatus. Follow the instructions provided and ask your demonstrator if you require advice. Compare the melting point you measure to that in the literature.

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2-9 Experiment 2: IODINATE, IODINATE

Student No

Name: _____________________ Date: ________

I have completed AND submitted my pre-lab questions on Blackboard at least 30 minutes before coming to class. I have completed the ChemWatch tables and flowchart in my laboratory manual. I have my safety glasses, lab coat and appropriate footwear with me.

Using the ChemWatch database, provide the severity rating for each of these properties for the following solvents/reagents: (Minimum/Nil = 0, Low = 1, Moderate = 2, High = 3, Extreme = 4) Note that these entries are for the pure compounds, but the hazards associated with dilute solutions will be much less. 4'-hydroxyaceto-

sodium iodide

phenone

Compound

CAS 7681-82-5 CAS 99-93-2 Flammability Toxicity Body Contact Reactivity Chronic Effects

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sodium hypochlorite 12.5% w/v CAS 33-3352

2-10 Flow Chart

As part of your preparation for the lab, draw a flow chart of the steps that will take 4'-hydroxyacetophenone through to produce and analyse the pure iodinated product. Include cartoon sketches of any glassware or apparatus that you will use. If there is any apparatus that you are unfamiliar with, then you can use the LabSkills link (Blackboard, CHEM1200, Laboratory) to find out more by consulting the glossary or viewing the other materials available. It should take up no more than this page (feel free to draw ‘landscape’). Upload a photo of your flow chart to the Pre-lab quiz. The library photocopiers c an also be used as s...