12 minutes reading time (2340 words)

    IN SEARCH OF MORE SOLUTIONS 2000

    J Taylor
    The Royal Society of Chemistry

    The following three problems have been reproduced (with permission from The Royal Society of Chemistry) from 'In Search of More Solutions, More Ideas from Problem Solving Activities' published and distributed by the Royal Society of Chemistry.

    ANY GLUCOSE?
    Design a biosensor to detect glucose. Glucose can be detected by using two enzymes, glucose oxidase and horseradish peroxidase.

    Glucose oxidase breaks down glucose in the presence of oxygen into hydrogen peroxide and gluconic acid. Horseradish peroxidase catalyses the oxidation of potassium iodide by hydrogen peroxide (formed by the action of glucose oxidase) to iodine, which is brown. The intensity of this brown colour can be taken as a measure of the amount of iodine present.

    SAFETY NOTE
    Use forceps for handling the enzyme-soaked paper.

    A YELLOW SOLID
    The following preparation should produce a good yield of a rather unusual compound. Find out as much as you can about the compound by using the tests suggested

    PREPARATION
    Dissolve 10g copper(II) sulfate crystals in 50 mL of warm water. In a separate beaker dissolve 18 g sodium thiosulfate crystals in 30mL of warm water. Adjust the temperature of each solution to about 40°C, and then pour the solution of thiosulfate into the copper solution with continuous stirring. After a short time, a bright yellow solid begins to separate, and is fully deposited in about an hour. Filter off the solid using a filter-pump and Buchner funnel and wash the solid on the filter paper with cold water followed by propanone. Finally, allow the solid to dry in air at room temperature.

    Investigate small quantities of the solid as follows.

    1. Heat some of the powder in a dry test-tube and test any gas evolved with a piece of filter paper soaked in acidified potassium dichromate(VI) solution.
    2. Warm a little of the powder slowly with dilute nitric(V) acid until boiling.
    3. Put a little of the solid in a test tube followed by 10 drops of iodine solution and shake.
    4. Put a little of the solid in a test-tube followed by dilute ammonia solution. Leave to stand for some time.
    5. Repeat (4) but also add a spatula of ammonium persulfate crystals.

    AS SWEET AS? DETECTING ASPARTAME IN A TABLE-TOP SWEETENER
    A sample of a table top sweetener is provided. One teaspoonful of this would sweeten a cup of tea.

    Find out whether or not this substance contains the sweetening agent aspartame. Aspartame is a synthetic sweetening agent which tastes very similar to sucrose but is about 200 times sweeter.

    Aspartame is the methyl ester of a dipeptide containing the amino acids aspartic acid and phenylalanine. The chemical name is L-aspartyl-L-phenylalanine methyl ester. The diagram below shows its structure with the dotted lines dividing the structure into its component groups. 

    TEACHER'S GUIDEANY GLUCOSE?

    TIME
    1 hr.

    LEVEL
    A-level, Higher Grade or equivalent.

    CURRICULUM LINKS
    Catalytic action of enzymes. Simple carbohydrate chemistry.

    GROUP SIZE
    2-3.

    MATERIALS AND EQUIPMENT
    Materials per group

    • a few drops of glucose oxidase/horseradish peroxidase mixture.
    • Fermcozyme 952 DM.
    • a few drops of 2% w/v potassium iodide solution.
    • solutions of glucose at various concentrations to test the sensor for the comparison with a commercial product DiastixTM (available from pharmacies).
    Equipment per group
    • dropping pipettes or 1 mL plastic syringes (without needles) for dispensing liquids.
    • filter paper.
    • scissors.
    • forceps for handling enzyme-soaked paper.
    • safety glasses.

    SAFETY
    Safety guidelines are given in the Teacher's notes of the National Dairy Council Publication (1). (General guidance will be found in Microbiology - An HMI guide for schools and non-advanced further education. HMSO. London: 1985).
    Eye protection must be worn. Unnecessary contact with the enzyme or inhalation of dust from dried-up enzyme spills should be avoided. In case of spillage or contact with the eyes, rinse by flushing with water.

    RISK ASSESSMENT
    A risk assessment must be carried out for this activity.

    COMMENTARY
    The students can be given the worksheet, the reagents and no other information. Test strips for detecting the presence of glucose in urine can be purchased at pharmacies (eg. DiatixTM, which works on a principle similar to that described on the student sheet). The students may be interested to compare their glucose detectors with commercially available products such as DiastixTM.

    This investigation is based on an activity designed for a workshop at the Natural History Museum, London, in May 1992 (1). On this occasion the students worked in groups of three and each group was given a box containing all the necessary apparatus. The practical details are adapted from a National Dairy council publication (2).

    PRACTICAL DETAILS (3)
    Special care must be taken to ensure that cross-contamination of the enzyme mixture, potassium iodide and sugar solutions does not occur. Separate, clearly marked syringes or pipettes should be used for dispensing each liquid. A possible approach is as follows.

    1. Cut out a small square of filter paper, roughly 10 mm x 10 mm.
    2. Place a drop of potassium iodide solution on the paper, and allow to dry slightly, then add a drop of the enzyme mixture to the paper.
    3. Add a drop of the glucose solutions to the paper and note the colour change; this may take a few minutes, as oxygen has to diffuse into the solutions.

    EXTENSION
    During trialling several institutions extended the problem by getting students to investigate how sensitive the sensor was to the concentration of glucose. The activity can be extended to test the specificity of the sensor by using sugars other than glucose.

    REFERENCES

    1. Madden, D., National Centre for Biotechnology Education Newsletter, p12. Spring 1992.
    2. Dairy Biotechnology, National Dairy Council 5/7 John Princes Street, London W1M OAP.
    3. Madden, D., National Centre for Biotechnology Education Newsletter, p22, Spring 1992.

    ACKNOWLEDGEMENTS
    Dean Madden and John Schollar of the National Centre for Biotechnology gave advice on the development of this activity and the experimental procedures described were developed in their laboratories.A YELLOW SOLIDTIME
    The preparation requires 2 h but during this time the reaction mixture is left to stand for 1 h.
    The qualitative investigation requires 1 h.

    LEVEL
    A-level, Higher Grade or equivalent.

    CURRICULUM LINKS
    Chemistry of copper(I) and copper(II) ions.

    GROUP SIZE
    2.

    MATERIALS AND EOUIPMENT
    Materials per group

    • 10 g copper(II) sulfate.
    • 18 g sodium thiosulfate.
    • deionised water.
    • propanone.
    • 0.1M potassium dichromate(VI) solution.
    • dilute nitric acid.
    • iodine solution (0.2M iodine in aqueous potassium iodide).
    • dilute ammonia solution.
    • ammonium persulfate.

    Equipment per group
    • two 100mL beakers.
    • 250mL beaker or flask.
    • magnetic stirrer or glass rod.
    • filter pump.
    • Buchner funnel and flask.
    • arrangement for heating solutions to about 40°C.
    • test-tubes. test-tube rack.
    • filter paper.
    • safety glasses.

    SAFETY
    Eye protection on must be worn.

    RISK ASSESSMENT
    A risk assessment must be carried out for this activity.

    COMMENTARY
    The bright yellow solid is the complex salt sodium copper(I) thiosulfate. W.G. Palmer (1,2) describes a quantitative investigation of this compound. The results quoted agree with the formula 3Cu2S2O3.2Na2S2O3.6H2O.

    The results of the investigations are as follows:
    1. Sulfur dioxide gas is produced. The filter paper soaked in acidified potassium manganate(VII) turns green.

    Na2Cr2O7(aq) + 3H2SO3(aq) + H2SO4(aq) → Na2SO4(aq) + Cr2(SO4)3(aq) + 4H2O(l)

    2. Sulfur dioxide is again produced.

    Na2S2O3(aq) + 2HNO3(aq) → SO2(g) + H2O(l) + S(s) + 2NaNO3(aq)

    3. Iodine solution is decolourised and a colourless solution of sodium tetrathionate is formed.

    2Na2S2O3(aq) + I2(aq) → Na2S4O6(aq) + 2NaI(aq)

    4. A blue copper-based complex is formed.

    5. Oxygen gas is released, indicating displacement of S2O32-(aq) from the copper complex.

    EXTENSION
    The quantitative analysis described by W.G Palmer could be carried out.

    REFERENCE

    1. Palmer, W.G., Experimental Inorganic Chemistry. Cambridge: CUP, 1965.
    2. Vowles, R.S., Bona, N., School Sci Rev.,1985, 66, 476.

    ACKNOWLEDGEMENT
    This activity is based on a suggestion by Colin Johnson.AS SWEET AS? DETECTING ASPARTAME IN A TABLE-TOP SWEETENERTIME
    Stage 1 Hydrolysis of Canderel®: 1-1.5 h (includes 0.5-1 h refluxing).
    Stage 2 Paper chromatography: 2.5 h (includes 1-2 h for running chromatograms.

    LEVEL
    A-level, Higher Grade or equivalent.

    CURRICULUM LINKS
    Amino acids, peptide linkage, Paper chromatography.

    GROUP SIZE
    Stage 1 Hydrolysis of Canderel®: If time is limited one batch could be hydrolysed to provide material for all the groups to use in preparing chromatograms.

    Stage 2 Paper chromatography: 2-3.

    MATERIALS AND EOUIPMENT
    Materials per group
    Stage 1 Hydrolysis of Canderel(R)

    • 12 g Canderel(R).
    • 200 mL of 6M hydrochloric acid.

    Stage 2 Paper chromatography
    • about 5mL of the solution produced in Stage 1 (acid hydrolysis of Canderel(R)).
    • 100mg activated charcoal.
    • about 50mL of solvent mixture for chromatography (ethanol:water:880 ammonia in the ratio 80:10:10).
    • ninhydrin, 0.2% solution in propanone, stored in a spray bottle (Ninhydrin is also available from BDH in a spray can as a 0.5% solution in butanol).
    Reference amino acids
    • 1mL of a DL-aspartic acid 0.01M solution dissolved in 10% v/v propan-2-ol/water
    • 1mL of a DL-phenylalanine 0.01M solution dissolved in 10% v/v propan-2-ol/water.

    Equipment per group
    Stage 1 Hydrolysis of Canderel(R).
    • 500mL round-bottomed flask.
    • condenser.
    • Bunsen burner or heating mantle.
    • Safety glasses.

    Stage 2 Paper chromatography
    • Pasteur pipette.
    • 5mL measuring cylinder.
    • test-tubes.
    • small funnel and filter paper.
    • chromatography tank or 1L beaker and cling film to cover.
    • chromatography paper (Whatman No. 1).
    • 25 or 50mL measuring cylinder depending on size of tank.
    • clips for paper, pencil.
    Access to:
    • fume cupboard.
    • oven at 110°C.
    • spray bottle containing 0.2% ninhydrin solution in propanone.

    SAFETY
    Eye protection must be worn.

    The ninhydrin spray should be used only in a fume cupboard. The chromatogram must be hung up inside the fume cupboard to be sprayed.

    RISK ASSESSMENT
    A risk assessment must be carried out for this activity.

    COMMENTARY
    The idea of applying the techniques of chromatography to the analysis of Canderel® is based on an experiment described by A.D. Heaton (1). If the students follow the approach suggested below they should obtain clear results as only two amino acids are involved.

    Aspartame
    The sweet taste of aspartame was discovered accidentally in 1965 by James Schatter who was synthesising a product for treating ulcers. He was heating aspartame in a flask with methanol when the mixture bumped onto the outside of the flask. He later detected a strong sweet taste on his fingers which he traced back to powdered aspartame on the flask. This method of discovery is not an example of good laboratory practice! Aspartame is valued because it has a clean sweet taste similar to that of sucrose. The aspartame in Canderel® is bulked out with carbohydrate so that one teaspoonful is perceived to be as sweet as one teaspoonful of sugar.

    Aspartame is one of the most thoroughly tested food additives (3). Aspartate, phenylalanine and methanol are produced when it is metabolised. The safety of aspartame has been called into question because high blood levels of each of these compounds is associated with toxicity. Because aspartame is approximately 200 times sweeter than sugar very little is needed to provide the equivalent sweetness. The amounts of the amino acids and methanol provided by a normal diet are much larger than those likely to be ingested as aspartame. A teaspoonful of Canderel® contains 20mg phenylalanine while an 8oz glass of milk provides 542mg.

    An infant suffering from the genetic disease phenylketonurea (PKU) is likely to be on a phenylalanine-restricted diet as this can prevent the onset of mental retardation that is associated with the disease. In such cases aspartame should be avoided.

    PROCEDURE
    The procedure for paper chromatography is adapted from a manual by Smith and Feinberg. An experiment on the chromatographic analysis of amino acids forms part of the Nuffield chemistry course (5).

    Stage 1 Hydrolysis of Canderel®
    12g of Canderel® is placed in a round-bottomed flask and 200mL of hydrochloric acid (6M) is added. The mixture is then refluxed for 1/2-1hr. After a short time the mixture will begin to turn brown, by the time this stage is finished it will be black

    Stage 2 Paper chromatography
    Care should be taken to touch the chromatography sheets only at the top corners as fingerprints contain traces of amino acids.

    A small sample of the black mixture is first decolourised by using activated charcoal Pasteur pipette is used to transfer ca 5 mL of the hydeolysate to a clean test-tube, This is decolourised with ca 100 mg activated charcoal and filtered to give a clear solution for spotting onto the chromatogram.

    The solvent mixture (ethanol:water:880 ammonia) is placed in the tank which is covered to produce a saturated atmosphere.

    The paper is prepared and spots of each of the reference amino acids and also the sample are placed on the paper. Pencil identification marks are made at the top of the paper.

    The paper is formed into a cylinder and secured with clips. It is than placed, with the spotted end down, in the tank taking care not to let the paper touch the glass walls. 

     The tank is closed. No observations can be made while the chromatogram is running because the compounds used are colourless. The chromatogram is run for a minimum of 1hr and longer if possible. It is then removed from the tank, the solvent front is marked with a pencil, and the paper is allowed to dry.

    The paper is then hung up in a fume cupboard and sprayed sparingly with the ninhydrin solution. It is then heated in an oven at 110°C for 5-10 min when the amino acids should appear as purple spots.

    The colour is stable for some weeks if kept in the dark and can be photocopied to give a permanent record.

    EXTENSION
    Although aspartame tastes very similar to sucrose, food chemists have to take its chemical properties into account before it is included in a food product in place of sugar. Studies of the stability of aspartame in solution have shown that it is likely to be fully hydrolysed within 9 days at pH 7.4 (2,3). If the aspartame is in a food system when this happens a loss of sweetness will be perceived. This effect could be investigated by dissolving Canderel® in a buffer solution and leaving it for various lengths of time in a warm place.

    REFERENCES

    1. Heaton, H.D., School Sci. Rev., 1985, 66, 728.
    2. Aspartame: Physiology and Biochemistry Lewis, D., Stegink and Filer, L.J., (eds.) Marcel Dekker, 1984.
    3. Food Additive User's Handbook, Smith, J., (ed). London: Blackie, 1991.
    4. Smith, I., and Feinberg, J.G., Paper and Thin Layer Chromatography and Electrophoresis. London: Longman, 1972.
    5. Nuffield Advanced Science Chemistry Students' Book ll. London: Longman, 1984.
    Basic Aerosol Technology
    IN SEARCH OF MORE SOLUTIONS 99
     

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