17 minutes reading time (3315 words)


    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.


    Find out which of the three enzymes provide - rennet, genetically engineered chymosin and a fungal enzyme - is the best for coagulating milk to make curds and whey.

    Traditionally rennet essence, obtained from the stomachs of calves or adult cows, is used for this process but biotechnology has made other enzymes available. The curds are used to make cheese, which is an enormous sector of the food industry. The whey can be treated to produce a sweet syrup that is used in preparing many different sorts of food.


    The pale yellow or brown colour of a sandy beach is due to the presence of iron compounds, mainly iron(III) oxide. Sand is made up of small fragments of rock; the most common mineral in sand is quartz (silica)with small amounts of mica and feldspar. Interestingly, pure silica sand is white. The iron(III) oxide usually forms a thin film or skin around each individual sand grain and it can cause the sand to be yellow-brown or red. There are many fine gradations and differences in hue and these are produced by varying concentrations of the iron(III) oxide colouring material. In addition to this, variations in colour occur in sands of different grain sizes with the same content of colouring material. The iron(III) oxide is on the outside of the sand grains, hence it is relatively easy to remove.

    Find out which of the sand samples contains the most iron.

    Possible procedures
    Isolating the iron(III) oxide
    The removal of the iron(III) oxide is the first practical stage in a chemical analysis of the sand. You have a choice of methods. The notes below are based on methods which are known to work; it is up to you to find the best method. An appropriate sample size for each method is 1g.

    (i) Sublimation method
    A boiling tube with a cold finger condenser is used. A little sand is mixed thoroughly with an approximately equal volume of solid ammonium chloride. The mixture is heated in the boiling tube when the sand will lose most of its colour and a yellow-stained sublimate will be obtained on the surface of the cold-finger and on the walls of the boiling tube. The iron is now in the sublimate a soluble iron(III) chloride.

    (ii) Acid extraction method
    An alternative approach is to dissolve the iron(III) oxide from the surface of the grains by using an acid: 2M hydrochloric acid will give good results (8M sulfuric acid has been used).

    Once the iron(III) ions are in solution the problem is to find a way to compare the amounts extracted from different sand samples.


    Tannin is a substance found naturally in red wines. Wines that contain a lot of tannin are described as 'full' and are said to have body; however, too much tannin makes the wine taste bitter. As wine matures the single tannin molecules slowly polymerise to give six or eight-unit tannins that are less bitter, mellowing the wine.

    Winemaking is a more scientific business than the wine correspondents may lead you to believe. The tannin concentration can be measured by oxidation with potassium manganate(VII) using indigo carmine as the indicator. But the tannin concentrations determined by this method also include the pigments in wine. Like tannins, the pigments belong to a class of compounds known as flavonoids.

    Potassium manganate(VII) also oxidises the alcohol and other substances in the wine, including the indicator. To allow for the indicator and other oxidisable compounds a blank titration is carried out by gently boiling the wine to remove the alcohol. For the blank titration a sample of wine must be treated with activated charcoal to remove the tannins and the pigment.

    Determine the tannin concentration in the samples of wine. How do you account for differences between red and white wine?



    1 hr.
    Extension 1 hr.

    A-level. Higher Grade or equivalent.

    Curriculum links
    Use of enzymes in cheese making. Catalytic action of enzyme.

    Group size

    Materials and equipment

    Materials per group

    • 40mL of whole pasteurised milk.
    • 1mL of rennet essence (of bovine origin, containing both chymosin and pepsin)
    • 1mL of 'Maxiren' (pure chymosin) from genetically engineered yeast
    • 1mL of 'Rennilase' (microbial protease) from a fungus
    • 1mL of deionised water.

    For the extension

    Making a sweet syrup from the whey

    • 2mL of lactase enzyme (Novo 'Lactozym®')
    • 8mL of a 2% (w/v) sodium alginate solution (Note: sodium alginate is not readily soluble and requires both warm water and stirring to dissolve)
    • 100mL of a 1.5% (w/v) calcium chloride solution, DiastixTM available from pharmacies) or other glucose detector.
    Maxiren, Rennilase and Novo Lactozym® are available from the National Centre for Biotechnology Education, Department of Microbiology, University of Reading, Whiteknights, PO Box 228, Reading RG6 2AJ. (Telephone 10734 873743).

    Equipment per group

    • 10mL plastic syringes without needles
    • 4 boiling tubes or sample bottles
    • 4 glass rods
    • Thermometer
    • Stop-watch
    • Access to water bath maintained at 37°C
    • Safety glasses

    For the extension
    • Small piece (about 1 cm2) of nylon gauze eg. net curtain
    • 10mL plastic syringe without needle
    • 10 cm length of 4 mm diameter aquarium airline tubing to fit syringe
    • Aquarium airline tap or adjustable laboratory tubing clip (Hoffman clip)
    • Retort stand, boss and clamp
    • 2 small beakers (ca 100mL) or disposable plastic cups
    • Tea strainer.


    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. London: HMSO, 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.

    This investigation is based on a problem designed by Dean Madden and John Scholar of the National Centre for Biotechnology Education for a practical 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). If the extension is completed this problem will fit well with 'Any glucose?'

    The aim of this experiment is to compare the milk-clotting abilities of the three enzymes. The students should be able to design the experiment and appreciate that they may need to warm the milk to 37°C.

    The formation of a precipitate may then be observed within 5-15 minutes. It may be seen adhering to the sides of the tubes when they are gently rocked from side to side, or clinging to a glass rod dipped into the liquid.

    The activities of the three enzymes should be found to vary markedly.

    You can extend the problem by investigating the activity of the enzymes at different temperatures and at different pH values.

    Making a sweet syrup from the whey
    Whey contains lactose which can be split up by the enzyme lactase to the simpler sugars glucose and galactose. These sugars are sweet and are the basis of a syrup that is used widely in the food industry. An interesting extension is therefore to ask the students to make this sweet syrup, containing glucose, from the whey by using an immobilised enzyme column (see below).

    Practical details
    The enzyme lactase converts the whey to a sweet syrup and this enzyme can be immobilised by trapping it in calcium alginate beads. The beads are packed into a small column, over which the whey is passed. Instructions for setting up the column and treating the whey are given below.

    Immobilising the lactase enzyme and packing the column

    1. Draw up 2mL of enzyme in a syringe and transfer to a small beaker.
    2. Using the same syringe, transfer 8mL of sodium alginate solution to the beaker and mix.
    3. Draw the mixed solution into the syringe.
    4. Pressing the plunger gently, add the mixture to the calcium chloride solution drop by drop.
    5. Strain the beads.
    6. Rinse with distilled water.
    7. Pack the beads into the column
    8. Treat the whey by pouring it through the column. The syrup coming out of the syringe should be found to contain glucose.

    The tip of the syringe must not be allowed to come into contact with the calcium chloride solution. The immobilised enzyme beads should be allowed to harden for a few minutes before being separated from the liquid with a tea-strainer.

    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.


    This activity lends itself to an extended investigation. Once the method of analysis has been developed this could be used as a 2 h class experiment with another group.

    A-level, Higher Grade or equivalent.

    Curriculum links
    Inorganic chemistry of iron compounds. Access to suitable textbooks may be required.

    Group size

    Materials and equipment
    Materials per group

    • 5g of each sample of sand (samples of sand are likely to be available locally. Packets of different colours are available by post from The Needles Pleasure Park. Alum Bay, Isle of Wight PO39 0JD. Tel: 01983 752401.
    The reagents required will vary according to the method of analysis selected; those mentioned in the suggestions below are all cheap and widely available. They include:
    • 10 g ammonium chloride
    • 50mL of 0.5M sodium hydroxide
    • Conc. hydrochloric acid
    • 10mL of 2M hydrochloric acid
    • 10mL of 8M sulfuric acid
    • Deionised water
    • 1L standard iron(III) solution (0.1 g/L or 100 ppm). This solution is prepared by dissolving 0.863 g ammonium iron(III) sulfate (NH4FeSO4.12H2O) in 100mL of conc. hydrochloric acid. The solution is diluted and transferred to a 1L volumetric flask. It is than diluted up to the mark with deionised water and mixed well.
    • Potassium thiocyanate solution. This solution is prepared by dissolving 29.1g potassium thiocyanate (KSCN) in 100mL deionised water.

    Equipment per group

    For the cold finger method
    • Boiling tube
    • Small test-tube
    • Bunsen burner
    • Safety glasses
    • Retort stand and clamps.
    For the colorimetric method
    • Colorimeter with green filter (λ max 490 nm), optically matched cuvettes or colorimeter tubes
    • Four 10mL graduated pipettes
    • Eight 100mL volumetric flasks.
    For the titration
    • 50mL burette
    • Assorted pipettes
    • White tile
    • Conical flasks
    • Glass rod


    Eye protection must be worn.

    Risk assessment

    A risk assessment must be carried out for this activity.

    The origin of the natural colours in coastal landscapes is interesting. The students are invited to apply classical chemical techniques to the problem of analysing sand. The experiments could feature as part of a special study of geochemistry.

    The problem could be set in a vocational context by relating it to the manufacture of glass (4). The sand used in glass manufacture is taken from glass sand quarries. Even a trace of iron will give a greenish colour to the glass. Sand with a low iron(III) oxide content (less than 0.25%) is therefore used in the glass industry. To achieve this the sand is treated with hot sulfuric acid to remove the iron(III) oxide. Hydrochloric acid reacts more readily but it it's too corrosive to the equipment. Sulfuric acid is also available as waste from other processes hence it is cheaper. The iron(III) oxide content must not exceed 0.003% if the glassware produced is to be colourless.

    In the sublimation process, iron(III) chloride forms because the ammonium chloride dissociates, forming hydrogen chloride which reacts with the iron(III) oxide in the sand. The iron(III) chloride sublimes and condenses on the cold finger with the ammonium chloride. When the sublimate is dissolved the solution contains ammonium chloride which may interfere with subsequent analyses. The iron(III) can be precipitated as iron(III) hydroxide by adding sodium hydroxide solution. The mixture can then be filtered and the precipitate redissolved in acid.

    Possible approaches
    (i) Colorimetric method
    The red colour of the iron(III) thiocyanate complex can be used to determine the iron(III) ion concentration by a colorimetric method. The differences in the colours of the samples can be detected by eye, but it is also possible to make more precise measurements. A detailed account of a similar experiment has been given by John Sleigh who uses this method to determine the amount of iron(III) in a metre length of audio tape (5). A drawback to using the thiocyanate reaction is the fading of the colour with time, but this is not a problem with the procedure that he describes which is simple and rapid. The experiment is carried out in an acid medium which is an advantage because this suppresses the hydrolysis of the iron(III) thiocyanate complex. The calibration curve for the colorimeter may be constructed (6). Solutions containing suitable concentrations of iron(III) are prepared by diluting the stock solution (see below). At each concentration a large excess of the thiocyanate is added to transform all the iron(III) salt into the thiocyanate and so produce the maximum coloration.

    Preparation of stock iron(III) solutions
    Aliquots of 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5mL of stock iron(III) solution (0.1 g L-1) are pipetted into separate 100mL volumetric flasks. Each solution is diluted to about 50mL with deionised water, then 10mL of potassium thiocyanate and 10 mL of concentrated hydrochloric acid are added. The solutions are then diluted to the mark with deionised water and mixed well. (The solutions contain 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 ppm of iron(III) respectively),

    The absorbances of these standard solutions should be measured immediately, with a sample containing the same amount of potassium thiocyanate and hydrochloric acid in the reference cell. A calibration curve can be constructed showing absorbance against ppm of iron(III). As the coloration of the iron(III) thiocyanate is sensitive to the presence of other ions it is important that the sample solution should be diluted and treated in a similar way.

    Preparation of sample solution
    A known amount of the sample solution is transferred to a 100mL flask. It is diluted to about 50mL with deionised water. 10mL of the potassium thiocyanate solution and 10mL of concentrated hydrochloric acid are added and the solution is made up to the mark with deionised water. The absorbance is measured immediately and the concentration read off from the calibration curve.

    (ii) Titration
    It is also possible to determine the iron(III) concentration by volumetric methods. The amounts of iron(III) are small and the sensitivity of the various alternatives must be taken into account.

    (a) The iron(III) may be titrated against EDTA. In a method given in an A-level practical textbook (6) and 6% w/v solution of sodium 2-hyroxybenzoate (salicylic acid) in propanone may be used as an indicator. The indicator recommended in Vogel's Quantitative chemical analysis (7) is variamine blue. The solutions are buffered to pH 2-3.

    (b) Alternatively the iron(III) may be reduced to iron(II), by using zinc, and then titrated against potassium manganate(VII).

    Metal ions may be determined by using atomic absorption spectroscopy. The results from the analysis may be compared with those from this instrumental technique.

    Iron(III) oxides are used as pigments in cosmetics. The iron(III) oxide content of two shades of face powder could be compared by methods similar to those used with sand.

    This activity is based on suggestions from Colin Johnson, Eileen Barret and Richard Blair-Gould. Bob Mudd tested the experimental methods described above.



    A-level, Higher Grade or equivalent.

    Curriculum links
    Redox titrations using potassium manganate(VII).

    Group size

    Material and equipment
    Materials per group

    • 50mL samples of red wine (for white wine see below)
    • 50mL of 0.004M potassium manganate(VII)
    • 1g activated charcoal
    • Deionised water
    • 10mL of 0.5% indigo carmine indicator solution. (The indigo carmine indicator is made up by dissolving 0.5 g of the dyestuff in 60mL warm deionised water. The solution is filtered through a No. 42 Whatman paper).
    Equipment per group
    • 50mL burette
    • 5mL and 2mL pipettes
    • Funnel.
    • Filter paper
    • 10mL, 25mL and 250mL measuring cylinders
    • 250mL conical flasks
    • white tile
    • safety glasses
    Eye protection must be worn.

    Risk assessment
    A risk assessment must be carried out for this activity.

    The colour of red wine is due to the presence of anthocyanidins, a class of flavonoids (8). Tannin is a collective name for other largely colourless but bitter flavonoids which are also present in the wine. In making red wine the crushed grapes are put into vats. Some of the stems, the skins, and the pulp remain with the juice forming a residue which is known as the must. The alcohol in the fermenting juice extracts colour from the skins and the longer the juice is in contact with the must the darker the wine. In the process tannin is also extracted into the wine.

    White wine, which should not pick up any colour from the grape skins, is made by pressing the grapes as quickly as possible and the juice alone is then set to ferment. The level of tannin in white wines is only about one-tenth of that found in red wine.

    The procedure described below is based on that described by Professor G.W.A. Fowles of University of Reading (9). During trialling most students needed a lot of guidance to work through this procedure.

    Actual titration
    A funnel is placed in the neck of a 250mL conical flask. 5mL of wine is pipetted and 10mL of deionised water is added. The flask is heated gently until the volume of the wine and water is reduced to 5-7mL. The alcohol will now have boiled off. 25mL of cold deionised water is now added and 2mL of indigo carmine indicator is added by using a pipette. (As the indicator uses up some potassium manganate(VII) it is important to measure it out carefully so that it can be allowed for in the 'blank').

    0.004M KMnO4 is placed in the burette and the mixture is titrated. A golden yellow colour appears at the end point. Let this titre be A mL.

    Blank titration
    20-25mL of the wine is placed in a beaker with 1g of activated charcoal and the mixture is stirred thoroughly. The mixture is then filtered and 5mL of the decolourised wine is transferred to a 250mL conical flask using a pipette.

    The procedure described in 'Actual titration' is repeated. This blank titration will allow for the indicator and for any oxidisable substances in the wine apart from the anthocyanidins and the tannins.

    Let the volume of the blank titre be B mL.

    The amount of potassium manganate(VII) used in oxidising the tannins (and anthocyanidins) is (A - B)mL.

    Tannins are of variable composition. The titration is referred to a standard tannin solution for which 1mL of 0.004M KMnO4 = 0.0832 mg tannin. Therefore % tannin in wine = 0.01664 C.

    The level of tannin for burgundies and clarets will be in the range 0.15 - 0.4%.

    In his book Chemistry in the Marketplace Ben Selinger describes a series of experiments on wine analysis (10) with Australian wines which are designed for first year undergraduates.


    1. D Madden, National Centre for Biotechnology Education Newsletter, p12.Spring 1992.
    2. Dairy Biotechnology National Dairy Council 5/7 John Princes Street, London W1M 0AP.
    3. D Madden, National Centre for Biotechnology Education Newsletter, p22. Spring 1992.
    4. Barret, E., and Beskeen, L., Let's look at sand produced by the Mineral Industry Manpower and Careers Unit.
    5. Sleigh, J., School Sci. Rev.,1988,70,251.
    6. Ratcliff, B., A-level practical chemistry. Cambridge: CUP, 1990.
    7. Jeffery, G.H., et al, Vogel's Textbook of quantitative chemical analysis,5th edn. Harlow: Longman. 1989.
    8. Atkins, P.W., Molecules. New York: Scientific American Library Series, 1987.
    9. Fowles, G.F.W., Educ. Chem., 1978, 15, 89.
    10. Selinger, B., Chemistry in the Marketplace, 4th edn. London: Harcourt Brace Jovanovich, 1989.


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