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The
CLEAPSS
Recipe Book
Introduction to this Edition
Recipe Cards were first produced in 1991 but very quickly demand led to an increase in the scope and
range of the information included. Over the ensuing 20 years we have added more and more
information whilst retaining the card format which users found useful.
This 2011 edition sees a change from cards to A4 sheets. The A5 card has become too small to contain
all the information required for some topics, and we believe that there is no further scope for reducing
the size of the print.
We have extended the number of separate entries from 74 to 106 to allow for more information on
some topics and to make other information easier to locate. We have added colour and some
photographs, where these are useful. The loose leaf A4 format also allows plenty of room for you to
insert recipes and instructions of your own.
Risk Assessment
Where a risk assessment includes control measures these have been incorporated into the instructions,
as describe in our Guidance Leaflet G90, Making and Recording Risk Assessments in School Science.
Each recipe therefore includes model risk assessments but does not include factors that you have to
thinks about, such as technician and teacher experience, and prep room conditions.
As in the previous edition, there is no mention of general bench solutions. In the past such solutions
were often much more concentrated than required and posed unnecessary hazards and risks. The
principle we follow (as in COSHH) is that the concentration of any reagent should be the lowest at
which the procedure works satisfactorily to give the intended result. Sometimes, therefore, we suggest
solutions which may appear to be an odd concentration but this nevertheless is the most suitable. For
example, 0.4 M sodium hydroxide solution is IRRITANT, whereas 0.5 M is CORROSIVE.
Making solutions
The recipes also make use of the laboratory jug as a measuring tool. Although apparently not
particularly accurate, we routinely achieve concentrations of between 1.95 and 2.05 M when using a jug
to prepare 2M sulfuric(VI) acid from concentrated sulfuric(VI) acid during the Practical Techniques in
Chemistry course. Clearly, using a jug can produce solutions with concentrations that are sufficiently
accurate for many laboratory purposes.
You will find more detail about making solutions in section 7.6 of the Handbook. If you run into
difficulties not covered in either the Recipe Book or the Handbook, phone CLEAPSS on 01895 251496
but do try the index first.
Contents
Recipe sheet Number Recipe sheet Number
Agar 1 Cobalt(II) chloride solution and 30
Alcohol/water and 2 thermochromic liquid
propanone/water solutions Copper(II) solutions 31
Alginate beads 3 Crude oil alternative 32
Aluminium solutions 4 2,4-Dinitrophenylhydrazine 33
Ammonia gas 5 solution
Ammonia solution (ammonium 6 3,5-Dinitrosalicylic acid 34

hydroxide) Drosophila food base 35
Ammonium chloride 7 Electroplating solutions 36
Ammonium vanadate(V) solution 8 Enzymes 37
Azo dyes 9 Etching solutions 38
Barium solutions 10 Ethanoic acid 39
Benedicts qualitative reagent 11 Fehlings solutions 40
Benedicts quantitative reagent 12 Fixatives used before preserving 41
Biochemical indicators and tests 13 biological specimens
Gases less commonly used in 42
Bismuth nitrate(V) solution 14
schools
Biuret reagent 15
Hydrochloric acid 43
Brodies fluid 16
Hydrogen gas 44
Bromine water 17
Hydrogen peroxide 45
Buffer solutions 18
Indicators (acid-base) 46
Calcium chloride and nitrate(V) 19
Indicator (universal) 47
solutions
Indicators (carbon dioxide) 48
Calcium hydroxide solution 20
Indicators for redox, precipitation 49
Carbon dioxide 21
and complexometric titrations
Cerium(IV) solutions 22
Iodine solution 50
Chemiluminesence reactions 23
Iron(II) solutions 51
Chlorine gas 24
Iron(III) solutions 52
Chlorine water 25
Lead(II) nitrate(V) 53
Chromatography solvents and 26
Lithium chloride 54
locating agents
Magnesium sulfate(VI) 55
Chromium(III) chloride and 27
chrome alum solutions Manganese(II) sulfate(VI) 56
Citric acid 28 Mercury solutions 57
Clock reactions 29 Methanal solution 58

Methanoic acid 59
Recipe sheet Number Recipe sheet Number
Nickel sulfate(VI) 60 Sodium hydrogencarbonate 84
Nitric(V) acid 61 Sodium hydroxide 85
Nylon rope experiment 62 Sodium silicate, the crystal 86
Oscillating reactions 63 (chemical) garden and silicate
gels
Oxygen gas 64
Sodium thiosulfate 87
Phosphoric(V) acid 65
Stains for bacterial activity 88
Plant mineral requirement 66
solutions Stains for cell contents 89
Potassium and sodium 67 Stains for electrophoresis 90

phosphates Stains for fungal material 91
Potassium chloride 68 Stains for metabolic activity 92
Potassium chromate(VI) 69 Stains for plant material 93
Potassium dichromate(VI) 70 Standard solutions for titration 94
Potassium hydroxide 71 Strontium chloride 95
Potassium iodide 72 Sulfur dioxide 96
Potassium manganate(VII) 73 Sulfur dioxide solution 97
Preservatives used after fixing 74 Sulfuric(VI) acid 98
biological specimens Testing for gases 99
Ringers and other saline 75 Testing for negative ions 100
solutions for physiological use
Testing for positive ions 101
Sandells solution 76
Testing for organic functional 102
Silver nitrate(V) 77 groups
Slime 78 Tin(II) chloride 103
Soap and bubble solutions 79 Water (sea and hard) 104
Sodium carbonate 80 Winklers method for dissolved 105
Sodium chlorate(I) solution 81 oxygen
Sodium chloride 82 Zinc sulfate(VI) 106
Sodium ethanoate 83
CLEAPSS Recipe Book
1 Agar
For microbiological activities using purchased media, follow the instructions on the bottle.
The recipes are grouped into agars for microbiology and agars for other activities. All agars for
microbiological work need to be sterilised before and after use.
General Hazards Agar inhaled as a fine powder may cause an allergic reaction or other respiratory
problems. The use of agar that could isolate human pathogens (eg, blood agar) should
be avoided.
Control Use a balance in a non-working fume cupboard, ie, not switched on, with the sash
measures down, to weigh out agar. Use heatproof gloves to protect from scalding when handling
freshly-sterilised molten agar.
Procedure for Mix 1.5 g of agar with 10 ml of water into a paste. Slowly add more water with stirring
preparing until the volume is 100 ml. Heat the mixture with stirring on a boiling water bath to
technical agar 95 C in the required container. This preliminary heating can be omitted if the agar is
(also called going to be sterilised immediately, unless it is necessary to decant the agar into
agar-agar) smaller containers. In acid media, the amount of agar should be increased from 1.5 to
2 g. If the solidified agar in any recipe is too sloppy or too firm, repeat the procedure
using slightly more or less agar.
The agar gel is not stable in strongly alkaline solutions.
Usual sterilising (If required) Autoclave the container(s) with the made-up suspension(s) for 15 minutes
conditions at 15 psi (121 C).
Agars for microbiology

China blue lactose Use 3.6 g of China blue lactose agar powder in 100 ml of distilled water. If this mix
agar proves too thin for rough handling by students, then thicken by adding 0.5 g of agar-
agar (just thickener, no nutrients).
Crystal violet In a fume cupboard which is not switched on, add 0.005 g of crystal violet (HARMFUL,
agar. A selective DANGEROUS FOR THE ENVIRONMENT) to 1 litre of liquid nutrient agar solution. The
(against Gram resultant solution is low hazard.
positive) medium
for soil bacteria
Glucose nutrient Add 0.5% w/v of glucose to molten nutrient agar.
agar
Malt agar for fungi Mix 2 g of malt extract with 2 g of agar with 10 ml of water into a paste. Slowly add
more water with stirring until the volume is 100 ml. Autoclave the suspension at
10 psi (115 C) for 10 minutes.
Mannitol yeast Mix 10 g agar in 1 litre of water, and dissolve in a boiling water bath. Add 0.5 g
extract agar for K2HPO4, 0.2 g MgSO4.7H2O, 0.2 g NaCl, 0.2 g CaCl2.6H2O,10 g mannitol, and 0.4 g
growing root yeast extract. Dispense as required and sterilise by autoclaving before use.
nodule bacteria
Nitrogen-free Dissolve 0.05 g FeCl3.6H2O in 500 ml distilled water. Add 2 g K2HPO4, 0.25 g
mineral salts agar MgSO4.7H2O and 10 g glucose. Check the pH and adjust, if necessary, to 8.3 using
for growing 0.1 M NaOH. Pour into a bottle containing 1 g CaCO3 and 7.5 g agar powder. Mix
nitrogen-fixing and autoclave at 121 C for 20 minutes. Before pouring plates, swirl to thoroughly
bacteria mix the CaCO3 and agar.
Nutrient agar for Mix 2 g of Bovril, 0.5 g of sodium chloride and 1.5 g of agar with 10 ml of water into
bacteria a paste. Slowly add more water with stirring until the volume is 100 ml. Heat/sterilise
the suspension as in Usual sterilising conditions above.
Starch malt agar Mix 3 g of light malt powder (from home-brewing shops), 0.5 g of peptone (to
for growth of promote growth) in 20 ml of water. Also make a paste containing 1 g of soluble
fungi and starch in 10 ml of hot water. Add these two solutions to 1.5 g of agar with stirring and
digestion of slowly add more water with stirring until the volume is 100 ml. Stir before decanting
starch into smaller containers (if required) and sterilising. Autoclave the suspension at
10 psi (115 C) for 10 minutes.
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Agars for other activities

These should be made up as required, and not stored for long periods to avoid any unwanted microbial
activity. Dispose of as soon as possible after the activity. Sterilise in an autoclave any which are suspected of
microbial contamination.
Agar for starch Mix 0.5 g of glucose-1-phosphate and 1.5 g of agar with 10 ml of water. Slowly add
synthesis more water with stirring until the volume is 100 ml. Boiling, not sterilising, should be
sufficient.
Electrolytic agar Add one gram of sodium sulfate(VI) or other electrolyte to the hot agar solution
before pouring.
Ferroxyl agar gel Add 1.4 g of agar, 2 g of sodium chloride, 0.1 g of potassium hexacyanoferrate(III)
for rusting and 1 ml of phenolphthalein solution to the 100 ml of water and warm, with stirring, to
experiments 95 C. Pour the solution into Petri dishes.
Indicator agar Add 1 ml of the chosen indicator solution to the agar solution before pouring.
Mayonnaise agar Dilute 4 g salad cream or mayonnaise with 5 ml water and add 1 ml 0.1 M sodium
for lipase activity hydroxide solution (IRRITANT). Add about 1 ml of this alkaline mixture to a solution of
(1) bromocresol green dye (about 0.003 g in 100 ml water) until the mixture just turns
blue-green and stir to ensure even distribution. Boil the resultant mixture with 2 g
agar, cool to 50-60 C then pour thin layers in Petri dishes. The plates will need to be
incubated at 30 C for 24 hours before being examined for orange-yellow areas
produced by lipases.
Mayonnaise agar Alternatively, mayonnaise agar can be made up without the dye and, after
for lipase activity incubation, the plates can be flooded with 0.4 M copper(II) sulfate solution and left for
(2) 30 minutes before being examined. Clear areas in the blue-green matrix indicate
where lipases have broken down the fatty acids in the mayonnaise.
Milk agar for Stir together 2 g low-fat milk powder (Marvel is recommended as it contains very little
protease activity fat), 1 g agar and 100 ml water. Heat as for technical agar and pour into Petri dishes
in very thin layers. Proteases should produce clear patches by breaking down
proteins in the milk within 30 minutes or so.
Phenolphthalein Use 2 g agar in 100 ml boiling distilled (or deionised) water in a beaker. Add 10 ml of
indicator agar 0.2 M sodium carbonate solution and 5 ml of phenolphthalein into the beaker and stir
well.
Carbon dioxide in the atmosphere causes the colour to fade on storage, so the agar
is better prepared shortly before it is required.
Starch agar for Mix a paste containing 1 g of soluble starch in 10 ml of cold water. Add 1.5 g of agar,
amylase activity stir well and slowly add more water with stirring until the volume is 100 ml. Heat as
for technical agar above.
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2 Alcohol/water and propanone/water solutions

Quoted flash points appear to vary slightly between sources.
To prepare molar solutions, liquids can be weighed in tared containers.
2 M propanone solution is used in a rate of reaction experiment called the iodination of propanone.
To prepare 100 ml of an x% (v/v) solution of ethanol in water

Add x ml of ethanol to a 100 ml measuring cylinder.
Add water up to the 100 ml mark.
Label the solution. If it is highly flammable, then it needs the hazard warning label but, if flammable, a
label is not needed. However, the hazard classification needs to be written on any risk assessment.
% ethanol 0 10 20 30 40 50 60 70 80 90 100
Flash point (C) - 49 36 29 26 24 22 21 20 17 13
Density (g cm-3) at 20 C 1.00 0.98 0.97 0.96 0.95 0.93 0.91 0.89 0.86 0.83 0.78
Hazard - FLAMMABLE HIGHLY FLAMMABLE
To prepare 1 litre of 1 M methanol solution

Weigh out 32 g of methanol in a tared container or measure out 41 ml of methanol in a measuring
cylinder.
Add this to a 1000 ml measuring cylinder or 1 litre measuring jug.
Add water up to the 1 litre mark.
Label the solution harmful.
To prepare 1 litre of 1 M ethanol solution

Weigh out 46 g of ethanol in a tared container or measure out 58 ml of ethanol in a measuring
cylinder.
The solution is low hazard.
To prepare 1 litre of 2 M propanone solution

Weigh out 116 g of propanone in a tared container or measure out 147 ml of propanone in a
measuring cylinder.
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3 Alginate beads
In studies of enzymes or the physiology of yeast cells, a valuable technique is to immobilise the enzyme or
cells inside beads of sodium alginate. The beads containing the enzyme/cells can then be used as usual or
packed into a column (eg, a syringe barrel) and a suitable substrate passed over them. The products are
collected at the bottom of the column and the immobilised enzymes or cells can be used again.
When making up the alginate and enzyme solutions it is essential to use purified water; otherwise calcium
ions in the water will cause the alginate to set prematurely.
Alginate beads can usually be stored overnight, covered and refrigerated but are unlikely to keep longer than
their non-immobilised components.
Always trial practicals to confirm activity of organisms or enzymes.
Preparing immobilised enzymes/cells in alginate beads

Make up a solution of the enzyme to be studied (see Recipe sheet 37 for enzymes), or a suspension
of yeast cells using purified water.
Sprinkle 2 g of sodium alginate in 100 ml of warm, purified water and mix using a mechanical stirrer.
Allow the solution to cool. Initially the mixture will form glutinous lumps, but it becomes smooth over
time.
Dissolve 3 g of calcium chloride-6-water in 200 ml of purified water in a 250 ml beaker.
Mix 2 ml of the enzyme/suspension with 8 ml of the 2% sodium alginate solution. Variations on these
proportions may be used.
Draw this up into a 10 ml syringe.
Add the sodium alginate/enzyme (or cell) mixture one drop at a time to the calcium chloride solution
making sure the tip of the syringe is held above the solution in the beaker.
Allow the beads to harden for a few minutes before straining them out of the beaker.
Hardened alginate beads in calcium chloride solution
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4 Aluminium solutions
Hydrated aluminium salts such as the chloride (AlCl6.6H2O, M = 241.5 g mol-1), sulfate(VI) (Al2(SO4)3.16H2O,
M = 630 g mol-1) and nitrate(V) (Al(NO3)3.9H2O, M = 375 g mol-1) absorb water (ie, they are hygroscopic) and
become damp on storage. Do check these chemicals before use.
Aluminium potassium sulfate(VI), also known as potassium aluminium sulfate, alum and potash alum is
easily stored and suitable for all activities where aluminium ions are required for testing. However, it is not all
that soluble in water, although it does make large octagonal crystals. Aluminium solutions are acidic.
Aluminium potassium sulfate(VI)

Formula: AlK(SO4)2.12H2O Molar mass: 474.39 g mol-1 Solubility: 11 g per 100 ml
General Hazards See Hazcard 2B. Aluminium salts in water are acidic.
Never use anhydrous aluminium chloride to make solutions. It reacts violently with
water producing toxic fumes of hydrogen chloride.
Mass (g) of solid to be used

Concentration Volume (ml) of solution required Hazard warning
required 100 250 1000 label
0.01 M Ten-fold dilution of the 0.1 M solution -
0.1 M 4.74 11.86 47.44 -
0.2 M 9.49 23.72 94.88 -
Saturated (20 C) 12 29 114 -
Aluminium chloride (hydrated)

Formula: AlCl3.6H2O Molar mass: 241.43 g mol-1 Solubility: 83 g per 100 ml
General Hazards See Hazcard 2A. Aluminium salts in water are acidic.

0.1 M 2.41 6.03 24.14 -
0.5 M 12.07 30.18 120.72 -
Saturated (20 C) 90 225 900 IRRITANT
Preparing solutions of aluminium salts

Wear eye protection.
Measure out the indicated quantity of the aluminium salt.
Add the solid to about two thirds of the final volume of water in a beaker or laboratory jug.
Stir to dissolve, warming if necessary.
Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
water to the required level.
Pour into a labelled bottle.
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5 Ammonia gas
Ammonia is less dense than air and very soluble in water so it has to be collected by the downward
displacement of air (upward delivery).
Theoretically, 2.1 ml of fresh concentrated (880) ammonia solution produces 1 litre of gas, although this is
never achieved in practice, so always use an excess. Older stocks of concentrated ammonia will be less
concentrated.
If the fountain experiment is to be carried out, a round bottom flask is substituted for the gas jar in the
diagram below. Many standard text books use a calcium oxide drying tube. There is no real need for this.
General Hazards See Hazcards 5 & 6. Ammonia begins to bubble off from 880 ammonia at about
55 C so heat gently and use anti-bumping granules to allow the ammonia to boil
off gradually.
Preparing ammonia gas

Use a fume cupboard. Wear goggles.
Add 10 ml of concentrated ammonia to the boiling tube
(CORROSIVE).
Inverted gas jar
Set up the equipment as shown on the right. held by a clamp
Warm the boiling tube gently for about 5 minutes.
Knowing when the gas jar is full is not easy. Placing
moist red litmus at the neck of the inverted flask is not a
good indicator that the flask is full of gas. It is a matter of
judgement and experience. It might be better to use a
fresh boiling tube of concentrated ammonia for each gas
jar or flask required.
When the collection is finished, place a cover slip or,
better still, a large bung into the opening of the gas jar.
Store the gas jar upside down because ammonia is
lighter than air. Anti-bumping
The residual ammonia solution can be poured down the granules 10 ml of concentrated
ammonia solution
sink in a fume cupboard with plenty of water.
Heat
gently
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6 Ammonia solution (ammonium hydroxide)

The concentration of 35% (w/v) ammonia solution, (also known as 880 ammonia), is 18.1 mol dm-3.
Educational suppliers commonly supply this concentration although other concentrated solutions, eg,
25% (w/v) are available.
If kept for long periods, the concentration of ammonia solutions decreases because of leakage of gas from
the container.
If the concentrated ammonia is several years old it would be wise to test the concentration of the solution
before diluting further.
It is sensible not to store diluted ammonia solutions for long periods. It is better to prepare diluted solutions
only when required.
Solutions less than 1 M should be made by further dilution of 1 M ammonia solution and are best made fresh
before use.
If you have other concentrations of concentrated ammonia, then contact CLEAPSS for more advice. Some of
the values below are different from previous Recipe Cards as more precise information is now available.
Formula: NH3 Molar mass: 17.03 g mol-1 Solubility: infinite
General Hazards Concentrated ammonia; see Hazcards 5 & 6.

Take great care when opening bottles of concentrated ammonia on hot days.
Volume of 880 ammonia to be used

0.1 M Ten-fold dilution of the 1 M solution -
0.5 M Two-fold dilution of 1 M solution -
1M 28 55 138 -
2M 55 111 276 -
5M 138 276 691 IRRITANT
Preparing ammonia solutions

Use a fume cupboard if concentrated ammonia or solutions more concentrated than 5 M ammonia are
used. Wear goggles (a face shield is preferable when handling large volumes) and chemical resistant
gloves.
Measure the indicated quantity of ammonia solution in an appropriate measuring cylinder.
Add the liquid to about two thirds of the final volume of water in an appropriate beaker or laboratory
jug.
Pour into a labelled bottle and mix well. Add a hazard warning if appropriate.
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7 Ammonium chloride
Formula: NH4Cl Molar mass: 53.5 g mol-1 Solubility: 36 g per 100 ml
General Hazards See Hazcard 9A.

0.5 M 2.68 6.69 26.75 -
1.0 M 5.34 13.38 53.50 -
Saturated (20 C) 40 100 400 -
Preparing ammonium chloride solution

Measure out the indicated quantity of ammonium chloride.
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8 Ammonium vanadate(V) solution

Also known as ammonium polytrioxovanadate(V) and ammonium metavanadate.
Ammonium vanadate solution is used to illustrate the various oxidising states exhibited by vanadium.
Ammonium vanadate is not very soluble in pure water and has to be dissolved in alkali first, before making
acidic for use.
Formula: NH4VO3 Molar mass: 117 g mol-1 Solubility: 0.52 g per 100 ml
General Hazards Ammonium vanadate(V) is TOXIC. See Hazcards 9B, 91 and 98A.
Preparing 100 ml of 0.1 M ammonium vanadate solution

Wear goggles.
Dissolve 1.17 g of ammonium vanadate(V) in 20 ml of 2 M sodium hydroxide in a beaker. (The odour
of ammonia may be detected but it will cause no harm.)
Transfer the solution to a 100 ml measuring cylinder.
Add 1 M sulfuric(VI) acid to bring the total volume to 100 ml.
The yellow solution is irritant because of the presence of sulfuric(VI) acid.
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9 Azo dyes
It is a myth that all azo dyes are carcinogens. The dyes can be prepared in schools as long as sensible
laboratory procedures are observed. On the whole, the more soluble a dye and the presence of sulfonic acid
groups on the benzene ring, then the safer the dye (eg, Orange II and methyl orange).
See Guide G195 for preparation of azo dyes from ethyl 4-aminobenzoate.
The recipes below can be used safely in schools.
General Hazards Sulfanilic acid (4-aminobenzenesulfonic acid), see Hazcard 4B. Sodium nitrate(III)
(sodium nitrite); see Hazcard 93. Naphthalen-2-ol; see Hazcard 70.
N,N-dimethylphenylamine; see Hazcard 4B. Sodium hydroxide; see Hazcard 91.
Ethanoic acid; see Hazcard 38A. Sodium carbonate; see Hazcard 95A.
Methyl orange; see Hazcard 32.
Preparing azo dyes as described is a two-stage process.

Stage 1: Preparing the diazonium salt of sulfanilic acid
Solution A: Solution B:
Add to a boiling tube, 0.6 g of sulfanilic acid, In another boiling tube, cool down 8 ml of
0.2 g of anhydrous sodium carbonate and 10 ml 1 M hydrochloric acid using an ice-water
of purified water. bath.
Warm the mixture to boiling and then cool under
the tap. Add 0.3 g of sodium nitrate(III) and
agitate the test tube until the salt dissolves.
Add solution A to solution B. The diazonium salt settles out. It is more stable then many others and
will keep for some hours.
Test for the presence of free nitrous acid with starch-iodide paper. If the paper does not turn blue,
add a little more sodium nitrate(III). Swirl the contents of the boiling tube and divide the contents into
two test tubes.
Stage 2 (a): Preparing Orange II (IRRITANT) and dying cotton with it

Wear goggles. Wear disposable nitrile gloves.
In a test tube, dissolve 0.25 g of naphthalen-2-ol* in 4 ml of 1 M sodium hydroxide solution by
warming.
Cool the solution in an ice-water bath and pour it into a Petri dish.
Using forceps, add white cotton wool or cotton cloth (eg, bandage material) to the Petri dish to soak up
the solution.
Add the contents of the test tube containing the diazonium salt onto the cotton. The dye will appear.
Remove the cotton with forceps from the Petri dish and rinse it under cold water to remove any solid
dye. This is a soluble dye so do not do this for too long. Allow the cotton to dry.
* Other phenols, benzene diols, cresols and naphthols can be used with the diazonium salt of sulfanilic acid to produce other dyes.
Stage 2 (b): Preparing methyl orange (TOXIC)

Wear goggles. Wear disposable nitrile gloves.
In a test tube, add 0.2 ml of N,N-dimethylphenylamine to 0.2 ml of glacial ethanoic acid and agitate the
mixture well.
Add the contents of the test tube containing the diazonium salt to the test tube containing
N,N-dimethylphenylamine. Leave for 5 minutes for the red dye to form.
Add 2 ml of 2 M sodium hydroxide solution, heat to boiling and allow the solution to cool.
The solid orange sodium salt of methyl orange should form.
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10 Barium solutions
Purified water should be used to avoid cloudiness caused by the precipitation of barium sulfate(VI).
Barium chloride is classified as TOXIC if swallowed and barium nitrate(V) as HARMFUL if swallowed.
All solutions of barium nitrate(V) are LOW HAZARD.
Barium chloride is more soluble in water than barium nitrate(V).
General Hazards Barium chloride; see Hazcard 10A. Barium nitrate(V); see Hazcard 11.
Barium chloride
Formula: BaCl2.2H2O Molar mass: 244.26 g mol-1 Solubility: 26 g per 100 ml

0.01 M Ten-fold dilution of 0.1 M solution -
0.1 M 2.44 6.11 24.43 HARMFUL
0.5 M 12.21 30.53 122.13 HARMFUL
Saturated (20 C) 36 90 260 TOXIC
Barium nitrate
Formula: Ba(NO3)2 Molar mass: 261.37 g mol-1 Solubility: 9 g per 100 ml

0.1 M 2.61 6.53 26.14 -
Saturated (20 C) 9 45 90 -
Preparing barium salts solutions

Wear eye protection. Wear disposable nitrile gloves when weighing and preparing the solution.
Measure out the indicated quantity of the solid barium salt.
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11 Benedicts qualitative reagent

Benedicts solution or DNSA (see Recipe sheet 34) should be used in place of Fehlings solution to test for
reducing sugars because it is less hazardous.
Glucose, lactose and maltose are reducing sugars and give a positive test. Sucrose is a non-reducing sugar
and does not give a positive result.
Benedicts solution is less satisfactory in testing for non-reducing sugars and aldehydes in organic chemistry
in which case Fehlings or Sandells solutions will be needed.
No hazard warning symbol is required on the bottle as the concentrations of each of the constituents are low.
This solution is not suitable for colorimetric work. See Recipe sheet 12 for Benedicts quantitative reagent or
Recipe sheet 34 for DNSA.
To differentiate between reducing sugars, enzyme tests are required.
General Hazards Sodium carbonate; see Hazcard 95A. Copper sulfate(VI); see Hazcard 27C.
Procedure to produce 1 litre of solution

Measure out 170 g of trisodium citrate-2-water and 100 g of anhydrous sodium carbonate (or 256 g of
hydrated sodium carbonate).
Add the solids to about 850 ml of purified water in a 1 litre beaker.
Add 17.4 g of copper(II) sulfate(VI)-5-water and stir to dissolve.
Pour the solution into an appropriate measuring cylinder and dilute to the final volume.
Filter if necessary.
Pour into a labelled bottle and mix well.
Procedure for carrying out the test

The material under test is mixed with about 1 ml of water in a test tube or vial, and about 3 ml of
Benedict's reagent is added.
Place the test tube in a boiling water bath for about 5 minutes.
The colour should progress from blue (with no glucose present) to green, yellow, orange, red, and
then brick red or brown as glucose concentration increases.
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12 Benedicts quantitative reagent

The procedure detects the loss of blue colour as the sugar reduces the copper(II) ions to copper(I).
It can be used either in volumetric or colorimetric methods.
The addition of thiocyanate forms a complex and prevents the precipitation of copper(I) oxide.
See also Recipe sheet 34 for DNSA which is an alternative test for reducing sugars.
General Hazards Sodium carbonate; see Hazcard 95A. Copper sulfate(VI); see Hazcard 27C.
Potassium thiocyanate; Hazcard 95C. Potassium hexacyanoferrate(II) see
Hazcard 79. The volumetric method uses hot liquids.
Preparing 1 litre of Benedicts quantitative solution (BQS)

Measure out 75 g of anhydrous (or 160 g of hydrated) sodium carbonate, 200 g of trisodium citrate-2-
water and 125 g of potassium thiocyanate. Add these solids to 700 ml of boiled distilled water in a 1
litre beaker. Stir to dissolve, reheating if necessary.
Measure out 18 g of copper(II) sulfate(VI), dissolve in 100 ml of purified water and add this, with
constant stirring, to the solution made in step 1.
Add 0.25 g of potassium hexacyanoferrate(II) and pour the solution into a suitable volumetric flask.
Dilute to the final volume with purified water and mix well. For the colorimetric procedure below, dilute
35 ml of this solution to 100 ml with water. (No hazard warning label is required at these
concentrations.)
Using the solution: volumetric procedure

Place a 0.3% solution of glucose in a burette.
Place 10 ml of BQS into a 100 ml conical flask, heat to boiling and add about 2 g of anhydrous sodium
carbonate.
Add the glucose solution in 0.5 ml amounts, boiling each time until the blue or green colour just
disappears. Record the total volume of glucose added. When nearing the endpoint it is advisable to
boil for 30 seconds to avoid overshooting.
Repeat the procedure to confirm your result.
To measure the concentration of glucose in an unknown solution, repeat the procedure with glucose
solutions of unknown concentration.
By comparing the volume obtained on titration of the known and unknown samples, the %
concentration of the unknown can be calculated.
0.3 x
%
A = titration volume for known glucose solution

B = titration volume for unknown glucose solution
(Some methods suggest adding a few drops of 1% aqueous methylene blue solution to act as an
indicator. It should be added when enough glucose solution has been added to turn the original
solution a very pale blue colour.)
Using the solution: colorimetric procedure

Place a 1% solution of glucose in a burette.
Place water in another burette and prepare a series of glucose solutions of varying concentration from
0 to 1%.
In a series of labelled fresh test tubes, add 1 ml of each solution prepared in step 2, to 10 ml of BQS.
Place these in a boiling water bath for a few minutes.
Allow the solutions to cool and any precipitates to settle.
Use a red or yellow filter in the colorimeter. Obtain the absorbance of light through each solution.
Use these readings to construct a glucose concentration calibration curve.
Use the calibration curve to identify unknown concentrations of glucose solution.
CLEAPSS 2011 13
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13 Biochemical indicators and tests

Amino acid, polypeptide and protein tests
See also Recipe Sheet 15 for the Biuret test.
Coles Test for Wear goggles and gloves. Dissolve 10 g of mercury(II) See Hazcards
modification soluble sulfate(VI) in 100 ml of 2 M sulfuric(VI) acid (solution A). 62, 93, 98A.
of Millons proteins Separately, dissolve 1 g of sodium nitrate(III) (nitrite) in Solution A and
reagent 100 ml of water (solution B). Before use, mix two volumes the combined
of solution A with one of solution B. A reddish-brown solution are
colouration or precipitate indicates the presence of soluble TOXIC.
proteins.
Marquiss Test for Wear goggles and gloves. Use a fume cupboard. Add 2 See Hazcards
reagent alkaloids drops of 40% methanal solution to 3 ml of concentrated 63 and 98.
sulfuric(VI) acid just before use. This is a spot test and Label the
various colours appear. See solution
http://en.wikipedia.org/wiki/Marquis_reagent. CORROSIVE and
TOXIC.
Ninhydrin For amine Wear eye protection. Dissolve 0.1 g of ninhydrin in 100 ml See Hazcard
groups of water. A purple colour, known as Ruhemann's purple, is 66. No hazard
produced when ninhydrin reacts with primary and warning is
secondary amines, indicating the presence of amino acids. required on the
Can be used as a spray or a dip, but needs to be heated, in solution.
an oven at 110 C or with a hairdryer, for the colours to
appear.
Sakaguchi Test for Wear goggles. Dissolve 5 g of sodium hydroxide in 100 ml See Hazcards
test proteins of water (solution A). Dissolve 1 g of napthalen-1-ol in 70, 89 & 91.
containing 100 ml of water (solution B). For the test, one drop of
arginine sodium chlorate(I) solution (10-14% available chlorine, see
Recipe sheet 81) is required as well. Proteins containing
arginine appear an intense red colour.
Carbohydrate tests
See also 3,5-dinitrosalacylic acid (DNSA), Benedicts solution, Sandells solution and Fehlings solution.
Molischs For all Wear goggles. Dissolve 5 g of napthalen-1-ol in 100 ml of See Hazcards
solution carbo- ethanol. 40A, 70 & 98.
hydrates The solution containing a possible carbohydrate is Label the
combined with a small amount of Molisch's reagent in a test solution HIGHLY
tube. After mixing, a small amount of concentrated FLAMMABLE &
sulfuric(VI) acid is slowly added down the sides of the HARMFUL .
sloping test tube, without mixing, to form a lower layer. Look
for a purple ring at the interface of the two layers.
Periodic For Wear goggles and chemical-resistant gloves. Dissolve 1 g Iodic(VII) acid is
acid Schiff poly- of iodic(VII) acid (periodic acid) in 100 ml of water. Used in CORROSIVE &
saccharides
(PAS) conjunction with Schiffs reagent. Changes from colourless OXIDISING. No
reaction to purple. hazard warning
is required on
the solution.
14 CLEAPSS 2011
CLEAPSS Recipe Book
Cellular respiration
Janus Wear eye protection and disposable nitrile gloves when making up the No hazard
green B solution. Dissolve 0.3 g of the dye in 100 ml of purified water. Dilute this label is
solution ten times with water before use. Colour changes are from blue to required on
salmon pink. the solution.
See Guidance leaflet PS88 for more details including a practical activity.
Methylene Wear eye protection, and gloves to avoid staining the skin. See Hazcards
blue Dissolve 1 g solid in 100 ml water and add 0.6 g sodium chloride. The blue 32 and 40. No
indicator turns colourless as the dye is reduced. hazard label is
required on
the solution.
TTC Dissolve 1 g of 2,3,5-triphenyl tetrazolium chloride (TTC) in 100 ml of water. Low hazard.
(A 0.5% solution is less expensive and gives just as good results but takes
longer. It works well with maize seedlings.) Turns red as the dye is reduced.
Tasting and genetics studies

Phenylthio- This is also called PTC, phenylthiourea or PTU. Dissolve 0.05 g in 100 ml of The solid is
carbamide water. Filter paper is soaked in the solution and then hung up to dry before VERY TOXIC;
cutting into strips. See Handbook 15.13 for details on tasting investigations. see Hazcard
35. The
solution is low
hazard.
Vitamin C
DCPIP Also called 2,6-dichlorophenol indophenol, and phenol-indodichlorophenol. Both are low
solution Dissolve 0.1 g of dye in 100 ml of water. The standard vitamin C solution hazard
should also be 0.1% (w/v). Add the blue dye until the colour does not chemicals.
disappear.
CLEAPSS 2011 15
CLEAPSS Recipe Book
14 Bismuth nitrate(V) solution

The solutions need to be kept acidic to avoid the formation of insoluble basic salts.
Formula: Bi(NO3)3.5H2O Molar mass: 395 g mol-1
General Hazards See Hazcards 67 & 73B.
Preparing 100 ml of 0.1 M of bismuth nitrate(V) solution

Wear goggles.
Dissolve 5.34 g of bismuth(III) nitrate(V)5-water in 70 ml of 1 M nitric(V) acid.
Make up to 100 ml with 1 M nitric(V) acid.
Label the solution CORROSIVE.
16 CLEAPSS 2011
CLEAPSS Recipe Book
15 Biuret reagent
Biuret reagent tests for proteins.
Previous methods and recipes for this solution have used more concentrated solutions than that described
below. This meant that Y7 and 8 pupils were precluded from doing this experiment according to the advice
on Hazcard 91 (sodium hydroxide). However, the procedure below uses 0.1 M sodium hydroxide and
0.01 M copper(II) sulfate(VI) solutions and teachers might find that further dilution is possible.
It may be necessary to make acidic samples alkaline before the test for proteins is carried out.
Coomassie blue can also be used to test for proteins. It is available in a test kit with instructions for use. Its
advantages are that it does not require heating and is very sensitive.
Qualitative Biuret Reagent

This does not keep so only prepare what is required.
General Hazards Sodium hydroxide (solid) and 2 M solution. See Hazcards 91. Copper sulphate, see
Hazcard 27C.
Preparing 1 litre of Qualitative Biuret reagent

Wear goggles.
Weigh out 0.75 g of copper(II) sulfate(VI)-5 -water.
Prepare 1 litre of 2 M potassium or sodium hydroxide solution.
Dissolve the copper(II) sulfate(VI) in the alkali and label the solution CORROSIVE.
Procedure and use of Biuret solution suitable for Y7 and 8

Wear suitable eye protection.
Prepare 0.01 M copper(II) sulfate(VI) solution (Recipe Sheet 31).
Prepare 0.1 M sodium hydroxide solution (Recipe Sheet 85).
Place the sample of a liquid to be tested in a test tube to a depth of 10 mm.
Add the same volume of 0.1 M sodium hydroxide solution and agitate the test tube.
Add a few drops of the 0.01 M copper(II) sulfate(VI) solution and agitate the test tube.
A purple or pink colouration indicates the presence of protein.
Quantitative Biuret Reagent

This does keep.
General Hazards Sodium hydroxide (solid) and a 2 M solution see Hazcard 91. Copper(II) sulfate(VI)
see Hazcard 27C.
Wear goggles.
Dissolve 1.5 g of copper(II) sulfate(VI)-5-water crystals and 6 g of potassium sodium tartrate-4-water in
500 g of purified water.
Add 375 ml of 2 M sodium hydroxide with stirring.
If a precipitate occurs, add 1 g of potassium iodide.
Pour this mixture into a 1000 ml volumetric flask and dilute to 1 litre. Mix well. Label this solution
CORROSIVE as it is a 0.75 M sodium hydroxide solution.
For quantitative analysis, a series of standards can be produced with solutions of varying % dilutions of a
protein such as albumen, using a colorimeter with a 540 nm (green) filter. Unknown proteins solutions can
then be compared against these standards.
CLEAPSS 2011 17
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16 Brodies fluid
Also known as manometer or manometric fluid.
Water strongly adheres to glass so requires an emulsifier to avoid water droplets sticking to the walls of the
manometer and affecting the readings, and to reduce capillary action in small bore manometers.
General Hazards Sodium azide is VERY TOXIC and contact with acids liberates a very toxic gas. (See
Hazcard 95B). For eosin, see Hazcard 32. Once in solution, the solution is LOW
HAZARD. Sodium azide may be omitted but the fluid will not keep as well and there
will be mould growth.
A. Preparing 1 litre of Brodies fluid

Weigh out 46 g of sodium chloride, 10 g of sodium tauroglycocholate (bile salts), 0.2 g sodium azide
(optional) and 0.5 g of eosin.
Make up to 1 litre with distilled or deionised water.
B. Brodies fluid substitute for short term use

44 g of sodium bromide.
1 g of liquid detergent (eg washing-up liquid).
0.3 g of Evans blue.
C. Simple version for short-term use

Use water with a food dye such as cochineal and a few drops of detergent.
18 CLEAPSS 2011
CLEAPSS Recipe Book
17 Bromine water
The solubility of bromine in water is 4 g, (ie, 1.25 cm3 ) in 100 g of water at room temperature. This would be
a 4% (w/v) solution. And its concentration would be 0.25 M.
Solutions equal to or greater than 0.06 mol dm-3 (ie, a 0.3% v/v solution) are TOXIC.
Solutions equal to or more concentrated than 0.006 mol dm-3 (0.1% w/v or 0.03% v/v) but more dilute than
0.06 mol dm-3 should be labelled HARMFUL.
Aqueous solutions of bromine should be prepared just before use. If stored for long periods, especially
though the summer, the solution becomes paler as bromine vapour is lost.
Do not make this solution for the first time without seeking practical advice from a more-experienced
colleague.
General Hazards Bromine is VERY TOXIC and CORROSIVE (see Hazcard 15A). Hazcard 15B deals with
bromine water. 0.025 M bromine water has a considerable vapour of bromine gas
above it. It should be dispensed from a fume cupboard. More-dilute solutions can
be used in a well-ventilated room but staff should discourage any direct inhalation
of the vapour.
Sodium chlorate(I) is CORROSIVE (see Hazcard 89), 2 M hydrochloric acid; see
Hazcard 47A, Potassium bromate(V) is TOXIC, (see Hazcard 80).
Preparing 1 litre of 0.02 M solution (HARMFUL) of bromine water

Method 1
Use a fume cupboard. Wear goggles or a face shield and chemical-resistant gloves.
Using a disposable plastic pipette, add 1 ml of bromine to 500 ml of water in a 1 litre beaker.
Add a stirrer bar and place on a magnetic stirrer to dissolve the bromine. This can take over
20 minutes.
Dilute to 1 litre with water.
Alternatively, an ampoule containing 1 ml of bromine can be crushed under 500 ml of water, stirred
using a magnetic stirrer until it dissolves and made up to 1 litre with water. The solution must be
decanted so that no broken glass is present.
Method 2
Use a fume cupboard. Wear goggles or a face shield. Consider wearing gloves.
Dissolve 4.76 g of potassium bromide in 76 ml of water, add 14 ml of 10% (available chlorine) sodium
chlorate(I) solution (CORROSIVE) and 10 ml of 2 M hydrochloric acid (IRRITANT). Dilute to 1 litre with
water.
Method 3
This reaction is slow and it is better to leave the solution 24 hours before it is used.
Use a fume cupboard. Wear eye protection.
-3
Add 1.12 g of potassium bromate(V) (TOXIC), 12 g of potassium bromide and 14 ml of 2 mol dm
hydrochloric acid (IRRITANT) into a 1 litre jug or measuring cylinder.
Add water to 1 litre.
CLEAPSS 2011 19
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18 Buffer solutions
Buffer solutions retain their pH on addition of small amounts of acid, alkali or on dilution.
Unless stated otherwise, buffers are low hazard but eye protection should be worn during preparation.
The pH value of a buffer is slightly altered by temperature but this is not usually significant. The values used
in the Recipe Sheet are for 20 C.
A definition for pH suitable for schools is minus the logarithm (to the base 10, ie log10) of the hydrogen ion
concentration in an aqueous solution. For a more advanced explanation, consult
http://en.wikipedia.org/wiki/PH.
There is also a pOH scale and pH + pOH =14.
A pH meter should be calibrated with standard buffers before it is used. These may be prepared from tablets
or commercial solutions.
Use distilled or deionised water to make up buffers.
Single-component buffers are very quick to make up but the values may not always be convenient.
The majority of buffers involve two components mixed in certain proportions. For accurate work, it is wise to
check their pH with a calibrated pH meter.
A universal buffer mixture can be used to obtain an array of buffer solutions from pH 2 to 11. The stock
solution can, usefully, be stored for several months.
Buffers (especially those between pH 4 and pH 7) do not keep well. Moulds develop which affect pH
readings and block the junctions on pH probes.
Commercial buffer solutions contain a mould inhibitor which allows longer storage.
To save time buffers can be stored as pre-weighed dry components or as frozen solutions. Before use, make
sure frozen solutions are returned to room temperature, that all components are fully dissolved, and check
the pH.
Special biological buffers (eg, TRIS) are available that do not use phosphate(V) or ethanoate ions which
might interfere with some biochemical processes. They are named Good buffers after their developer,
Norman Good. Different enzymes are inhibited by different reagents, so check the protocol and choose the
correct buffer system.
Single component buffers

pH 1.7 Dissolve 1.27 g of potassium hydrogen-ethanoate-1-ethanedioic Low hazard
buffer acid-2-water (potassium tetroxalate) in 10 ml of hot water and make See Hazcard 36A.
up to 100 ml with cold water.
pH 4 buffer Dissolve 1.01 g of potassium hydrogenphthalate in 10 ml of hot Low hazard
water and make up to 100 ml with cold water. The solution does not See Hazcard 13B.
keep well because of mould growth.
pH 7 buffer Dissolve 0.77 g of ammonium ethanoate in 100 ml of cold water. Low hazard
The solution does not keep well because of mould growth. See Hazcard 9B.
pH 9.2 Dissolve 0.38 g of sodium tetraborate-10-water in 100 ml of water. Low hazard
buffer See Hazcard 14.
pH 12.6 Use saturated limewater solution. Low hazard but wear
buffer eye protection.
See Hazcard 18.
Temperature effects on single component buffers

Temperature / C 5 10 15 20 25 30 40 50 60 70 80 90
pH 4 buffer 4.01 4 4 4 4.01 4.01 4.03 4.06 4.09 4.12 4.16 4.2
pH 9.2 buffer 9.39 9.33 9.27 9.23 9.18 9.14 9.07 9.02 8.97 8.93 8.89 8.85
20 CLEAPSS 2011
CLEAPSS Recipe Book
Buffers for pH probe calibration

pH 4 buffer Dissolve 1.012 g of potassium hydrogenphthalate in 10 ml of hot See Hazcard 13B
water and make up to 100 ml with cold water. The solution does not
keep well because of mould growth.
pH7 buffer 0.339 g of citric acid and 4.306 disodium hydrogenphosphate(V)- See Hazcard 9B.
12-water (or 1.707 g of anhydrous salt) dissolved in water and
made up to 100 ml with water.
pH10 buffer 0.477 g sodium tetraborate-10-water and 18.3 ml of 0.1 M sodium See Hazcards 14
hydroxide (from a burette) is made up to 100 ml with water. and 91.
NB Although masses are given to 3 decimal places, it would be acceptable to work to 2 decimal places.
Double component buffers

To prepare these buffers, make up the individual components, then mix the quantities given for the required
pH. For accurate work, confirm the values with a calibrated pH meter and probe.
Using a solid and a solution.

General Hazards As 0.1 M sodium hydroxide solution is IRRITANT, wear eye protection when
preparing the buffers. All the resulting buffers are low hazard. See Hazcards 13B,
36C and 91.
3 10.21 g potassium hydrogen phthalate and 223 ml of 0.1 M

hydrochloric acid
4 10.21 g potassium hydrogen phthalate
5 10.21 g potassium hydrogen phthalate and 226 ml of freshly-made

0.1 M sodium hydroxide solution
6 6.81 g potassium dihydrogen phosphate and 56 ml of freshly-made Dilute each mixture to 1
0.1 M sodium hydroxide solution litre solution with distilled or
7 6.81 g potassium dihydrogen phosphate and 291 ml of 0.1 M sodium deionised water.
hydroxide solution
8 6.81 g potassium dihydrogen phosphate and 467 ml of freshly-made
0.1 M sodium hydroxide solution
9 4.77 g sodium tetraborate-10-water and 46 ml of 0.1 M hydrochloric
acid
10 4.77 g sodium tetraborate-10-water and 183 ml of freshly-made 0.1 M
sodium hydroxide solution
11 2.1 g sodium bicarbonate and 227 ml of freshly-made 0.1 M sodium
hydroxide solution
CLEAPSS 2011 21
CLEAPSS Recipe Book
Mixing two prepared solutions Consult the relevant Recipe Sheets for the preparation of the solutions.
General All these buffers are low hazard. See relevant Hazcards for more information.
Hazards
The citric acid/disodium hydrogenphosphate(V) buffer system

A is 0.2 M disodium hydrogenphosphate(V) and B is 0.1 M citric acid.
pH A (ml) B (ml) pH A (ml) B (ml) pH A (ml) B (ml)
2.2 2.00 98.00 4.2 41.40 58.60 6.2 66.10 33.90
2.4 6.20 93.80 4.4 44.10 55.90 6.4 69.25 30.75
2.6 10.90 89.10 4.6 46.75 53.25 6.6 72.75 27.25
2.8 15.85 84.15 4.8 49.30 50.70 6.8 77.25 22.75
3.0 20.55 79.45 5.0 51.50 48.50 7.0 82.35 17.65
3.2 24.70 75.30 5.2 53.60 46.40 7.2 86.95 13.05
3.4 28.50 71.50 5.4 55.75 44.25 7.4 90.85 9.15
3.6 32.20 67.80 5.6 58.00 42.00 7.6 93.65 6.35
3.8 35.50 64.50 5.8 60.45 39.55 7.8 95.75 4.25
4.0 38.55 61.45 6.0 63.15 36.85 8.0 97.25 2.75
The ethanoic acid/sodium ethanoate buffer The ammonia/ammonium chloride buffer

system system
Add the stated volume of A (0.2 M sodium Add the stated volume of A (0.2 M ammonia) to
ethanoate solution) to the stated volume B the stated volume of B (0.2 M ammonium
(0.2 M ethanoic acid) chloride solution)
pH A (ml) B (ml) pH A (ml) B (ml)
3.8 12.0 88.0 8.4 12.5 87.5
4.0 18.0 82.0 8.6 18.5 81.5
4.2 26.5 73.5 8.8 26.0 74.0
4.4 37.0 63.0 9.0 36.0 64.0
4.6 49.0 51.0 9.25 50.0 50.0
4.8 60.0 40.0 9.4 58.5 41.5
5.0 70.5 29.5 9.6 69.0 31.0
5.2 79.0 21.0 9.8 78.0 22.0
5.4 85.5 14.05 10.0 85.0 15.0
The sodium hydrogencarbonate/sodium The phosphate(V) buffer system. (potassium

hydroxide) system buffer system salts may also be used)
Add the stated volume of A (0.1 M sodium Add the stated volume of A [0.2 M sodium
hydroxide) to 50 ml of 0.05 M sodium dihydrogenphosphate(V)] to the stated
hydrogencarbonate and dilute to 100 ml volume of B [0.2 M disodium
(Wear eye protection.) hydrogenphosphate(V)]
pH A (ml) pH A (ml) B (ml)
9.6 5.00 6.0 87.7 12.3
9.8 6.20 6.5 68.5 31.5
10.0 10.70 7.0 39.0 61.0
10.2 13.80 7.5 16.0 84.0
10.4 16.50 8.0 5.3 94.7
10.6 19.10
10.8 21.20
11.0 22.70
22 CLEAPSS 2011
CLEAPSS Recipe Book
Universal Buffer solutions
The Universal Buffer: Recipe 1

To prepare the stock solution, add 9.9 g of disodium hydrogenphosphate-12-water, (or 4.6 g disodium
hydrogenphosphate-2-water) (see Hazcard 72), 7.0 g of citric acid (see Hazcard 9) and 3.5 g of boric
acid (see Hazcard 14) to 250 ml of 1 M sodium hydroxide solution (see Hazcard 91) and dilute to
1000 ml with distilled/deionised water. This solution keeps well. Label the solution IRRITANT.
To make any buffer between 3.5 and 10

Place 0.1 M hydrochloric acid in a burette.
Place 20 ml of the stock solution in a 250 ml beaker on a magnetic stirrer. Clamp a pH probe into the
solution.
Add the acid to the solution in the beaker with constant stirring until the required pH is obtained. If too
much acid is added, use a little more stock solution to increase the pH value.
Add distilled/deionised water to make the final solution 100 ml.
The Universal Buffer: Recipe 2

To 500 ml of water, add 2 ml of concentrated ethanoic acid (see Hazcard 38A), 3 ml of 85%
phosphoric(V) acid (see Hazcard 72), and 2.4 g of boric acid (see Hazcard 14), and make the solution
up to one litre with distilled/deionised water.
Prepare 0.2 M sodium hydroxide solution (see Hazcard 91), and label the solution IRRITANT.
To make any buffer between 2 and 11

Put 0.2 M sodium hydroxide solution in a burette.
Place 100 ml of the acid solution in a 400 ml beaker on a magnetic stirrer. Clamp a calibrated pH
probe into the solution.
Add 0.2 M sodium hydroxide to the acid solution until the required pH is obtained.
Buffer solutions: Biological

Two component buffers are often used for biological systems. However, if an enzyme is affected by one, or
more, of the component ions, eg, phosphate(V), ethanoate or ammonium ions, specialist buffers are
required.
The cheapest specialist biological buffer is tris(hydroxymethylamino)methane (IRRITANT) known as TRIS. The
solutions made up from the data below are low hazard.
TRIS buffers
pH Volume of 0.1 M Volume of 0.1 M

TRIS solution hydrochloric acid
7.0 100 93.2
7.5 100 80.6
8.0 100 58.4
8.5 100 29.4
9.0 100 11.4
A full range of biological buffers can be found at

www.sigmaaldrich.com/life-science/core-bioreagebnts/bioogical-buffers.html.
For products and applications such as extraction of materials or electrophoresis, see protocols at
www.ncbe.reading.ac.uk.
CLEAPSS 2011 23
CLEAPSS Recipe Book
19 Calcium chloride and nitrate(V) solutions

Hydrated calcium chloride and nitrate(V) absorb water from the atmosphere. On occasions, the solid
completely dissolves to leave a clear solution.
Distilled or deionised water should be used to make solutions. In hard water areas, solutions may be cloudy
if tap water is used.
Do not use anhydrous calcium chloride to make solutions.
General Hazards See Hazcard 19A
Formula: CaCl2.6H2O Molar mass: 219.08 g mol-1 Solubility: 74 g per 100 ml
Preparing 100 ml of 0.1 M of calcium chloride solution

Dissolve 2.19 g of calcium chloride-6-water in 70 ml of water.
Make up to 100 ml with water.
Formula: Ca(NO3)2.4H2O Molar mass: 236.15 g mol-1 Solubility: 121 g per 100 ml
Preparing 100 ml of 0.1 M of calcium nitrate(V) solution

Dissolve 2.36 g of calcium nitrate-4-water in 70 ml of water.
24 CLEAPSS 2011
CLEAPSS Recipe Book
20 Calcium hydroxide solution (Limewater)

Saturated calcium hydroxide solution, commonly called lime water, is a 0.02 M aqueous solution of calcium
hydroxide with a pH of 12.4.
It does not keep for long periods in large bottles because it reacts with carbon dioxide from the atmosphere.
It would be wise to start afresh each year.
Lime water, in small bottles designed for class use, will quickly cease to work. Class sets need to be tested
before handing out to the class.
1 M hydrochloric acid can be used to clean bottles that previously contained limewater.
Formula: Ca(OH)2 Molar mass: 74.09 g mol-1 Solubility: 0.15 g per 100 ml
General Hazards Calcium hydroxide solid; see Hazcard 18. Splashed droplets of limewater in the eye
have caused quite severe irritation, so the solution should also be labelled and treated
as an IRRITANT even though strictly its dilution is such that it is not formally classed as
hazardous.
Preparing make 2.5 L of lime water in a bottle

Place 5 g of calcium hydroxide in a 600 ml beaker and half-fill it with water.
Stir the suspension and pour it via a funnel into a 2.5 litre bottle.
Fill the bottle with water, stopper and shake it.
Leave the bottle overnight for the suspension to settle.
When required, decant the limewater solution, slowly without agitating the sediment, into smaller
bottles for use in lessons.
Add more water and/or calcium hydroxide suspension when the level becomes low.
Preparing a large continual supply

If a large stock is required, keep an excess of calcium hydroxide in an aspirator protected by a soda-
lime tube, as shown below, and top up with distilled water as necessary.
Use about 100 g calcium hydroxide for a 10 litre aspirator. It might take a week to fully settle.
Absorption tube
filled with soda lime
Lime
water
Calcium
hydroxide
CLEAPSS 2011 25
CLEAPSS Recipe Book
21 Carbon dioxide
Carbon dioxide is prepared by the action of dilute hydrochloric or nitric acid on calcium carbonate, usually as
marble chips. If powdered calcium carbonate is used, the rate of gas production may be too rapid to be
easily controlled.
The gas is collected over water or by downward delivery. (Downward delivery uses a tube into the bottom of
an upright gas jar. It is also known as upward displacement of air and relies on the fact that carbon dioxide is
more dense than air.) Although carbon dioxide is slightly soluble in water (at room temperature, the solubility
of carbon dioxide is about 6.4 cm3 of carbon dioxide per 100 ml of water), the rate at which it dissolves is
slow. However, if necessary, the gas can be collected over warm water in which it is less soluble.
Collection by downward delivery is quicker but it is difficult to ascertain exactly when the gas jar is full.
However, downward delivery is necessary for burning magnesium in carbon dioxide, in order to avoid water
interfering with the reaction.
Soda water is a saturated solution of carbon dioxide in water (carbonic acid).
General Hazards See Hazcards 20 & 47A.

In the 250 ml Bchner flask, add several lumps of marble chips.
Add enough water to immerse the base of the thistle funnel tube.
Set up the apparatus as shown in the diagram.
The inverted measuring cylinder should be full with water.
Now add 5 ml of 5 M hydrochloric acid (IRRITANT) down the thistle funnel.
Once the measuring cylinder is full of gas, it indicates the apparatus has been completely purged.
Remove it and replace with an inverted gas jar full of warm water.
Keep collecting gas jars of gas. When full of gas, either slide a gas jar cover in place or put a large
bung into the gas jar.
If the rate of gas production slows down but more gas is needed, add further 5 ml portions of 5 M
hydrochloric acid.
Add 5 mol dm hydrochloric acid

-3
250 ml measuring cylinder
Warm water
26 CLEAPSS 2011
CLEAPSS Recipe Book
22 Cerium(IV) solutions
Cerium(IV) compounds have a variable water content, which makes preparing accurate concentration
solutions very difficult. This is why the mass given in the recipe is approximate.
Cerium(IV) solutions, which have an intense yellow colour, are used in redox titrations.
Cerium(IV) solutions are more stable in solution than potassium manganate(VII) solutions.
Cerium(IV) solutions need to be prepared in dilute sulfuric(VI) acid.
[Hydrochloric acid must be avoided as cerium(IV) ions oxidise the chloride ions slowly to chlorine.]
Cerium(IV) solutions should be standardised against sodium ethanedioate solution before use.
General Hazards The solids are irritating to the eyes, respiratory system and skin.
Preparing 1 litre of 0.1 M cerium(IV) ions

Measure out about 64 g of ammonium cerium(IV) sulfate(VI)-2-water or 40 g of cerium(IV) sulfate(VI)-
4-water.
Add the solid to about 500 ml of 1 M sulfuric(VI) acid in a beaker.
Pour the solution into a 1-litre volumetric flask and make it up to the mark with water.
CLEAPSS 2011 27
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23 Chemiluminesence reactions
Luminol (3-aminobenzene-1,2-dicarboxylic hydrazide or 3-aminophthalhydrazide) in alkaline solution is
oxidised to the 4-aminobenzene-1,2-dicarboxylate ion with the evolution of oxygen.
Goggles should be worn when preparing the solutions if using solid sodium hydroxide but spectacles are
suitable for the demonstrations using the solutions.
Recipe D is ideal for a chemiluminescent fountain demonstration using ammonia (see picture below).
Four recipes illustrating chemiluminesence reactions
A Solution A: Dissolve 0.4 g of luminol and 4 g of potassium hydroxide or sodium See Hazcards
hydroxide in 1000 ml of water. Label the container IRRITANT. 4B, 89, 91.
Solution B: Dilute 50 ml of fresh (10-14% available chlorine) sodium chlorate(I) Wear goggles
solution to 1000 ml. Label the container IRRITANT. to prepare the
In a darkened room, mix equal volumes of solutions A & B together. The addition solution.
of a pellet of potassium hydroxide or sodium hydroxide may produce more light.
B Dissolve 0.2 g of luminol and 1 g of potassium hydroxide or sodium hydroxide in See Hazcards
1000 ml of water. To a known volume of this solution add an equal volume of 4B, 50, 79, 91.
20 vol hydrogen peroxide solution. Wear goggles
In a darkened room, sprinkle a few crystals of potassium hexacyanoferrate(III) to prepare the
onto the surface of the solution and observe the trails of light. solution.
The addition of a pellet of potassium hydroxide or sodium hydroxide may
produce more light.
C Solution A: Dissolve 0.2 g luminol, 4 g of anhydrous sodium carbonate, 24 g of See Hazcards
sodium hydrogencarbonate, 0.5 g of ammonium carbonate and 0.4 g of hydrated 4B, 27C, 50,
copper(II) sulfate(VI) in 1000 ml water. 95A.
Solution B: Prepare 1000 ml of 5 vol hydrogen peroxide solution.
Mix equal volumes of A and B in a dark room or in a box with spy holes.
D Solution A: Mix together 0.2 g luminol, 11 g of anhydrous sodium carbonate, See Hazcards
8 g of sodium hydrogencarbonate, 0.5 g of ammonium carbonate and 0.4 g of 4B, 9A, 25,
copper(II) sulfate(VI) in 1000 ml water. Add 25 ml of 10 vol hydrogen peroxide. 27C, 50, 89, 93,
Solution B: Dissolve 0.1 g of cobalt(II) chloride and 0.1 g of sodium nitrate(III) 95A.
(ie, sodium nitrite) in 1000 ml of water.
Mix equal volumes of A and B in a dark room or in a box with spy holes.
The chemiluminescent fountain demonstration

The flask is originally filled with ammonia. Two containers labelled A and B which contain 1 litre each of
solution A & B in Recipe D are used to supply the solution in the fountain. The solutions are connected via a
T-piece into a single tube which is inserted into the flask.
28 CLEAPSS 2011
CLEAPSS Recipe Book
24 Chlorine gas
Version 1: Concentrated hydrochloric acid on potassium manganate(VII)
The reaction of concentrated hydrochloric acid on potassium manganate(VII) is the more common and
reliable of the two methods. However it uses more-hazardous starting chemicals.
It produces chlorine quickly.
The glassware is difficult to clean at the end and more-concentrated hydrochloric acid is required to remove
manganese(IV) oxide stains.
colleague.
General Hazards See Hazcards 22A, 20 & 81. Double check that you are using concentrated
hydrochloric acid and not concentrated sulfuric(VI) acid. Explosions have resulted from
using the wrong acid.
Use a fume cupboard. Make sure it is working. Wear goggles.

Set up the equipment as in the illustration below. Make sure that the thistle funnel tube nearly reaches
the base of the flask.
Place at least 5 g of potassium manganate(VII) (OXIDISING & HARMFUL) in the Bchner flask.
Pour the concentrated hydrochloric acid (CORROSIVE) down the thistle funnel.
Collect the gas in a gas jar. When the contents of the gas jar are clearly green, assume the gas jar is
full. Or (optional), plug the gas jar with a large wad of mineral wool, or cotton wool, and put some
damp blue litmus paper on top. It takes time for the chlorine to diffuse through the wool and bleach the
litmus. When the bleaching is complete the jar will be full. Remove it and replace the mineral wool with
a gas jar cover or a large bung.
More gas jars of gas can be collected.
To dispose of the reaction mixture, pour it down the fume cupboard sink with lots of water. The stained
glass may need treating with a little concentrated hydrochloric acid before washing further.
In a fume cupboard,
pour concentrated
hydrochloric acid down
the thistle funnel
Bchner flask
Moist blue litmus
Mineral wool
At least 5 g of
Potassium
Manganate(VII)
Gas jar
Wooden block
Delivery tube as far
down as possible
CLEAPSS 2011 29
CLEAPSS Recipe Book
Version 2: 5 M hydrochloric acid added to sodium chlorate(I) solution

This reaction is slower than first version and is easier to control. The glassware is easy to clean at the end of
the procedure.
Sodium chlorate(I) solution is difficult to store and must be less than 1 year old to obtain a good supply of
chlorine. Domestic bleach is not suitable.
As the reaction subsides, either the acid or the sodium chlorate(I) solution can be alternatively added to the
reaction vessel to supply more gas.
The gas preparation arrangement (shown below) is known as the Andrews method. It is an alternative
arrangement to using a thistle funnel. The arrangement in version 1 can also be used.
colleague.
General Hazards See Hazcards 22A, 20 & 89. Sodium chlorate(I) is a solution provided by the
supplier often as sodium hypochlorite. Sodium chlorate(V) is a solid. Do not get
them mixed up.

Place at least 50 ml sodium chlorate(I) solution (CORROSIVE) in the Bchner flask.
Set up the equipment as in the illustration below. The magnetic stirrer is optional but it does appear to
make the procedure more efficient.
Pour the 5 M hydrochloric acid (IRRITANT) into the separating funnel.
Turn the stirrer on and drip the acid into the flask. The solution will now bubble as the chlorine is
produced.
Collect the gas in a gas jar. When the contents of the gas jar are clearly green, assume the gas jar is
full. Or (optional), plug the gas jar with a large wad of cotton or mineral wool and put some damp blue
litmus paper on top. It takes time for the chlorine to diffuse through the cotton wool and bleach the
litmus. When the bleaching is complete the jar will be full. Remove it and replace the mineral wool with
a gas jar cover or a large bung.
To dispose of the reaction mixture, pour it down the fume cupboard sink with lots of water.
Separating funnel Rubber tubing
5 M hydrochloric acid
Moist blue litmus

Sodium
chlorate(i) Mineral wool
solution
Magnetic stirrer
Gas jar
Delivery tube as far
down as possible
30 CLEAPSS 2011
CLEAPSS Recipe Book
25 Chlorine water
The solubility of chlorine in water is about 0.6 g in 100 ml of water at room temperature. This would be a
0.6% (w/v) solution. Its concentration would be 0.085 M.
An aqueous solution of chlorine should be prepared just before use. It does not keep for more than just a few
days and should not be stored.
It is very difficult to make up solutions of known concentration. All that is required for displacement reactions
is that the solution works. It should, therefore, be trialled before use.
Do not make this solution for the first time without seeking practical advice from a more experienced
colleague.
Three methods are described below.
General Hazards Chlorine gas is TOXIC and CORROSIVE (see Hazcard 22A). Hazcard 22B deals with
chlorine water. Freshly-made chlorine water is formally LOW HAZARD but it has a
considerable vapour of chlorine gas above it. It is better dispensed from a fume
cupboard. More dilute solutions can be used in a well-ventilated room but staff
should discourage direct inhalation of the vapour.
Method 1
Bubble chlorine gas into 250 ml water in a gas jar until the solution goes light green. Use the
equipment for preparing chlorine gas but fill the gas jar half-full with water.
Method 2
Sodium chlorate(I) solution is an aqueous solution. Do not become confused with sodium chlorate(V), which
is a solid.
10% w/v available chlorine sodium chlorate(I) solution does not store well. Over two years it may become
completely useless.
Place 10 ml of 10% w/v available chlorine sodium chlorate(I) solution (CORROSIVE) in 1 litre beaker.
Add about 80 ml of water and 10 ml of 2 M hydrochloric acid. Stir well.
Dilute to a suitable volume with more water.
Method 3
Sodium dichloroisocyanurate is used for purifying water.
Use a fume cupboard. Wear eye protection.
3
Add 3 g of sodium dichloroisocyanurate (OXIDISING; HARMFUL), to 100 cm of water. When the solution
3 -3
is clear, add 100 cm of 1 mol dm hydrochloric acid.
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26 Chromatography solvents and locating agents

A mixture of compounds is placed on a stationary phase such as paper, or silica on thin layer plates.
Chromatography involves passing a solvent through the stationary phase which separates the components
of the mixture due to subtle differences in a compound's partition coefficient between the mobile solvent and
the stationary phase.
A locating agent is used to emphasise where the components of the mixture end up after the
chromatography has finished. This is particularly important for colourless components of mixtures.
Control measures Solutions are better made up in a fume cupboard.

for all of the Wear eye protection.
following solutions There must be no sources of ignition in the vicinity.
Substances
separated
Amino acids Solvent: Butan-1-ol, glacial ethanoic acid, water; 6:1:2 by See Hazcards 38A,
volume. 66, 84B. Label
Locator: Apply ninhydrin and heat in an oven at 110 C or with a solvent HARMFUL and
hairdryer. CORROSIVE.
Analgesics, Solvent: Ethyl ethanoate, hexane, ethanoic acid; 10:9:1 by Label the solvent
eg, aspirin, volume. HIGHLY FLAMMABLE
paracetamol Locator: UV light or iodine vapour. Do not look directly at UV and CORROSIVE. See
radiation sources. Hazcards 43A, 54A.
Anthocyanins Solvent: 100 ml 50% aqueous methanol solution with 1 ml of Label the solvent
(plant ethanoic acid or HIGHLY FLAMMABLE
pigments) 1 ml concentrated hydrochloric acid in 100 ml ethanol. and TOXIC. See
Locator: Natural colour. (The components are light sensitive so Hazcards 38A, 40B.
run the chromatograms in the dark if possible and
quickly note or photograph the results.)
Inks from Solvent: Butan-1-ol, ethanol, water; 3:1:1 by volume. The Label the solvent
Biro pens addition of a few drops of 880 ammonia is said to HIGHLY FLAMMABLE
produce a better chromatogram. and HARMFUL. See
Locator: Natural colour. Hazcards 6, 40A,
84B.
Chlorophyll Solvent: Propanone, petroleum spirit (100-120 C); 1 :9 by Label the solvents
volume or HIGHLY FLAMMABLE.
cyclohexane, propanone, ethoxyethane; 2:1:1 by See Hazcards 45A,
volume. 85 or 42, 45B, 85.
Locator: Natural colour of dyes and UV light. Do not look directly
at UV light. (The components are light sensitive so the
results should be noted or photographed quickly.)
Lipstick Solvent: 3-Methylbutan-1-ol,propanone and water; 2:1:9 by The solvent is LOW
volume. HAZARD See
Locator: Natural colour. Hazcards 84C, 85.
Metal ions Solvent: Propanone, hydrochloric acid (conc), Distilled Label solvent HIGHLY
water; 17:2:1 by volume. FLAMMABLE and
Locator: Use conc ammonia solution followed by 0.1% dithio- IRRITANT Label the
oxamide (rubeanic acid). locator CORROSIVE.
See Hazcards 35,
47A, 85.
Nitration of For separating the crude products obtained from the nitration of Label the solvent
methyl methyl benzoate. HIGHLY FLAMMABLE
benzoate Solvent: Ethoxyethane, pet ether (80/100 C); 1:9 by volume. and HARMFUL. See
Locator: UV light. Do not look directly at UV radiation sources. Hazcards 42, 45A.
Sugars Solvent: Ethyl ethanoate, pyridine, water, 8:2:1 by volume. Label the solvent
Locator: Dab Benedicts solution on the chromatogram and dry in HIGHLY FLAMMABLE.
an oven at 110 C. See Hazcards 4C, 8,
43A.
32 CLEAPSS 2011
CLEAPSS Recipe Book
27 Chromium(III) chloride and chrome alum solutions

The colour of chromium(III) solutions varies with temperature and storage.
Use cold water to dissolve the salt and do not heat the solutions.
Prepare just before use.
General Hazards Chromium (III) salts are IRRITANT. See Hazcard 24.
Formula: CrCl3.6H2O Molar mass: 266.5 g mol-1 Solubility: 58 g per 100 ml
Preparing 100 ml of 0.1 M chromium(III) chloride solution

Dissolve 2.67 g of chromium(III) chloride-6-water in 70 ml of cold pure water.
Make up to 100 ml with pure water.
Formula: CrK(SO4)2.12H2O Molar mass: 499.4 g mol-1 Solubility: 22 g per 100 ml
Preparing 100 ml of 0.1 M chromium(III) potassium sulfate(VI) solution

Dissolve 5.0 g of chromium(III) potassium sulfate(VI)-12-water in 70 ml of cold pure water.
CLEAPSS 2011 33
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28 Citric acid
Also known as 2-hydroxypropane-1,2,3-tricarboxylic acid.
It can purchased in either an anhydrous or monohydrate form.
Values in italics below are for the monohydrate form.
General Hazards See Hazcard 36C.
Formula: C6H8O7 Molar mass: 192.12 g mol-1 Solubility: 133 g per 100 ml
Formula: C6H8O7.H2O Molar mass: 210.14 g mol-1 Solubility: 133 g per 100 ml

0.1 M 1.92 (2.10) 4.80 (5.35) 19.21 (21.01) -
0.5 M 9.60 (10.51) 24.02 (26.27) 96.06 (105.07) -
1.0 M 19.21 (21.01) 48.03 (53.54) 192.12 (210.14) -
Procedure
Measure out the indicated quantity of the solid citric acid.
34 CLEAPSS 2011
CLEAPSS Recipe Book
29 Clock reactions
These are used for rate of reaction investigations.
Solutions should be made a day before use and stored in the place where they will be used to attain room
temperature.
Clock reactions need to be tried out beforehand so that concentrations can be tweaked to give reasonable
times. Summer and winter temperature variations can also alter reaction times.
The suggested amounts are for student activities. These reactions, however, make excellent demonstrations
and the volumes can be significantly increased.
The asterisk by the name of a solution indicates that this reagent is involved in the main reaction and its
concentration can be varied. However, the amount of each solution must remain as specified.
Varying reaction temperature will alter the time lapse for the clock.
The addition of transition metal ions, eg, copper(II) ions, as a catalyst can also be investigated. However, you
need to trial the experiment as the sequence that components are added is very important.
The hydrogen Solution A 1% starch solution (indicator) See Hazcards 47A

peroxide/potassium Solution B* 4 vol hydrogen peroxide & B, 67, 95C.
iodide reaction Solution C* 0.1 M hydrochloric acid
(The Harcourt- Solution D 0.01 M sodium thiosulfate Wear eye
Essen reaction) Solution E* 0.05 M potassium iodide protection.
Add 5 drops of solution A to a suitable beaker plus 10 ml
each of solutions B to E in that order.
The thiosulfate/acid Solution A 0.1 M sodium thiosulfate See Hazcards 47A,
reaction Solution B 1 M hydrochloric or 0.05 M sulfuric(VI) acid 95C & 98.
(disappearing Add 1 ml of the acid for every 10 ml of sodium thiosulfate.
cross) Once the run is complete, dispose of the contents in 0.5 M Wear eye
sodium carbonate solution to stop the reaction and neutralise protection. Do not
sulfur dioxide. use temperatures
above 50 C.
The potassium Solution A 1% starch solution (indicator) See Hazcards 80,
iodate/sodium Solution B* 0.025 M potassium iodate(V) solution (5.35 g 92.
metabisulfite per 1000 ml of solution)
reaction Solution C* 0.025 M sodium metabisulfite solution (4.75 g Wear eye
(The Landolt iodine per 1000 ml of solution. Prepare in a fume cupboard.) protection.
clock) Add 5 drops of solution A to a suitable beaker plus 10 ml
each of solutions B and C.
The reaction Solution A 1% starch solution (indicator) See Hazcards 41,
between iron(III) Solution B 0.01 M sodium thiosulfate 47B, 55, 67, 95C.
ions and iodide Solution C* 0.025 M iron(III) chloride using 0.1 M
ions hydrochloric acid to dilute a more concentrated iron(III) Wear eye
solution protection.
Solution D* 0.025 M potassium iodide
Mix 5 drops of solution A followed by 10 ml each of solutions
B, C and D in that order.
The potassium Solution A Dissolve 0.25 g of sodium thiosulfate in 100 ml See Hazcards 47B,
iodide/potassium of 1% starch solution 95B, 95C.
persulfate reaction Solution B* Dissolve 13.50 g of potassium persulfate in
1000 ml of solution Wear eye
Solution C* 0.1 M potassium iodide protection.
Mix 5 ml of solution A with 50 ml each of solutions B and C in
that order.
CLEAPSS 2011 35
CLEAPSS Recipe Book
30 Cobalt(II) chloride solution and thermochromic liquid

General Hazards Cobalt salts are TOXIC (See Hazcard 25). Reported carcinogenic and sensitisation
warnings about cobalt compounds have become more severe in recent years.
CLEAPSS has not received any reports from educational establishments of cobalt
salts causing harm. The concerns for health are in mining and metallurgical
applications used on a daily basis and in large doses.
Formula: CoCl2.6H2O Molar mass: 237.9 g mol-1 Solubility: 97 g per 100 ml
Preparing 100 ml of 0.1 M cobalt(II) chloride solution

Wear eye protection. Wear disposable gloves and consider weighing the solid in non-working fume
cupboard with the sash window low enough to stop any particles being inhaled.
Dissolve 2.38 g of cobalt(II) chloride-6-water in 70 ml of water.
The solution should be labelled TOXIC.
Formula: Co(NO3)2.6H2O Molar mass: 291 g mol-1 Solubility: 155 g per 100 ml
Preparing 100 ml of 0.1 M cobalt(II) nitrate solution

Use the method above but dissolve 2.91 g of cobalt(II) nitrate-6-water in 70 ml of water.
Formula: CoSO4.7H2O Molar mass: 281.1 g mol-1 Solubility: 97 g per 100 ml
Preparing 100 ml of 0.1 M cobalt(II) sulfate solution

Use the method above but dissolve 2.81 g of cobalt(II) sulfate(VI)-7-water in 70 ml of water.
Thermochromic liquid
Used in models of hot-water systems.
Dissolve 40 g of hydrated cobalt(II) chloride in 1 litre of ethanol without heating.

Add drops of water and stir until pink. A drop or two of ammonia solution will precipitate the hydroxide
which shows the flow in the model.
Label the solution TOXIC & HIGHLY FLAMMABLE.
36 CLEAPSS 2011
CLEAPSS Recipe Book
31 Copper(II) solutions
It is advisable to use distilled or deionised water to make these solutions.
Solutions of copper(II) salts are sometimes cloudy. If this is the case, add 1 ml of 1 M sulfuric acid and stir.
Continue this procedure until the solution is clear.
The Health & Safety Executive provide hazard classifications for solutions by % (w/v). We generally measure
concentration in mols per dm-3 rather than % (w/v), which leads to inconsistencies in hazard classification
between different copper salts with the same concentration. In the tables below the hazard classifications in
brackets are consistent with that for copper sulfate(VI), the most commonly used copper(II) salt, but are not
strictly correct.
General Hazards Copper(II) salts are HARMFUL if swallowed. See Hazcard 27A, B & C.
Formula: CuSO4.5H2O Molar mass: 249.68 g mol-1 Solubility: 32 g per 100 ml

0.1 M 2.50 6.24 24.97 -
0.5 M 12.49 31.21 124.84 -
1.0 M 24.97 62.42 249.68 HARMFUL
Saturated (20 C) 40 100 400 HARMFUL
Formula: CuCl2.2H2O Molar mass: 170.48 g mol-1 Solubility: 76 g per 100 ml

0.1 M 1.71 4.26 17.05 -
0.5 M 8.52 21.31 85.24 -
1.0 M 17.05 42.62 170.48 (HARMFUL)
Formula: Cu(NO3)2.3H2O Molar mass: 241.6 g mol-1 Solubility: 138 g per 100 ml
0.1 M 2.42 6.03 24.15 -
0.5 M 12.08 30.19 120.75 -
1.0 M 24.15 60.34 241.50 (HARMFUL)
Procedure
Measure out the indicated quantity of copper(II) salt.
Add the solid to about two thirds of the final volume of water in a beaker or jug.
CLEAPSS 2011 37
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32 Crude oil alternative

Real crude oil (and petrol) contain benzene in concentrations greater than 0.1% and must not be used in
school science (COSHH Regulations).
A synthetic mixture can be prepared using mainly aliphatic hydrocarbons to illustrate the principle of
fractional distillation of crude oil in industry.
An alternative name for petroleum spirit is petroleum ether.
On storing for several months, the lighter fractions evaporate away so the procedure should be tested to see
that enough low boiling point fractions can be distilled off.
Further details on the procedure can be found in L195, Safer Chemicals, Safer Reactions and section 13 of
the Handbook.
Standard fractional distillation equipment using a fractionating column is unsuitable as very high
temperatures are required. Use apparatus similar to the diagram below.
General Hazards Petroleum spirit is HIGHLY FLAMMABLE and HARMFUL (see Hazcard 45A).
Preparing 100 ml of synthetic crude oil

Wear eye protection. Do not prepare the mixture near sources of ignition.
Mix together 55 ml of liquid paraffin (medicinal), 20 ml of paraffin oil (kerosene), 11 ml of white spirit,
4 ml of petroleum ether (100-120 C), 4 ml of petroleum ether (80-100 C) and 6 ml of petroleum ether
(60-80 C).
Add a squeeze of black oil paint (eg, Winsor and Newtons Ivory Black) from a tube and stir well.
After adding to a labelled bottle, shake the mixture well. Label the container HIGHLY FLAMMABLE &
HARMFUL.
Always shake the mixture well before use. The paint will separate out if the bottle is left still for some
time.
Note
The recipe is not written in stone. Another possibility, if you have an even lower fraction of petroleum spirit,
is:
Mix together 50 ml of liquid paraffin (medicinal), 20 ml of paraffin oil (kerosene), 10 ml of white spirit,
5 ml of petroleum ether (100-120 C), 5 ml of petroleum ether (80-100 C), 5 ml of petroleum spirit
(60-80 C) and 5 ml of petroleum spirit (40-60 C).
0 360 C thermometer
Side-arm boiling tube

75 x 10 mm test tubes
to collect the fractions
Mineral fibre with

6 ml of crude oil 1 ml of water in a
test tube to compare volumes
(optional)
Block of wood to
Heat hold the test tubes
38 CLEAPSS 2011
CLEAPSS Recipe Book
33 2,4-Dinitrophenylhydrazine solution
The solid is also known as 2,4-DNP and the solution is called Bradys reagent.
It is used to identify organic compounds with carbonyl groups by producing orange or yellow insoluble
derivatives which, if purified by recrystallisation, give sharp melting points.
Solid 2,4-DNP is supplied moist as there may be an explosion risk if very dry solid is handled. Unopened
bottles of 2,4-DNP already contain 20% to 33% water.
Two recipes for preparing Bradys reagent are given below. The new recipe using phosphoric acid has been
shown to work and is safer.
General Hazards 2,4-DNP is EXPLOSIVE and TOXIC by inhalation, skin contact and if swallowed
(see Hazcard 30). For information about the alcohols see Hazcard 40A. Both
concentrated sulfuric(VI) acid and phosphoric(V) acid are CORROSIVE (see
Hazcards 98A and 72 respectively).
Formula: (NO2)2C6H3NHNH2 Molar mass: 198.14 g mol-1
Procedure for making about 100 ml of Bradys reagent

Traditional method New method
Wear eye protection, gloves and work in a fume Wear eye protection and gloves.
cupboard. Stir 2 g of 2,4-DNP with 50 ml of 85%
Stir 2.7 g of 2,4-DNP with 96 ml of methanol. phosphoric(V) acid.
Cautiously and slowly add 4 ml of concentrated Add 50 ml of ethanol.
sulfuric(VI) acid.
Usually the solid dissolves to form a clear
solution with a little stirring. If cloudy, filter.
Label the solution TOXIC and HIGHLY FLAMMABLE.
Procedure for using Bradys reagent

Wear eye protection, and gloves.
Place 0.5 ml (if liquid) or 0.5 g (if solid) of the carbonyl compound to be identified in a test tube.
Add 5 ml of Bradys reagent. [If crystals do not appear then add a little 2 M sulfuric(VI) acid.]
Cool the solution in an ice bath for 15 to 30 minutes.
Filter off the crystals (vacuum filtration is quicker).
Wash the crystals with water and recrystallise them before obtaining a melting point.
CLEAPSS 2011 39
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34 3,5-Dinitrosalicylic acid
Also called DNSA or DNS.
3,5-Dinitrosalicylic acid is an alternative to Benedicts solution for testing glucose and other reducing sugars.
It can be used either qualitatively (yellow to red) or quantitatively using colorimetry with a green filter
absorbing at 565 nm.
It is more sensitive to glucose than Benedicts solution.
General Hazards 3,5-Dinitrosalicylic acid is HARMFUL by ingestion and irritating to the eyes and skin.
Preparing DNSA reagent

Wear goggles.
Add 1.0 g of 3,5-dinitrosalicylic acid to 50 ml of water and warm gently to dissolve.
Slowly add 30 g sodium potassium tartrate tetrahydrate, (KNaC4H4O64H2O). The mixture thickens
(like custard).
Add 20 ml of 2 M sodium hydroxide solution and dilute the mixture with water to a final volume of
100 ml.
Label the solution IRRITANT.
Experimental procedure
Mix 0.3 ml of the sample with 3 ml of DNSA reagent. Glass vials are ideal for this.
Heat in hot water (from a kettle) for 5 minutes till a yellow colour develops. The samples do not need
to be boiled.
Remove the vial with tongs and allow to cool in a cold water bath.
The colorimeter will need to be set up with the green filter (about 565 nm).
Use reagent with water as a blank.
There are no further colour changes beyond a glucose concentration of 0.04 M, ie 0.72% (w/v).
Therefore, prepare a glucose standard curve with this concentration as the maximum.
Samples which are believed to contain higher amounts of glucose will need to be diluted before
testing.
40 CLEAPSS 2011
CLEAPSS Recipe Book
35 Drosophila food base

The food-base should pour easily when hot but set firmly when cold. It should also remain firm when the
adult flies emerge.
Preparing the food base

Soak 72 g of oatmeal in 120 ml of pure water.
Dissolve 35 g of treacle (black molasses) in 40 ml of pure water.
Boil 6 g of agar in 400 ml of pure water.
The mould inhibitor is made by dissolving 0.1 g of methyl 4-hydroxybenzoate (Nipagin; see
Hazcard 52) in a small volume of 95% (v/v) industrial denatured alcohol.
Mix all the ingredients and heat in a boiling-water bath, with constant stirring, for at least 15 minutes.
Add more water if the mixture does not pour easily.
Using a funnel, pour the mixture into either milk bottles or specimen tubes, making sure that the
medium does not drip down the sides of the vessels.
Autoclave at 15 psi (121 C) for 15 minutes.
Allow the autoclave to cool naturally after sterilisation is complete; otherwise the medium will boil up
and contaminate the walls of the vessels.
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36 Electroplating solutions
There are many formulations for plating solutions. Some are hazardous and where they are not covered by a
model (general) risk assessment, either here or elsewhere, a special risk assessment will be needed before
use by teachers / technicians or students. Help can be obtained from CLEAPSS.
Use Recipe for 100 ml of solution Notes

Copper Use 1 M copper(II) sulfate(VI) solution See Hazcard 27C. Label the
electroplating solution HARMFUL.
Nickel Dissolve 5 g of ammonium nickel(II) See Hazcard 65B. Label the
electroplating sulfate(VI) in 80 ml of water and dilute to solution HARMFUL.
100 ml.
Silver Dissolve 1.6 g of silver nitrate(V) and 32 g of See Hazcards 87 and 98A. Label
electroplating potassium iodide in 100 ml of water. Add 3 the solution IRRITANT.
drops of concentrated sulfuric(VI) acid. Use a
carbon rod as the anode.
Zinc electroplating Dissolve 33 g of zinc sulfate(VI)-7-water in See Hazcards 14, 98A and 108B.
50 ml of water and dilute to 100 ml. Add five It would be advisable to label the
drops of 2 M sulfuric(VI) acid and 2 spatulas solution HARMFUL.
full of boric acid. Boric acid crystals dissolve
more easily than the powder.
This is an old recipe and the word spatula is
used which is not helpful. The solubility of
boric acid is 6 g per 100 ml of water. 6 g is
probably a sensible amount to add.
42 CLEAPSS 2011
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37 Enzymes
This sheet is applicable to amylase, lipase, trypsin, pepsin etc.
Enzyme preparations are available in the form of powders, capsules, tablets or concentrated solutions.
Stock enzymes should be stored in the refrigerator and discarded when out of date or no longer active.
Enzyme activity varies considerably with age, source and storage conditions. It is therefore essential to
perform preliminary trials to check whether the enzyme is working and whether the endpoint is reached in an
appropriate time. Allow time to order fresh stock in case the sample does not yield satisfactory results.
If the concentration required is not specified on the protocol for the activity or suggested by the supplier,
begin with 1% (w/v), test, and adjust accordingly.
It is often preferable to obtain enzymes from a natural source. Purchased enzymes may have a microbial
origin and are sometimes very heat stable or have unusual pH profiles so that investigations of the influence
of temperature or pH on enzyme activity yield unexpected results. For example, saliva is often more reliable
as a source of amylase than commercial products, which may also be contaminated with sugar. Health risks
are minimal if each pupil uses his/her own saliva and rinses out contaminated glassware afterwards, prior to
soaking in sodium chlorate(I) solution before normal washing up. Follow procedures in the Handbook.
Note that for many enzymes, the pH will need to be adjusted to the optimum; check the details provided or
the protocol.
Enzyme powders may, in extreme cases, cause sensitisation. It is best to make up solutions in a fume
cupboard which is not switched on. This helps to protect the operator from breathing in the powder in case it
disperses. An operating fume cupboard may cause the powder to blow around. In case of a spill, the fume
cupboard should be switched on to vent the powder.
General Hazards See Hazcard 33. All enzyme powders are HARMFUL and may cause sensitisation.
Any solutions greater than 1% should be considered as IRRITANT.
Preparing 100 ml solution of 1% enzyme solution

Wear eye protection and disposable nitrile gloves when making up solutions from the powders.
Avoid inhaling the powder. It may be wise to carry out the weighing and preparation in a fume
cupboard (with the fan switched off to avoid the draught affecting the balance).
Measure out 1 g of the enzyme.
Add the solid to about 70 ml of water or buffer (if needed for the activity) in a beaker.
Stir to dissolve (do not warm the solution).
Pour the solution into an appropriate measuring cylinder/volumetric flask.
Dilute to the final volume with pure water.
Pour into a labelled bottle and mix well. Store in the refrigerator or on ice during use.
Carry out the procedure that the students will undertake and consult with the teacher to confirm that
the results are satisfactory. It may be necessary either to dilute the solution further with more water or
add more of the enzyme.
Sodium tauroglycocholate (Bile salts)

General Hazards See Hazcard 95C. Although low hazard, bile salts have an unpleasant odour and
may be better prepared in the fume cupboard.
Preparing 100 ml of 3% bile salt solution

Weigh out 3 g of bile salts.
Dissolve the salt in 70 ml of water.
Make up to 100 ml.
CLEAPSS 2011 43
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38 Etching solutions
There are many formulations for etchants. Some are hazardous and where they are not covered by a model
(general) risk assessment, here or elsewhere, a special risk assessment will be needed before use by
teachers / technicians or students. Help can be obtained from CLEAPSS.
Use Recipe for 100 ml of solution Notes

Aluminium Use 2 M sodium hydroxide solution at 60-70 C. See Hazcard 91, Wear goggles.
etching
Copper etching Dissolve 20 g of iron(III) chloride-6-water in See Hazcards 47A and 55C.
80 ml of water and 20 ml of concentrated Label the solution IRRITANT.
hydrochloric acid (36% w/v).
Iron etching 1 Dissolve 59 g of copper(II) choride-2-water in See Hazcards 27A and 47A. Label
(Frys reagent) 40 ml of water and add 60 ml of concentrated the solution CORROSIVE.
hydrochloric acid (36% w/v).
Iron etching 2 Add 2 ml of concentrated nitric(V) acid to 98 ml See Hazcards 40A and 67. Label
(Nital solution) of ethanol. Under no circumstances use more the solution HIGHLY FLAMMABLE &
than 2 ml of nitric(V) acid in making up 100 ml HARMFUL. Do not store.
of solution.
Lead etching Wear goggles and chemical resistant gloves. See Hazcards 9A and 67. Label
Before use, mix together 50 ml of 5 M nitric(V) the solution CORROSIVE.
acid and 50 ml of 15% (w/v) ammonium
molybdate(VI) solution. Apply with a swab for
30 s before rinsing.
44 CLEAPSS 2011
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39 Ethanoic acid
Also known as glacial acetic acid.
The concentration of this solution is 17.4 mol dm-3.
On cold days, the acid freezes to form a glacial solid. In this case, place the bottle in warm water for several
hours before making the diluted sample, but make sure the label does not come off.
Formula: CH3COOH Molar mass: 60.05 g mol-1
General Hazards See Hazcard 38A. Ethanoic acid is CORROSIVE. It has a very sharp odour.
Volume (ml) of concentrated acid to be used

0.1 M Ten-fold dilution of 1 M solution -
0.5 M 14 29 71 -
1M 29 57 143 -
2M 57 114 286 IRRITANT
Procedure
Wear goggles (a face shield is preferable for large volumes) and chemical resistant gloves.
Use the fume cupboard.
Measure out the indicated volume of ethanoic acid in a measuring cylinder.
Add the liquid to about two thirds of the final volume of water in a beaker or laboratory jug.
Stir the mixture well.
CLEAPSS 2011 45
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40 Fehlings solutions
Benedicts solution or DNSA are safer alternatives when testing for sugars.
Fehling's solution can only be used to test for aliphatic aldehydes, whereas Tollen's reagent (see Recipe
Sheet 102) can be used to test for both aliphatic and aromatic aldehydes.
Fehling's solutions A & B need to be made and stored separately. They are mixed just prior to use.
Sandells solution (which is little known and not cited in exam school texts; see Recipe Sheet 76) can be
made and stored for long periods. The concentration of sodium hydroxide is only 0.4 M in the solution
(Fehlings solution B is close to 4M).
General Hazards Copper(II) sulfate(VI) crystals are HARMFUL if swallowed (see Hazcard 27C). 2M
sulfuric(VI) acid, solid sodium hydroxide and 0.5 M sodium hydroxide solution are
CORROSIVE (see Hazcards 91 and 98A).
Preparing 100 ml of Fehlings solution
Fehlings solution A
Measure out 7 g of copper(II) sulfate(VI)-5-water and dissolve this in 60 ml of water.
If the solution appears cloudy add 10 ml of 2 M sulphuric acid.
Make up to 100 ml with water. Label the bottle.
Fehlings solution B
Wear goggles.
Measure out 15.4 g of sodium hydroxide and 35 g of potassium sodium tartrate.
Add the solids to 60 ml of water on a magnetic stirrer. Make up to 100 ml with water. Label the bottle
and add a CORROSIVE warning.
Solutions A and B should be mixed in equal volumes, just prior to use. During the test, do not heat directly
over a Bunsen burner: use a boiling water bath.
46 CLEAPSS 2011
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41 Fixatives used before preserving biological specimens

Fixatives kill cells whilst retaining their structure.
Although safer preservatives are available as alternatives to methanal, it is necessary to use methanal in
many fixatives. (See Recipe Sheet 74 for preservatives.)
More information about the procedures for fixing and preserving specimens can be found in the Laboratory
Handbook, section 15.
Methanal solution Dilute 10 ml of 40% methanal solution to For methanal, see Hazcard 63. Label
for fixing animal 100 ml. Leave specimen in this solution the final solution HARMFUL. Use a
tissue for 16-48 hours before preserving. fume cupboard, wear eye protection
and disposable nitrile gloves.
Formalin-alcohol Mix together 90 ml of ethanol with 10 ml For ethanol; see Hazcard 40A. For
fixative for plant of 40% methanal solution. Leave methanal, see Hazcard 63. Label the
tissue specimen in this solution for 12 hours solution HIGHLY FLAMMABLE and
before preserving. HARMFUL. Use a fume cupboard,
wear eye protection and disposable
nitrile gloves.
Formalin-aceto- Mix together 5 ml of 40% methanal and For ethanol; see Hazcard 40A. For
alcohol (FAA) 5 ml of glacial ethanoic acid in 90 ml of methanal, see Hazcard 63. For
fixative for plant 70% ethanol. Leave specimen in this ethanoic acid, see Hazcard 38.
tissue solution for 12 hours before preserving. Label the solution HIGHLY FLAMMABLE
& HARMFUL. Use a fume cupboard,
wear eye protection and disposable
nitrile gloves.
Acetic alcohol Mix 25 ml of glacial ethanoic acid with For ethanol; see Hazcard 40A. For
(Clarkes fluid) (a 75 ml of absolute alcohol (Industrial ethanoic acid, see Hazcard 38.
cytological fixative denatured alcohol is normally Label the solution HIGHLY FLAMMABLE
especially for adequate). and CORROSIVE. Use a fume
chromosomes) cupboard, wear eye protection and
disposable nitrile gloves.
CLEAPSS 2011 47
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42 Gases less commonly used in schools
Gas Hazard Minimum amount of reagents to prepare 1000 ml of gas

information
Carbon See Hazcard 21 Concentrated sulfuric(VI) acid is slowly added to 2.9 g of solid sodium
monoxide methanoate. Collect over water.
Dinitrogen See Hazcard 68 5.8 g of hydroxyammonium chloride in 30 ml of water is added to a warm
monoxide solution containing 81 g of ammonium iron(III) sulfate(VI) in 160 ml of
water. Collect over warm water.
Hydrogen See Hazcard 49 2.1 ml of concentrated sulfuric(VI) acid is slowly added to excess solid
chloride sodium chloride. Collect by downward delivery (a tube into the bottom of
an upright gas jar).
Hydrogen See Hazcard 51 42 ml of 2 M hydrochloric acid is added to excess iron(II) sulfide. Collect
sulfide over hot water.
Nitrogen See Hazcard 93 2.9 g of sodium nitrate(III) (nitrite) dissolved in a little water is warmed
gently with excess ammonium chloride. Pass the gas through water to
remove any oxides of nitrogen formed as a side reaction, and then collect.
Nitrogen See Hazcard 68 8 ml of concentrated nitric(V) acid (70%) is added to an excess of copper
dioxide turnings. Collect by downward delivery (a tube into the bottom of an
upright gas jar).
Nitrogen See Hazcard 68 30 ml of half-concentrated nitric(V) acid (35%) is added to an excess of
monoxide copper turnings. Collect over water.
OR
2.9 g of sodium nitrate(III) (nitrite) dissolved in 6 ml of water is added to
12 g of hydrated iron(II) sulfate(VI) and enough 5 M hydrochloric acid is
added to cover the crystals. Collect over water.
48 CLEAPSS 2011
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43 Hydrochloric acid
Hydrochloric acid is available from all major educational suppliers as 35 - 38% (w/w) solution with a specific
gravity of 1.18. If you wish to know the dilution factors for other commercial concentrations (eg, 32%), then
contact CLEAPSS.
For accurate work, the concentration should always be found by titrating against a standard solution of
sodium carbonate.
The concentration of concentrated hydrochloric aid is about 11.7 mol dm-3.
If kept for long periods, the concentration of 35 - 38% (w/w) hydrochloric acid decreases as the gas diffuses
into the atmosphere.
Do not make dilute solutions for the first time without seeking practical advice from a more experienced
colleague.
Formula: HCl Density: 1.18 g cm-3 Molar mass: 36.46 g mol-1
General Hazards See Hazcard 47A. Hydrochloric acid is CORROSIVE. It has a very sharp odour.

0.4 M 17 34 84 -
0.5 M 21 42 105 -
1M 42 84 209 -
Procedure
Wear goggles (a face shield is preferable when handling large volumes) and chemical resistant
gloves.
Use a fume cupboard. Take care opening a bottle on a hot day.
Measure out the indicated quantity of concentrated hydrochloric acid in a measuring cylinder.
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44 Hydrogen gas
General Hazards See Hazcards 27C, 48 & 98. Hydrogen is extremely flammable. There must be no
Bunsen burner being used within 1 m of the apparatus.
Note that the first gas collected consists of air and hydrogen. This is an explosive
mixture and should be discarded.
Wear goggles.
Place several granules of zinc in the
generator. 1 to 5 ml of 1M
The reaction needs to be speeded up at the copper(II) sulfate(VI) plus
2M sulfuric acid
start, to flush out air quickly.
250 ml measuring
Add about 5 ml of 1 M copper sulfate(VI) cylinder
(HARMFUL) down the thistle funnel. This reacts
with zinc, coating it with copper which acts as
250 ml
a catalyst. plastic bottle
Add 2 M sulfuric(VI) acid (CORROSIVE) down
the thistle funnel. Hydrogen
generator
Collect the gas in the 250 ml measuring
cylinder. Once the measuring cylinder is full, Zinc
its contents (largely air) must be discarded.
Now gas jars, boiling tubes, syringes and soap
bubbles of pure hydrogen can be prepared.
Dry hydrogen [for burning or reducing copper(II) oxide]

Use a U-tube filled loosely with calcium
chloride granules (not powder) and collect the
first gases in measuring cylinder one size A 500ml measuring
larger than the hydrogen generator. cylinder;
Once the measuring cylinder is full, disconnect to ensure
at A, and attach other equipment but before all air is flushed
through
using the hydrogen, leave time to flush the
U-tube
air out of this newly-attached equipment.
The gas collected in the measuring cylinder Calcium chloride
can be discarded.
50 CLEAPSS 2011
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45 Hydrogen peroxide
In the UK, the concentration of hydrogen peroxide is given in terms of the volume of oxygen it can produce;
thus 1 ml of 10 vol hydrogen peroxide is capable of producing 10 ml of oxygen gas at 0 C and 1atm.
The maximum concentration of purchased hydrogen peroxide is 100 vol and even this solution will become
more dilute with time.
The concentration of 100 vol solution is 8.3 mol dm-3.
Commercial hydrogen peroxide solutions contain an inhibitor to slow down decomposition. During the
preparation of diluted solutions, the inhibitor is diluted so solutions will deteriorate quite quickly. They should
be prepared when required and not stored for long periods.
For accurate work you will need to standardise the solution against a known solution of potassium
manganate(VII).
For activities with catalase, start with a 1 M solution and adjust subsequent concentrations depending on
observed results.
Formula: H2O2 Molar mass: 34.02 g mol-1
General Hazards See Hazcard 50. 100 vol hydrogen peroxide is HARMFUL.
Concentration Volume (ml) of solution required Hazard

required 250 1000 2500 warning
label
0.08 M 1 vol 0.29% Twenty-fold dilution of the 20 vol solution -
0.1 M 1.2 vol 0.34% Ten-fold dilution of the 1 M solution -
0.34 M 4 vol 1.14% Five-fold dilution of the 20 vol solution -
1M 12 vol 3.3% 29 115 288 -
1.7 M 20 vol 5.7% 50 200 500 IRRITANT
3.4 M 40 vol 11.4% 100 400 1000 IRRITANT
Procedure
Wear eye protection and gloves.
Measure out the indicated quantity of 100 vol hydrogen peroxide in a measuring cylinder.
Add the liquid to about two thirds of the final volume of water in a beaker or laboratory jug and stir.
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46 Indicators (acid-base)
General Hazards See Hazcard 32. The hazards of many dyes and indicators are not well known.
Many are made up in ethanol which is HIGHLY FLAMMABLE & HARMFUL (see
Hazcard 40A).
Indicator Quantity Volume of Acid Alkali pH range

(g) IDA
Bromophenol blue 0.4 200 Yellow Blue 3.0 - 4.5

Methyl orange 0.4 200 Red Yellow 3.0 - 4.5
Bromocresol green 0.4 200 Yellow Blue 4.0 - 5.4
Methyl red 0.2 300 Red Yellow 4.0 - 6.0
Bromothymol blue 0.4 200 Yellow Blue 6.0 - 7.5
Phenol red 0.4 200 Yellow Red 6.6 - 8.0
Neutral Red 0.4 200 Red Yellow 6.8 - 8.0
Cresol red 0.2 200 Yellow Purple 7.2 - 8.8
Thymol blue 0.4 200 Yellow Violet 8.0 - 9.6
Phenolphthalein 1.0 600 Colourless Mauve 8.0 - 10.0
Thymolphthalein 2.0 1000 Colourless Blue 9.3 - 10.5
Procedure to prepare 1000 ml of indicator solution

Wear eye protection and use a fume cupboard when dispensing from bottles of solid indicators to
avoid raising dust. Use gloves to avoid staining the skin.
Dissolve the quantity of indicator in the volume of industrial denatured alcohol (IDA) given in the table
in a suitable beaker.
Transfer solution to a 1000 ml measuring cylinder and make up to 1000 ml with water.
Litmus solution
Dissolve 1 g of litmus in 1000 ml of water. Other recipes suggest up to 10 g of litmus but this may
depend upon the age of the sample. Filter if necessary.
Home-made indicators from fruits and vegetables

Coloured aqueous solutions can be made from red cabbage, flower petals, blackberries, beetroot, etc, frozen
in ice-cube trays and stored for months.
Screened methyl orange

The normal colour change of some indicators is not always clear and may be altered by screening. The
most common screened indicator used in schools is screened methyl orange.
Dissolve 1 g of methyl orange and 2.6 g of xylene cyanol FF in 1000 ml of water. The end point is a grey
colour.
52 CLEAPSS 2011
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47 Indicator (universal)
Ethanol is HIGHLY FLAMMABLE & HARMFUL (see Hazcard 40A).
Procedure to prepare 1000 ml of Yamada Universal indicator solution

Wear eye protection and use a fume cupboard (not switched on) to avoid raising dust when
dispensing from bottles of solid indicators. Use gloves to avoid staining the skin.
Place 0.25 g of bromothymol blue, 0.025 g of thymol blue, 0.063 g of methyl red and 0.5 g of
phenolphthalein in an appropriate beaker.
Add 500 ml of ethanol and stir to dissolve the dyes.
Add 500 ml of pure water.
Pour into a labelled 1000 ml bottle and mix well.
Label this solution (which is red) FLAMMABLE.
The colours of this solution when added to buffers are shown below.
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11
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48 Indicators (carbon dioxide)
Many are made up in ethanol which is HIGHLY FLAMMABLE & HARMFUL (see
Hazcard 40A).
Hydrogencarbonate (bicarbonate) indicator: Preparation of the stock solution:

Use a fume cupboard (not switched on) to avoid raising dust when dispensing from bottles of solid
indicators. Use gloves to avoid staining the skin.
Dissolve 0.20 g of thymol blue and 0.1 g of cresol red in 20 ml of ethanol.
Dissolve 0.85 g of sodium hydrogencarbonate in about 200 ml of freshly-boiled distilled water.
Add the ethanolic dye solution and dilute to 1000 ml with water.
For use:
Dilute the stock solution ten times with freshly-boiled distilled water.
Bubble air through the diluted solution to equilibrate it with atmospheric carbon dioxide.
When ready for use, the solution should be a deep cherry red colour in a bottle (in a test tube the
colour of the solution is less intense). If difficulties are experienced (eg, if distilled water is too acidic),
try adding a pinch more sodium hydrogencarbonate. Avoid breathing over open vessels of the diluted
indicator; the carbon dioxide exhaled may alter its pH.
Bromothymol blue indicator

This can be used as an alternative to the above indicator in carbon dioxide uptake/release studies in biology.
Some authorities report fewer difficulties in obtaining good results. The colour change is to yellow (more
acid) and blue (more alkaline).

Use a fume cupboard (not switched on) to avoid raising dust when dispensing from bottles of solid
indicators. Use gloves to avoid staining the skin.
Dissolve 0.02 g of bromothymol blue in 20 ml ethanol before diluting to 1 litre with water.
54 CLEAPSS 2011
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49 Indicators for redox, precipitation and complexometric titrations

Cerium(IV) titrations
Dissolve 1.49 g of 1:10-phenanthroline in 100 ml of water and add Wear eye protection. 1:10-
0.70 g of iron(II) sulfate(VI). (Ferroin indicator). phenanthroline is TOXIC. The solution is
low hazard.
Dissolve 0.1 g of N-phenylanthranilic acid in 5 ml of See Hazcard 78.
0.1 M sodium hydroxide. Dilute to 100 ml with water.
Dichromate(VI) titrations
Dissolve 0.1 g of N-phenylanthranilic acid in 5 ml of See Hazcard 78.
0.1 M sodium hydroxide. Dilute to 100 ml with water.
Dissolve 0.2 g of sodium or barium diphenylamine-4-sulfonate See Hazcard 10B. Sodium
[diphenylamine-4-sulfonic acid (sodium salt)] in 100 ml of water. diphenylamine-4-sulfonate is an
IRRITANT. Wear eye protection.
Starch indicator for thiosulfate/iodine titrations (and detecting iodine)

Mix 1 g of soluble starch with a small amount of pure water and This solution (it is really a suspension)
stir it vigorously to form a paste. does not keep. Dispose of it if it starts to
Boil 100 ml of pure water and add it to the paste with stirring and go cloudier or lumpy.
allow to cool.
Silver nitrate(V) titrations

Dissolve 5 g of potassium chromate(VI) in 100 ml of pure water. See Hazcard 78. Wear goggles. Label
this solution TOXIC.
Dissolve 0.1 g of eosin in a mixture of 70 ml of ethanol and See Hazcard 32 & 40A. Label this
30 ml of water. solution HIGHLY FLAMMABLE & HARMFUL.
Precipitate becomes pink.
Dissolve 0.1 g of fluorescein (sodium salt) in 100 ml of water. See Hazcard 32 & 40A. Label this
solution HARMFUL. Precipitate becomes
pink.
(For Volhards method.) Dissolve 10 g of ammonium iron(III) See Hazcard 55C. Colour change: white
sulfate(VI).12-water in 90 ml of water and add enough 2 M ppt to orange red colouration.
nitric acid to clear the solution and prevent hydrolysis. Make up to
100 ml. Use 1 ml per titration.
EDTA titrations
Dissolve 1 g of the Eriochrome black T (Solochrome black) in See Hazcard 32 & 40A. Label this
100 ml of ethanol or grind 1 g of the solid dye with 100 g of solution HARMFUL. (Colour change is red
sodium chloride. Use about 0.2 g of this solid mixture for each to blue.)
titration.
Grind 1 g of murexide with 100 g of sodium chloride. Use 0.2 g for See Hazcard 32 & 40A. Label this
each titration. solution HARMFUL. (Colour change is red
to blue.)
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50 Iodine solution
Iodine is only sparingly soluble in water (0.3 g/L).
It is usual to dissolve it in aqueous potassium iodide solution (KI) or organic solvents such as ethanol.
The procedure will take time even with stirring.
A 0.01 M solution is suitable as a test reagent for starch.
The concentration of solutions decreases with storage. Check that the solutions work before use in the
laboratory.
Formula: I2 Molar mass: 253.80 g mol-1
General Hazards See Hazcard 54. The organic solvents used are also hazardous and the relevant
Hazcard should be consulted.

required 100 250 1000 warning
label
0.1 M 8 g of KI + 2.54 g of I2 24 g of KI + 6.35 g of I2 80 g of KI + 25.38 g of I2 -
Procedure
Wear eye protection and work in a well-ventilated room. Wear disposable nitrile gloves.
Measure out the indicated quantity of potassium iodide (KI) into an appropriate beaker.
Moisten the potassium iodide with a few drops of water.
Measure out the indicated quantity of iodine and add it to the moistened potassium iodide.
Add a small volume of water and stir. When no more iodine appears to dissolve, add some more water
and stir. Keep repeating this procedure until all the iodine has dissolved.
Pour the solution into a measuring cylinder and dilute to the final volume. Make sure there are no bits
of iodine remaining. If there are, return the solution to the beaker and leave it on a magnetic stirrer for
several minutes.
Add the solution to a labelled bottle and mix well.
56 CLEAPSS 2011
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51 Iron(II) solutions
Comments about iron(II) salts
Iron(II) sulfate(VI)-7-water crystals are green but they sometimes turn white through loss of water of
crystallisation (efflorescence) or brown through oxidation during storage.
Iron(II) chloride-4-water should also be green but it is even more susceptible to oxidation than iron
sulfate(VI), changing to a brown solid. It is often quoted in experimental worksheets for displacement
reactions but iron(II) sulfate(VI) or diammonium iron(II) sulfate(VI) work just as well.
Neutral solutions of iron(II) salts turn brown very quickly on standing because of oxidation by air.
Iron(II) salts are best prepared in an acidic solution rather than water. It is safer to use this procedure than to
start adding concentrated sulfuric(VI) acid at the end, as is often stated in older recipe books.
Diammonium iron(II) sulfate(VI) is more stable to air during storage. It is the preferred salt for titration
experiments (see Recipe sheet 52).
Do not to store these acidic iron(II) solutions for long periods. A clear green colour gradually turns to dirty
green because of air oxidation.
General Hazards See Hazcards 55B and 98A. Label the final solution IRRITANT because of the acid
present.
Iron(II) sulfate(VI)-7-water
Also known as ferrous sulfate heptahydrate.
Formula: FeSO4.7H2O Molar mass: 278.01 g mol-1 Solubility: 48 g per 100 ml

0.01 M Ten-fold dilution of a 0.1 M solution with 0.1 M sulfuric(VI) acid -
0.1 M 2.78 6.95 27.80 IRRITANT
1M 27.80 69.50 278.01 IRRITANT
Iron(II) chloride-4-water
Do not use the anhydrous salt.
Formula: FeCl2.4H2O Molar mass: 198.8 g mol-1 Solubility: 68 g per 100 ml

0.01 M Ten-fold dilution of a 0.1 M solution with 0.1 M hydrochloric acid -
0.1 M 1.99 4.97 19.88 IRRITANT
1M 19.88 49.7 198.80 IRRITANT
Procedure
Measure out the indicated quantity of iron(II) salt.
Add the solid to about two thirds of the final volume of 1 M sulfuric(VI) acid for the sulfate(VI) or 1 M
hydrochloric acid for the chloride in a beaker or laboratory jug.
either 1 M sulfuric(VI) acid for the sulfate(VI) or 1 M hydrochloric acid for the chloride to the required
level.
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Diammonium iron(II) sulfate(VI)

Also known as ammonium ferrous sulfate(VI) or Mohrs salt.
Formula: (NH4)2SO4.FeSO4.6H2O Molar mass: 392.14 g mol-1 Solubility: 36 g per 100 ml
General Hazards 1 M sulfuric(VI) acid; Hazcard 98A. Iron(II) salts; Hazcard 55B.

0.01 M Ten-fold dilution of a 0.1 M solution with 0.1 M sulfuric(VI) acid IRRITANT
0.1 M 3.92 9.80 39.21 IRRITANT
0.5 M 19.61 49.02 196.07 IRRITANT
Procedure
Measure out the indicated quantity of diammonium iron(II) sulfate(VI).
Add the solid to about two thirds of the final volume of 1 M sulfuric(VI) acid in a beaker or laboratory
jug.
1 M sulfuric(VI) acid to the required level.
Pour into a labelled bottle and mix well. Add hazard warning.
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52 Iron (III) solutions

Aqueous solutions of iron(III) salts do not keep if made up solely in water. If required for a longer period, an
acid should be added. Even in acid solution, there is gradual deterioration and a solid often separates out on
storage.
Iron(III) chloride (also known as ferric chloride) is available in two forms, hydrated iron(III) chloride and
anhydrous iron(III) chloride. The anhydrous solid should not be used to make iron(III) solutions. It does not
dissolve but reacts exothermically with water, to form iron(III) hydroxide and hydrogen chloride gas.
Iron(III) chloride-6-water does not store well as it absorbs water (hygroscopic).
Iron(III) sulfate(VI) has a variable amount of water of crystallisation (9-water is just one of the varieties on
sale). It does not dissolve completely and the solution will need to be filtered (or the solid allowed to settle)
before use.
Ammonium iron(III) sulfate(VI) is much easier to use and is suitable for most applications. The lilac salt
stores quite well but the surface sometimes becomes brown/yellow if the solid has become damp. The
brown/yellow solid should be removed before weighing. The solid also deteriorates if exposed to heat and
light. The solution is brown, Solutions should not be heated to accelerate dissolving as colloidal solutions can
form.
See the etching solution on Recipe sheet 38 if the iron(III) chloride is to be used for this purpose.
Iron (III) chloride

Also known as ferric chloride.
Formula: FeCl3.6H2O Molar mass: 270.30 g mol-1 Solubility: 92 g per 100 ml
General Hazards See Hazcard 55C. Do make sure you are using then hydrated salt and not the
anhydrous salt.

0.01 M Ten-fold dilution of a 0.1 M solution with 0.2 M hydrochloric acid -
0.1 M 2.70 6.76 27.03 IRRITANT
1M 27.03 67.58 270.30 IRRITANT
Procedure
Measure out the indicated quantity of iron(III) chloride-6-water.
Add the solid to about two thirds of the final volume of 1 M hydrochloric acid in a beaker or laboratory
jug.
Pour the solution from the beaker into an appropriate measuring cylinder or a laboratory jug and add
1 M hydrochloric acid to the required level.
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Iron(III) sulfate(VI)
Formula: Fe2(SO4)2.9.H2O Molar mass: 562 g mol-1
General Hazards See Hazcards 55C and 98. 1 M sulfuric(VI) acid. Label the final solution IRRITANT
because of the acid present.

0.1 M 5.6 14.1 56.2 IRRITANT
0.5 M 28.1 70.3 281 IRRITANT
1M 56.2 140.5 562 IRRITANT
Ammonium iron(III) sulfate(VI)
Formula: NH4Fe(SO4)2.12.H2O Molar mass: 482.19 g mol-1 Solubility: 124 g per 100 ml
General Hazards See Hazcards 55C and 98. 1 M sulfuric(VI) acid. Label the final solution IRRITANT
because of the acid present.

0.1 M 4.82 12.05 48.22 IRRITANT
0.5 M 24.11 96.4 241.10 IRRITANT
Procedure
Measure out the indicated quantity of the iron(III) salt.
Add the solid to about two thirds of the final volume of 1 M sulfuric(VI) acid in a beaker or laboratory
jug.
1 M sulfuric(VI) acid to the required level.
Ammonium iron(III) citrate and blueprints

Wear eye protection. Dissolve 3 g of iron(III) chloride-6-water and 3 g of citric acid in 50 ml of water in a
250 ml plastic beaker. Add about 4.5 g of ammonium carbonate in small amounts, stirring each time. There
is quite a lot of frothing and the solution turns green. This solution will keep.
Blue print solution: Add 5 g of potassium hexacyanoferrate(III) and stir. This addition has to be made just
before it is required.
Procedure
The solution is painted onto paper in dull indoor light (preferably in a cupboard) and hung to dry over paper
towels or blotting paper. An opaque object (eg, scissors) is placed on the dry paper and left in the light for a
day or two although, on a sunny day, 20 minutes may well be enough for light to convert soluble iron(III) salts
to insoluble iron(II) salts.
Wearing gloves, rinse the paper in a bowl of water to remove the solution leaving a colourless image on a
Prussian Blue background. This can be messy and may stain clothing and unprotected hands. The paper is
again hung to dry over absorbent paper.
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53 Lead(II) nitrate(V)
Tap water contains sulfate and carbonate ions which will precipitate out as insoluble lead compounds if lead
ions are added. Lead salts should, thereore, be prepared in distilled or deionised water.
Lead(II) nitrate(V) is used in preference to lead(II) ethanoate (acetate) as it appears to be slightly less
hazardous.
Disposal issues: see section 7.5 of the Handbook.
Formula: Pb(NO3)2 Molar mass: 331.21 g mol-1 Solubility: 56 g per 100 ml
General Hazards See Hazcards 57A & B.

0.005 M Twenty-fold dilution of a 0.1 M solution with water -
or ten-fold dilution of 0.05 M solution
0.01 M Ten-fold dilution of a 0.1 M solution with water TOXIC
0.05 M 1.66 16.55 TOXIC
0.1 M 3.31 8.28 33.12 TOXIC
1M 33.12 82.80 331.21 TOXIC
Saturated (20 C) 56 140 560 TOXIC
Procedure
Wear goggles and disposable nitrile gloves. If necessary, weigh materials on a balance placed in a
fume cupboard which is not switched on and the sash pulled down.
Measure out the indicated quantity of lead(II) nitrate(V).
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54 Lithium chloride
Formula: LiCl Molar mass: 42.39 g mol-1 Solubility: 84 g per 100 ml
General Hazards Lithium salts are HARMFUL if swallowed and irritants. See Hazcards 47A and 58.

0.5 M 2.12 5.30 21.20 -
1.0 M 4.24 10.60 42.39 -
Procedure
Measure out the indicated quantity of lithium chloride.
Pour into a labelled bottle. Add a hazard warning if appropriate.
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55 Magnesium sulfate(VI)
Magnesium sulfate(VI)-7-water loses water of crystallisation slowly on standing to the air and becomes
powdery (efflorescent). However, it is more convenient to make solutions from the hydrated salt than the
anhydrous salt which is also available from suppliers.
The anhydrous salt readily absorbs water from the atmosphere during storage.
Formula: MgSO4.7H2O Molar mass: 246.47 g mol-1 Solubility: 71 g per 100 ml
General Hazards See Hazcard 59B.

0.1 M 2.47 6.16 24.65 -
0.5 M 12.32 30.81 123.24 -
1.0 M 24.65 61.62 246.47 -
Saturated (20 C) 75 190 750 -
Procedure
Measure out the indicated quantity of hydrated magnesium sulfate(VI).
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56 Manganese(II) sulfate(VI)
Manganese(II) sulfate(VI) forms many hydrates. The 4 water and 1 water varieties appear to be the most
readily available.
The values in brackets in the table below are for the monohydrate.
Formula: MnSO4.4H2O Molar mass: 223.07 g mol-1 Solubility: 95 g per 100 ml

Formula: MnSO4.H2O Molar mass: 169.02 g mol-1 Solubility: 40 g per 100 ml
General Hazards Manganese(II) sulfate(VI) is HARMFUL. See Hazcard 60.

0.1 M 2.23 (1.69) 5.58 (4.23) 22.31 (16.90) -
0.5 M 11.15 (8.45) 27.89 (21.13) 111.54 (84.51) -
1.0 M 22.31 (16.90) 55.77 (42.26) 223.07 (169.02) HARMFUL
Saturated (20 C) 100 (45) 250 (115) 1000 (450) HARMFUL
Procedure
Measure out the indicated quantity of hydrated manganese(II) sulfate(VI).
Pour into a labelled bottle. Add a hazard label if appropriate.
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57 Mercury solutions
Only make what is needed. Diluting to 0.04 M reduces the level of hazard to TOXIC.
The main issue with mercury salts is that they should not be put down the drain. The waste should be
collected, stored as toxic waste and removed to sealed landfill by a registered hazardous waste collector.
Although labelled toxic and having a notorious history, using an inorganic salt will not kill you. However, the
risk assessment for the activity must establish that the use of the mercury compound is really necessary and
that no alternative is possible.
General Hazards Mercury salts are VERY TOXIC. See Hazcards 62 & 67.
Mercury(I) nitrate [also known as dimercury(I) nitrate(V)]
Make up in dilute nitric(V) acid to produce a clear solution.
Formula: Hg2(NO3)2.2H2O Molar mass: 561.22 g mol-1 Solubility: 2 g per 100 ml
Procedure for preparing 100 ml of 0.02 M of dimercury(I) nitrate(V)-2-water solution [which is 0.04 M
with respect to the mercury(I) ion]
Wear goggles.
Dissolve 1.12 g of the solid in 70 ml of 1 M nitric(V) acid.
Make up to 100 ml with 1 M nitric(V) acid.
Label the solution CORROSIVE and TOXIC.
Mercury(II) chloride
Formula: HgCl2 Molar mass: 271.54 g mol-1 Solubility: 7.4 g per 100 ml
Procedure for preparing 100 ml of 0.04 M of mercury(II) chloride solution

Wear goggles.
Dissolve 1.09 g of mercury(II) chloride-2-water in 70 ml of water.
Label the solution TOXIC.
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58 Methanal solution
Also known as formalin or formaldehyde solution.
The commercial solution is a 40% (w/v) solution of methanal in water with stabiliser added.
Methanal is a gas at room temperature but it dissolves in water. If the bottle top is not secure then it will
gradually lose gas and its concentration will fall.
The concentration of the 40% solution is 11.3 mol dm-3.
Formula: HCHO Molar mass: 30.03 g mol-1
General Hazards See Hazcard 63. It can cause burns, conjunctivitis or sensitisation by skin contact.
Volume of liquid required

0.3% 0.1 M Ten-fold dilution of a 0.1 M solution with water IRRITANT
1% 0.3 M 13 25 63 HARMFUL
3% 1M 38 75 190 HARMFUL
4% 1.3 M 50 100 250 HARMFUL
Procedure
Use a fume cupboard to handle the concentrated solution; wear goggles (a face shield is preferable
when handling large volumes) and wear disposable nitrile gloves.
Measure out the indicated volume of 40% methanal solution in a measuring cylinder.
Add the liquid to about two thirds of the final volume of water in a suitable beaker or laboratory jug and
stir.
Pour into a labelled bottle and mix well. Add the appropriate hazard warning.
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59 Methanoic acid
Also known as formic acid.
It is usually supplied as a 90% (w/w) solution.
Formula: HCOOH Density: 1.22 g cm-3 Molar mass: 46.02 g mol-1
General Hazards See Hazcard 38A. Methanoic acid is CORROSIVE. It has a sharp odour.

required 250 500 1000 2500 warning label
1M 10.5 21 42 105 -
2M 21 42 84 210 IRRITANT
Procedure
Wear goggles (a face shield is preferable for large volumes) and chemical-resistant gloves.
Work in a fume cupboard.
Measure out the indicated quantity of methanoic acid in a measuring cylinder.
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60 Nickel sulfate(VI)
Nickel sulfate(VI)-6-water is the most common nickel sulphate salt sold now, but do check the label on the
bottle because the 7-water hydrate is also available.
Formula: NiSO4.6H2O Molar mass: 262.86 g mol-1 Solubility: 65 g per 100 ml
General Hazards The hazards of nickel salts are being constantly reviewed. Sensitisation may be a
problem for some people. See Hazcard 65B.
Procedure for preparing 100 ml of 0.1 M of nickel(II) sulfate(VI) solution

Dissolve 2.63 g of nickel(II) sulfate(VI)-6-water in 70 ml of water.
The solution should be labelled TOXIC.
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61 Nitric(V) acid
The commercial solution is about 70% (w/v) and has a specific gravity of 1.42. Its concentration is
15.8 mol dm-3.
Concentrated nitric(V) acid should be a colourless solution and stored in dark bottles. When exposed to light,
the solution goes brown and TOXIC nitrogen dioxide gas is produced (see Hazcard 68). Nitrogen dioxide will
leak from bottles and the solution will become less concentrated.
Also available from some suppliers is 95-100% (fuming) nitric(V) acid, which is even more hazardous as it is
unstable. It is unsuitable for schools to store this for long periods. Contact CLEAPSS if you find some.
Do not make dilute solutions for the first time without seeking practical advice from a more-experienced
colleague.
Formula: HNO3 Molar mass: 63.01 g mol-1
General Hazards See Hazcard 67. If in contact with the skin, this becomes yellow and layers of skin
peel off.

0.01 M Ten-fold dilution of a 0.1 M solution with water -
0.1 M Ten-fold dilution of a 1 M solution with water IRRITANT
0.4 M 13 25 63 IRRITANT
1M 32 63 158 CORROSIVE
Procedure
Wear goggles (a face shield is preferable when handling large volumes) and chemical-resistant
gloves.
Work in a fume cupboard.
Measure out the indicated volume of concentrated nitric(V) acid in a measuring cylinder.
Add the liquid to about two thirds of the final volume of water in suitable beaker or laboratory jug.
Stir the solution (which will become warm).
Pour the solution into a labelled bottle and mix well. Add a hazard warning if appropriate.
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62 Nylon rope experiment

Although used in the past, tetrachloromethane (carbon tetrachloride) (see Hazcard 100) and
1,1,1-trichloroethane (see Hazcard 103), should no longer be chosen as the solvent. An alternative is
cyclohexane.
Hexanedioyl chloride (adipoyl chloride) may be used but is more unstable in storage, so it is normally better
to buy decanedioyl (sebacoyl) chloride.
The acid chlorides are liquids at room temperature. They can be weighed out in a suitable container.
Suppliers also provide the prepared solutions. These are used directly. Do check the labels.
General Hazards The acid chlorides and hexane-1,6-diamine are CORROSIVE (see Hazcards 3B & 41)
and cyclohexane is HIGHLY FLAMMABLE & HARMFUL (see Hazcard 45B).
Procedure using cyclohexane as the solvent

Wear eye protection. Wear disposable nitrile gloves when pulling out the nylon thread. The room
should be well ventilated and there must be no sources of ignition.
Dissolve 2.2 g of hexane-1,6-diamine in 50 ml of distilled water in a beaker (label it A).
Dissolve 1.5 g of decanedioyl chloride or hexanedioyl chloride in 50 ml of cyclohexane in another
beaker (label it B) and add HIGHLY FLAMMABLE & HARMFUL warning signs.
Large-scale version
Carefully pour the solution from beaker B onto solution A down a glass rod. The two liquid phases do
not mix; resist any attempt to stir the mixture at all.
Use forceps to pull out the nylon formed at the interface of the two solutions.
Disposal: see Handbook section 7.5.
Small-scale version
Place sufficient solution A to cover the base in a Petri dish.
Carefully pour solution B onto solution A in a Petri dish down a glass rod. The two liquid phases do not
mix; resist any attempt to stir the mixture at all.
Use forceps to pull out the nylon formed at the interface of the two solutions.
The thread can pulled over glass rods or pulleys smeared with a lubricant such as WD40 (see pictures
below.)
Disposal: see Handbook section 7.5.
It is also possible to reduce the scale further and use 5 ml beakers. This is particularly suitable for use by
students.
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63 Oscillating reactions
More information about these reactions can be found in Chemical Demonstrations (Vol 2), Shakashiri, 1985,
University of Wisconsin Press, ISBN 0299101304. The entire four volume series is a good source of
information.
The blue-bottle reaction

Wear goggles to prepare the sodium hydroxide solution. See Hazcards 32 &
Wear eye protection for the experiment. 91. The solution
In a 500 ml flask, dissolve 10 g of glucose in 300 ml of 0.4 M sodium hydroxide should be labelled
solution. Add 6 drops of a 0.2% aqueous methylene blue solution. Fit a rubber IRRITANT.
bung.
When the solution becomes colourless, it can be shaken to turn it blue again after
which it will again turn colourless.
Other dyes and mixtures of dyes that can be used are:
1% resazurin solution (pale blue to purple pink).
1% indigo carmine (yellow to red brown and to green with vigorous shaking).
0.2% phenosafranine (pink to colourless).
Phenosafranine and methylene blue (pink to purple and to blue with vigorous
shaking) - amounts need to be found by trial and error.
Methyl red and methylene blue (yellow to green) amounts need to be found by
trial and error.
The Belousov-Zhabotinski (B-Z) reaction

All reagents in the Belousov-Zhabotinski (B-Z) reactions must be free of chloride ions, See Hazcards 6B,
which inhibit the reaction.
80 & 98A.
Place 100 ml of 1 M sulfuric(VI) acid in a beaker on a magnetic stirrer.
Add 2.86 g of malonic acid. Let it dissolve.
Add 1.04 g of potassium bromate(V). Let it dissolve.
Add 0.11 g of cerium(IV) ammonium nitrate(V) (IRRITANT). Let it dissolve.
Add about 0.5 ml of ferroin indicator (see Recipe Sheet 49). If the red/green
oscillations slow down, more potassium bromate(V) can be added.
The Briggs-Rausher (B-R) reaction

All reagents in the Briggs-Rausher (B-R) reaction must be free of chloride ions, which See Hazcards 6B,
inhibit the reaction. 50 & 80. The
Solution A: 10 vol hydrogen peroxide solution. diluted solutions do
Solution B: 6% (w/v) potassium iodate(V) solution in 0.1 M sulfuric(VI) acid. not require a
Solution C: 1% starch solution. hazard warning.
Solution D: a solution containing 2% (w/v) malonic acid and 0.45% (w/v)
manganese sulfate(VI).
Add the solutions to a beaker on an magnetic stirrer in the following order and
quantities: 20 ml of solution A, 20 ml of solution B, 1 ml of solution C and 20 ml of
solution D.
D. The nitrogen gas oscillating reaction

Use a fume cupboard (nitrogen dioxide, VERY TOXIC, is produced). See Hazcards 68 &
Wear eye protection. 93.
Solution A: Dissolve 2.6 g of ammonium sulfate(VI) in 10 ml of 0.2 M sulfuric(VI)
acid.
Solution B: Dissolve 2.8 g of sodium nitrate(III) (nitrite) in 10 ml of water.
Mix the two solutions in a 100 ml beaker and stir quickly. Transfer to a 50 ml
measuring cylinder and after a minute the oscillations become quite distinct.
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64 Oxygen gas
Oxygen is prepared by adding hydrogen peroxide solution onto a catalyst, manganese(IV) oxide.
The reaction is very vigorous and exothermic and chemicals have been known to shoot up the thistle funnel
or the bung has been forced out of the flask. The rate of the reaction depends on the surface area of the
catalyst. It is better to use granules rather than a fine powder. If only powder is available, then only a small
amount is required.
Potassium iodide (1 g) produces oxygen at a steadier rate. The solution first saturates itself with oxygen and
then the gas is released.
Hydrogen peroxide decomposes slowly during storage. Suppliers add an inhibitor to slow the process, but
once this is used up, decomposition accelerates. If more hydrogen peroxide than expected is required, then
this is a sure sign that the hydrogen peroxide is old and new stock is required.
5 ml of 100 vol solution of hydrogen peroxide produces 500 ml of oxygen, ie, 5 x 100 ml.
General Hazards See Hazcards 50, 60 & 69.

In the 250 ml Bchner flask, add 0.1 g of manganese(IV) oxide (HARMFUL) powder or 1 g of granules.
Fill the measuring cylinder with water ready to collect the first 250 ml of gas produced. The measuring
cylinder allows you to be confident that apparatus is well flushed out before oxygen is collected.
Pour at least 25 ml of cold water, enough to cover the bottom, into the thistle funnel.
Now add 5 ml of 100 vol hydrogen peroxide (HARMFUL).
Once the measuring cylinder is full of gas, remove it and replace with a gas jar full of water.
Keep collecting gas jars of gas. When full of gas, place a glass cover on the gas jar.
If the rate of gas being produced slows down, add further 5 ml portions of 100 vol hydrogen peroxide.
Add 5 ml of 100 vol

hydrogen peroxide
250-ml
Bchner
Flask
250 ml measuring
cylinder
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65 Phosphoric(V) acid
Also known as orthophosphoric acid.
The usual concentrated commercial solution is about 85% (w/v).
Formula: H3PO4 Density: 1.7 g cm-3 Molar mass: 98 g mol-1
General Hazards See Hazcard 72.

required
500 1000 2500 label
0.1 M 3.5 7.0 17.0 -
0.5 M 17 34 85 -
Procedure
Wear goggles and chemical-resistant gloves.
Measure out the indicated volume of concentrated phosphoric(V) acid in a measuring cylinder.
Add the liquid to about two thirds of the final volume of water in a suitable beaker or laboratory jug.
Stir the solution.
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66 Plant mineral requirement solutions

A Sachs culture solution (complete recipe)
Dissolve the following salts in 1000 ml of pure water:
0.25 g of calcium sulfate(VI)-2-water

0.25 g of calcium phosphate(V)-2-water [CaH4(PO4)2.2H2O]
0.25 g of magnesium sulfate(VI)-7-water
0.08 g of sodium chloride
0.70 g of potassium nitrate(V)
0.005 g of iron(III) chloride-6-water
B Sachs culture solution with mineral deficiencies

Deficient in calcium: 0.2 g of potassium sulfate(VI) replaces calcium sulfate(VI)-2-water and
0.71 g of sodium dihydrogenphosphate(V)-2-water replaces calcium
phosphate(V) in recipe A.
Deficient in iron: Omit iron(III) chloride-6-water in recipe A.
Deficient in magnesium: 0.17 g of potassium sulfate(VI) replaces magnesium sulfate(VI) in recipe A.
Deficient in nitrogen: 0.52 g of potassium chloride replaces potassium nitrate(V) in recipe A.
Deficient in phosphorus: 0.16 g of calcium nitrate(V)-4-water replaces calcium phosphate(V) in
recipe A.
Deficient in potassium: 0.59 g of sodium nitrate(V) replaces potassium nitrate(V) in recipe A.
Deficient in sulfur: 0.16 g of calcium chloride replaces calcium sulfate(VI) and 0.21 g of
magnesium chloride-6-water replaces magnesium sulfate(VI) in recipe A.
C Knops culture solution

Solution 1
Dissolve the following salts in 1000 ml of pure water:
1 g of magnesium sulfate(VI)
1 g of potassium nitrate(V)
1 g of dipotassium hydrogenphosphate(V) [K2HPO4]
Solution 2
Dissolve 4 g of calcium nitrate(V) in 1000 ml of pure water.
Add solution 1 to solution 2 before use. This recipe is suitable for algae. For flowering plants, further dilution
by 2, 3 or 4 times may produce better results. This should be determined by experiment.
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67 Potassium and sodium phosphates

These solutions are used to make buffer solutions (see Recipe Sheet 18 for Buffers). Masses are given to 3
decimal places but for many purposes, such accuracy is not required. Adjust to 2 decimal places.
Make sure you are using the correct salt. It is best to check with the formula (see below).
Sodium and potassium dihydrogen phosphate(V) solutions are best prepared just before use. During storage
there may be mould growth.
Potassium dihydrogen KH2PO4 0.2 M Dissolve 27.218 g of the solid in 500 ml of water
phosphate(V) and add water to 1000 ml.
Sodium dihydrogen NaH2PO4 0.2 M Dissolve 23.996 g of the solid in 500 ml of water
Sodium dihydrogen NaH2PO4.H2O 0.2 M Dissolve 27.598 g of the solid in 500 ml of water
phosphate(V)-1-water and add water to 1000 ml.
Dipotassium hydrogen K2HPO4 0.2 M Dissolve 34.836 g of the solid in 500 ml of water
Dipotassium hydrogen K2HPO4.3H2O 0.2 M Dissolve 45.644 g of the solid in 500 ml of water
Disodium hydrogen Na2HPO4 0.2 M Dissolve 28.392 g of the solid in 500 ml of water
Disodium hydrogen Na2HPO4.7H20 0.2 M Dissolve 53.614 g of the solid in 500 ml of water
Potassium K3PO4 0.2 M Dissolve 42.454 g of the solid in 500 ml of water

Sodium phosphate(V) Na3PO4 0.2 M Dissolve 32.788 g of the solid in 500 ml of water
and add water to 1000 ml.
Sodium phosphate(V)- Na3PO4.12H2O 0.2 M Dissolve 76.024 g of the solid in 500 ml of water
12-water and add water to 1000 ml.
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68 Potassium chloride
Formula: KCl Molar mass: 74.55 g mol-1 Solubility: 11 g per 100 ml

1.0 M 7.46 18.64 74.55 -
3.0 M (used for 22.34 55.91 223.65 -
filing pH probes)
Saturated (20 C) 40 100 400 -
Procedure
Measure out the indicated quantity of potassium chloride.
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69 Potassium chromate(VI)
Formula: K2CrO4 Molar mass: 194.19 g mol-1 Solubility: 64 g per 100 ml

0.01 M Ten-fold dilution of the 0.1 M solution TOXIC
0.1 M 1.94 4.86 19.42 TOXIC
1.0 M 19.42 48.58 194.19 VERY TOXIC
Saturated (20 C) 68 158 630 VERY TOXIC
Procedure
Wear chemical-resistant gloves and goggles. Use a fume cupboard if the solid is a fine powder.
Measure out the indicated quantity of potassium chromate(VI).
Pour into a labelled bottle. Add the hazard warning.
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70 Potassium dichromate(VI)
Potassium dichromate solutions are best made up in 0.1 M sulfuric(VI) acid rather than in pure water.
This can be used as acidified potassium dichromate(VI) solution.
The solution is used to test for the presence of alcohols. See Recipe Sheet 102 for Test reagents for organic
functional groups.
Formula: K2Cr2O7 Molar mass: 294.18 g mol-1 Solubility: 12 g per 100 ml

0.01 M Ten-fold dilution of the 0.1 M solution with 0.1 M sulfuric(VI) acid TOXIC
0.1 M 2.94 7.36 29.42 TOXIC
Saturated (20 C) 12 30 120 VERY TOXIC
Procedure
Measure out the indicated quantity of potassium dichromate(VI).
Add the solid to about two thirds of the final volume of 0.1 M sulfuric(VI) acid in a beaker or laboratory
jug.
0.1 M sulfuric(VI) acid to the required level.
Pour into a labelled bottle. Add the hazard warning.
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71 Potassium hydroxide
Solutions absorb carbon dioxide on standing. Solutions lower than 0.1 M are most significantly affected and
should, therefore, be freshly made.
Potassium hydroxide solution is best stored in plastic screw-cap bottles. It is known to etch glass when
stored for long periods and polystop caps can allow carbon dioxide to enter.
Equipment with glass joints, eg, burettes and bottles, can seize up over time.
Formula: KOH Molar mass: 56.11 g mol-1 Solubility: 112 g per 100 ml
General Hazards See Hazcard 91. When added to water so much heat is evolved that boiling could
occur.
Do not make this solution for the first time without seeking practical advice from a
more experienced colleague. A choking mist is often formed as the solid dissolves in
water. While this is not a serious safety risk, it is unpleasant and it is wise to use a
fume cupboard where possible. If saturated potassium hydroxide has to be prepared
after carrying out an exhaustive risk assessment, then start from 5 M potassium
hydroxide solution and add pellets a little at a time. Do not store this solution.

0.1 M Ten-fold dilution of the 1 M solution IRRITANT
0.4 M 2.24 g 5.61 g 22.44 g IRRITANT
1.0 M 5.61 g 14.03 g 56.11 g CORROSIVE
5.0 M 28.06 g 70.14 g 280.55 g CORROSIVE
Procedure
Measure out the indicated quantity of potassium hydroxide.
Add the solid in stages to about two thirds of the final volume of water in a beaker or laboratory jug. If
concentrated solutions are being made, ice should be used in place of water.
Stir carefully to dissolve before adding the next group of pellets. It may be necessary to cool the
solution between additions. (Ice could be added.)
Pour into a labelled plastic bottle. Add a hazard warning if appropriate.
A 10% (w/v) alcoholic solution of potassium hydroxide

Measure out 10 g of potassium hydroxide.
Place 70 ml of the alcohol (ethanol or methanol) in a glass 250 ml beaker.
Insert a magnetic stirrer bar and place the beaker on a magnetic stirrer.
Add the potassium hydroxide solution in 3 parts, allowing the solid to dissolving before adding the next
amount. The process is exothermic but not as extreme as that with water.
Place the solution in a 100 ml measuring cylinder and make up to the 100 ml mark with the alcohol.
Pour into a labelled plastic bottle. Label the solution CORROSIVE and HIGHLY FLAMMABLE.
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72 Potassium iodide
Solutions of potassium iodide often go yellow on keeping. If this occurs, add 0.1 M sodium thiosulfate
solution dropwise, or a crystal of the solid with stirring, until the solution becomes colourless.
Formula: KI Molar mass: 166.00 g mol-1 Solubility: 144 g per 100 ml

0.1 M 1.66 4.15 16.60 -
1.0 M 16.60 41.50 166.00 -
Saturated (20 C) 150 375 1500 -
Procedure
Measure out the indicated quantity of potassium iodide.
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73 Potassium manganate(VII)
Also known as potassium permanganate.
Solutions must be acidic. Solutions are safer made up in dilute sulfuric(VI) acid rather than adding
concentrated sulfuric(VI) acid at a later stage.
Solutions do not keep well unless the container is scrupulously clean. They slowly reacts with water, forming
manganese(IV) oxide which badly stains glass and plastic equipment.
Solutions are best kept in dark bottles, shielded from light. Light increases the rate of decomposition and
staining of equipment.
Equipment can be cleaned by filling the bottle either with 1 M sulfuric(VI) acid with a small amount of
hydrogen peroxide solution (20 or 100 vol) or, if this does not work, add 2 M hydrochloric acid and leave it for
several hours (or days) in a fume cupboard.
Use a 0.002 M solution for carrying out tests for unsaturation in alkenes.
It is not possible to make a 1 M solution.
Formula: KmnO4 Molar mass: 158.03 g mol-1 Solubility: 6 g per 100 ml

Concentration Conc of acid Volume (ml) of solution required Hazard
required for dilution 500 1000 2500 warning label
0.002 M 0.1 M Ten-fold dilution of the 0.02 M solution -
with 0.1 M sulphuric(VI) acid
0.02 M 0.1 M 1.58 3.16 7.90 -
0.1 M 1M 7.90 15.80 39.51 IRRITANT
Saturated 1M 32 64 160 IRRITANT
Procedure
It would be advisable to wear gloves as the chemical stains the skin brown.
Measure out the indicated quantity of potassium manganate(VII).
Add the solid to about two thirds of the final volume of the suggested dilute sulfuric(VI) acid in a
beaker or laboratory jug.
Stir to dissolve. It is better not to heat but dissolving can take some time, and it is difficult to see when
the solid has all dissolved.
dilute sulfuric(VI) acid to the required level.
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74 Preservatives used after fixing biological specimens

Preservatives prevent chemical decomposition of the fixed material (see Recipe Sheet 41).
More information about the procedures for fixing and preserving specimens can be found in the CLEAPSS
Handbook, section 15.8.
Preserving animal Add 10 ml of odourless preservative Wear eye protection. See

tissue (Recipe 1) (eg, Phenoxetol or Opresol) to 50 ml Hazcard 37.
of propane-1,2-diol. Dilute this Animal tissue should not be
mixture to 100 ml with purified water. preserved for more than 1 month in
(Another version of this recipe these solutions unless it is fixed
includes and additional 1 g of sodium first. Label the solution IRRITANT.
chloride.)
Preserving animal Mix together 10 g of 4-methoxybenz- Wear eye protection. See
tissue (Recipe 2) aldehyde (p-anisaldehyde), 50 ml of Hazcard 37.
Teepol and 50 ml of propane-1,2-diol Animal tissue should not be
and make up this mixture to 1000 ml preserved for more than 1 month in
with purified water. these solutions unless it is fixed
first. Label the solution IRRITANT.
4-Methoxybenzaldehyde is
HARMFUL if swallowed. Label the
solution HARMFUL.
Preserving plant tissue Use the formalin-aceto-alcohol (FAA) Use a fume cupboard. Wear eye
recipe on the fixative recipe sheet 41. protection.
To preserve the red or green colour For ethanol; see Hazcard 40A.
add copper(II) sulfate(VI)-5-water For methanal, see Hazcard 63.
crystals; the amount is not too For ethanoic acid, see Hazcard 38.
critical. For copper(II) sulfate(VI), see
Hazcard 27C. Label the solution
HIGHLY FLAMMABLE, IRRITANT and
HARMFUL.
Kaiserlings Add 36 g of sodium ethanoate, 5 ml Wear eye protection.
preservative of a 5% camphor solution in ethanol Camphor is HARMFUL. For propane-
and 72 ml of propane-1,2,3-triol 1,2,3-triol, see Hazcard 37. For
(glycerol). Add this mixture to 120 ml sodium ethanoate see Hazcard
of purified water. 38A. for ethanol, see Hazcard 40A.
Label the solution HARMFUL and
HIGHLY FLAMMABLE.
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75 Ringers and other saline solutions for physiological use

Saline solutions are suitable for temporary microscopial preparations and short experiments.
For more prolonged investigations, a suitable Ringers solution should be used.
Ringers solutions
For frogs Dissolve 6.50 g of sodium chloride, 0.12 g of calcium chloride-6-water (IRRITANT), 0.14 g of
potassium chloride and 0.20 g of sodium hydrogencarbonate in 1000 ml of water.
For mammals Dissolve 8 g of sodium chloride, 0.2 g of calcium chloride-6-water (IRRITANT), 0.2 g of
potassium chloride, 0.19 g of magnesium chloride-6-water, 0.05 g of sodium
dihydrogenphosphate and 1 g of sodium hydrogencarbonate in 1000 ml of water.
For locusts Dissolve 7.60 g of sodium chloride, 0.22 g of calcium chloride-6-water, 0.75 g of potassium
and other chloride, 0.19 g of magnesium chloride-6-water, 0.48 g of sodium dihydrogen phosphate,
insects and 0.37 g of sodium hydrogencarbonate in 1000 ml of water.
For Dissolve 6 g of sodium chloride, 0.2 g of calcium chloride-6-water, 0.12 g of potassium
earthworms chloride and 0.1 g of sodium hydrogencarbonate in 1000 ml of water.
For marine Dissolve 31 g of sodium chloride, 1.37 g of calcium chloride-6-water (IRRITANT), 0.99 g of
crustaceans potassium chloride, 2.35 g of magnesium chloride-6-water, 0.48 g of sodium
dihydrogenphosphate, and 0.22 g of sodium hydrogencarbonate in 1000 ml of water.
Saline solutions
For invertebrate tissues Dissolve 7.5 g of sodium chloride in 1000 ml of water.
For amphibian tissues Dissolve 6.4 g of sodium chloride in 1000 ml of water.
For mammalian tissues Dissolve 9 g of sodium chloride in 1000 ml of water.
For mammalian blood Dissolve 6 g of sodium chloride in 1000 ml of water.
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76 Sandells solution
Sandells solution is a safer alternative than Fehlings testing for sugars.
Sandells solution (which is little known) can be stored for a long time.
The advantage in its use is that the concentration of sodium hydroxide is 0.4 M in the solution, much lower
than in Fehlings solution B, which is close to 4M.
The method, by A. Sandell, was published in the Journal of Chemical Education, April 1994, p346. SSERC
has shown that this solution is stable enough to store and works with both aldehydes and monosaccharides.
It is used in the same way as Benedicts solution.
General Hazards Copper(II) sulfate(VI) crystals are HARMFUL if swallowed (see Hazcard 27C).
2M sulfuric(VI) acid, solid sodium hydroxide and 0.5 M sodium hydroxide solution
are CORROSIVE (see Hazcards 91 and 98A). EDTA (disodium salt) is low hazard
(see Hazcard 3B).
Procedure to make 100 ml of Sandells solution

Wear goggles when preparing the solution.
Measure out 0.80 g of copper(II) sulfate(VI)-5-water and 1.80 g of EDTA (disodium salt), and dissolve
in 80 ml of water.
Add 20 ml of 2 M sodium hydroxide (CORROSIVE).
Make up to 100 ml with water. Label the bottle IRRITANT.
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77 Silver nitrate(V)
This is probably the most expensive chemical used in schools; use it with care, retrieving unused stock.
Pure water (ie, distilled or deionised) should be used to avoid cloudiness of solutions.
Tollens reagent [ammoniacal silver nitrate(V) solution], (see Recipe sheet 102) should not be stored. It
should be made and disposed of in situ by the students.
Formula: AgNO3 Molar mass: 169.87 g mol-1 Solubility: 216 g per 100 ml
General Hazards See Hazcard 87. The solid and more-concentrated solutions will stain skin and other
organic material black. This stain is difficult to remove. Work surfaces also stain.

0.01 M Ten-fold dilution of the 0.1 M with water -
0.1 1.70 4.25 16.99 -
0.2 3.40 8.50 33.98 IRRITANT
0.5 8.49 21.23 84.94 CORROSIVE
Saturated (20 C) 220 550 2200 CORROSIVE
Procedure
Measure out the indicated quantity of silver nitrate(V).
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78 Slime
Different sources of polyvinyl alcohol vary in their average molar mass; some are about 115000 g mol-1,
while others are much less, at around 17000 g mol-1 (and less expensive). The larger the average molar
mass, the better the slime.
PVA wood glue is cheaper still but contains polyvinyl acetate and other chemicals such as binders. This will
affect the nature of the slime and it may become more solid.
Custard powder can also be used with PVA glue to form bouncing custard balls.
Slime is a gel. It is thought that borate ions react with hydroxyl groups in the polymer of vinyl alcohol to form
cross links, with the elimination of water. These cross links probably involve hydrogen bonds which
continually form and break under flow.
The sliminess of the gel can be adjusted by altering the amount of sodium borate used. Viscosity changes
can be detected by timing the passage of a ball bearing through the slime in a measuring cylinder.
If acid is added to the slime, the gel collapses to give a free-flowing solution.
Although the slime can be stored in a labelled sealed bag and placed in a refrigerator, it can develop mould if
kept for long periods.
Disposal: Add enough 1 M sulfuric(VI) acid to just allow the solution to become free-flowing, dilute and pour
down a foul-water drain.
These chemicals, and the made-up slime, should not be taken home.
General Hazards See Hazcard 14. However, since publication of Hazcards, the toxicity of boron
compounds has been the subject of a considerable review and debate in the
European Union. Some suppliers will now label the bottle TOXIC with R63 (Possible
risk of harm to the unborn child) warning. However only solutions above 8% (w/v)
will be labelled TOXIC.
Sodium tetraborate should be weighed in a fume cupboard to avoid raising dust.
Procedure using polyvinyl alcohol (PVA)
PVA with a molar mass above 85,000 g mol-1 PVA with a molar mass below 85,000 g mol-1
Wear eye protection. Hot water from a kettle may be useful to start with. Those with sensitive skin may be
affected by the weak alkalinity of the solutions. In this case, disposable gloves should be worn.
Pour about 100 ml of water (no hotter than 90 C) Pour about 100 ml of water (no hotter than 90 C)
into a 400 ml beaker, add 4 g of high-mass into a 400 ml beaker, add 8 g of low-mass
polyvinyl alcohol, stirring rapidly. Add food polyvinyl alcohol, stirring rapidly. Add food
colouring and/or fluorescent dye, eg, fluorescein, colouring and/or fluorescent dye, eg, fluorescein,
for added (disgusting) effect. for added (disgusting) effect.
Heat to 90 C (but do not boil) and keep stirring Heat to 90 C (but do not boil) and keep stirring as
as required until the polymer dissolves. Allow the required until the polymer dissolves. Allow the
solution to cool. All of this may take some time solution to cool. All of this may take some time
(Solution A.). (Solution A.).
Dissolve 0.80 g of sodium tetraborate-10-water Dissolve 1.6 g of sodium tetraborate-10-water
(0.42 g of anhydrous sodium tetraborate) in (0.84 g of anhydrous sodium tetraborate) in
20 ml of water. (Solution B.) 20 ml of water. (Solution B.)
Add solution B to A with vigorous stirring. Let the gel form before removing it and washing with water.
Using PVA glue:

Dilute it to about 25%; there is no need to heat it but mix it well with water. Add sodium tetraborate as
prepared above for use with low-mass PVA. Change the concentration of PVA glue to alter the slime
quality.
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79 Soap and bubble solutions

Alcoholic soap solution can be titrated against tap water in a stoppered bottle until a froth, stable for 15
seconds, is obtained. The concentration of soap solution and/or volume of water involved in the titration will
depend on the hardness of the local tap water. Solutions should be tested before being presented to a class
so that minor adjustments to the formulation can be made.
The alcoholic soap solution is called either Clarkes or Wanklyns soap solution.
General Hazards See Hazcards 37 & 40A. Do not heat soap solutions made up in ethanol with a
naked flame. Use a hot plate or surround the beaker with hot water.
Procedure to make a soap solution suitable for titrating

Dissolve 5 g of soap flakes (eg, Lux) in 500 ml of ethanol by stirring the suspension on a hot plate.
This may take some time.
After cooling, pour the solution into a 1 litre volumetric flask and dilute to 1000 ml with pure water. Mix
the solution well.
The titration with tap water to obtain a permanent froth should be rehearsed so that adjustments to the
volume of tap water or concentration of the soap solution can be made.
Bubble mixture
Recipes may differ with different makes of liquid detergent.
The recipe below works best using Fairy Liquid. (other detergents will also work but you may need to
experiment with quantities.
Procedure to make a bubble solution

Mix together by volume, 10 parts liquid detergent, 85 parts pure water and 5 parts propane-1,2,3-triol
(glycerol).
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80 Sodium carbonate
Sodium carbonate is sold both as anhydrous sodium carbonate or sodium carbonate-10-hydrate.
Anhydrous sodium carbonate absorbs water from the atmosphere (deliquescence).
Hydrated sodium carbonate loses water to the atmosphere (efflorescence); look for a white powder as
opposed to a glassy solid.
Do not use technical grade sodium carbonate to make up solutions. There is a lot of sediment.
Solutions may be cloudy if hard tap water is used.
Formula: Na2CO3 Molar mass: 105.99 g mol-1 Solubility: 22 g per 100 ml

Formula: Na2CO3.10H2O Molar mass: 286.14 g mol-1 Solubility: 50 g per 100 ml
Anhydrous sodium carbonate

0.05 M Two-fold dilution of 0.1 M solution -
0.1 M 2.65 5.30 10.60 -
0.5 M 13.25 26.50 53.00 -
1M 26.50 53.00 105.99 -
Saturated 55 110 220 IRRITANT
Hydrated sodium carbonate

0.05 M Two-fold dilution of 0.1 M solution -
0.1 M 7.15 14.31 28.61 -
0.5 M 35.77 71.54 143.07 -
1M 71.54 143.07 286.14 -
Saturated 125 250 500 IRRITANT
Procedure
Measure out the indicated quantity of anhydrous or hydrated sodium carbonate.
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81 Sodium chlorate(I) solution

Also known as sodium hypochlorite solution, sodium chlorate(I) or bleach.
Do not get confused with the solid sodium chlorate(V).
The solution sold by laboratory suppliers and described as 10-14% available chlorine contains 100 000 to
140 000 ppm of chlorine and has a molarity of 1.5 M.
It has a limited shelf-life and may not be as active after a year in storage. Some technicians use domestic
products such as bleach but please read the containers carefully as some bleaches use hydrogen peroxide
and not sodium chlorate(I). Domestic bleach is usually 0.5 M or less and represents poorer value per mole
but may be suitable if only needed infrequently. However, there are also thickeners and detergents in
domestic bleach that may cause problems. See also Handbook section 15.12. Solutions used for disinfecting
should always be freshly prepared.
If using this as a disinfectant, purchase or prepare fresh stocks after 3 months or so.
Formula: NaClO Molar mass: 74.45 g mol-1
Concentration required Preparation Use

1% 10000 ppm 0.15 M See below for procedure. Disinfectant for blood spills or
No need for further dilution. dirty conditions.
0.25% 2500 ppm 0.038 M Mix, by volume, 1 part of the 1% Disinfectant for micro-biological
solution with 3 parts of water. discard pots.
0.1% 1000 ppm 0.015 M Mix by volume, 1 part of the 1% Disinfectant for general use, eg,
solution with 9 parts of water. sterilising solution.
0.01% 100 ppm 0.0015 M Dilute the 0.1% solution further Disinfectant for sterilising
by mixing by volume, 1 part of mouthpieces, swabbing skin.
the 0.1% solution with 9 parts of
water.
Procedure to make 1000 ml of 1% available chlorine solution

Wear gloves and goggles or a face shield.
Measure out 100 ml of 10-14% sodium chlorate(I) solution into a 1000 ml measuring cylinder.
Add water to the 1000 ml level.
Pour the solution into an appropriate labelled bottle and mix well. The solution is low hazard.
Depending on use, dilute, if necessary, from a 1% solution according to the table above.
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82 Sodium chloride
Formula: NaCl Molar mass: 58.44 g mol-1 Solubility: 36 g per 100 ml

0.5 M 2.92 7.31 29.22 -
1.0 M 5.84 14.61 58.44 -
Saturated (20 C) 37 93 370 -
Procedure
Measure out the indicated quantity of sodium chloride.
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83 Sodium ethanoate
Sodium ethanoate is sold as the anhydrous salt or as the trihydrate.
Anhydrous sodium ethanoate

Formula: NaCH3COO Molar mass: 82.03 g mol-1 Solubility: 46 g per 100 ml

0.2 M 5-fold dilution of the 1 M solution -
1.0 M 8.20 20.51 82.03 -
Saturated (20 C) 50 125 500 -
Hydrated sodium ethanoate

Formula: NaCH3COO.3H2O Molar mass: 136.08 g mol-1 Solubility: 76 g per 100 ml

0.2 M 5-fold dilution of the 1 M solution -
1.0 M 13.61 34.02 136.08 -
Saturated (20 C) 80 200 800 -
Procedure
Measure out the indicated quantity of sodium ethanoate.
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84 Sodium hydrogencarbonate
Formula: NaHCO3 Molar mass: 84.01 g mol-1 Solubility: 10 g per 100 ml

0.5 4.20 11.50 42.00 -
1.0 M 8.40 21.00 84.01 -
Saturated (20 C) 12 30 120 -
Procedure
Measure out the indicated quantity of sodium hydrogencarbonate.
Stir to dissolve. Do not warm as decomposition may take place.
Sodium hydrogencarbonate - alkaline pyrogallol solution for absorbing or removing

oxygen gas
Although safer than using sodium hydroxide with pyrogallol (benzene-1,2,3-triol), the absorption of oxygen is
slower and may be too slow for some procedures In such circumstances, there is no alternative to the use of
sodium hydroxide. The procedure described below limits the absorption of oxygen from the air.
Procedure
Wear eye protection when preparing the solution.
Prepare a saturated solution of sodium hydrogencarbonate using freshly-boiled pure water.
Place a wide-bore glass tube into the water and then pour liquid paraffin onto the waters surface.
Add a few crystals of benzene-1,2,3-triol (pyrogallol) down the glass tube and then slowly withdraw it.
(1 g of benzene-1,2,3-triol is capable of absorbing 190 cm3 of oxygen.)
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85 Sodium hydroxide
Sodium hydroxide solution is better stored in plastic screw-cap bottles. It is know to etch glass when stored
for long periods and polystop caps can allow carbon dioxide to enter.
Equipment with glass joints, eg, burettes and bottles, can seize up over time.
Formula: NaOH Molar mass: 40.00 g mol-1 Solubility: 108 g per 100 ml
General Hazards See Hazcard 91. When added to water, heat is evolved such that boiling could
occur. Do not make this solution for the first time without seeking practical advice
from a more-experienced colleague. A choking mist is often formed as the solid
dissolves in water. While this is not a serious safety risk, it is unpleasant and it is
wise to use a fume cupboard where possible. If after carrying out an exhaustive risk
assessment, there is no alternative to preparing saturated sodium hydroxide, then
start from 5 M sodium hydroxide solution and add pellets a little at a time. Do not
store this solution.

0.1 M Ten-fold dilution of the 1 M solution IRRITANT
0.4 M 4.00 16.00 40.00 IRRITANT
1.0 M 10.00 40.00 100.00 CORROSIVE
5.0 M 50.00 200.00 500.00 CORROSIVE
Procedure
Measure out the indicated quantity of sodium hydroxide.
Add the solid in stages to about two thirds of the final volume of water in a beaker or laboratory jug. If
concentrated solutions are being made, ice should be used in place of water.
Stir carefully to dissolve before adding the next group of pellets. It may be necessary to cool the
solution between additions. (Ice could be added.)
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86 Sodium silicate, the crystal (chemical) garden and silicate gels

The solution is also known as water glass.
It is more convenient to buy a solution which has a specific gravity of about 1.5, rather than the solid, and
dilute it down.
It is, however, possible to make a solution from the solid.
Expensive glassware can be very difficult to clean after using this reagent. In this case, it is better to use old
jam or coffee jars and dispose of them after use.
General Hazards See Hazcard 95B. The solution is strongly alkaline. Consult the relevant Hazcards
when using metal salts to add to the chemical garden.
Procedure to make 250 ml of sodium silicate solution for a chemical garden from the
commercially-available solution
Add 200 ml of commercial sodium silicate to 600 ml of pure water in a 1-litre beaker.
Pour into a labelled bottle. Although the solution is deemed low hazard, it might be prudent to label the
bottle IRRITANT as the solution is strongly alkaline.
Procedure to make 250 ml of 8% sodium silicate solution from the solid

Add 20 g of sodium silicate to a 600 ml beaker and add 250 ml of pure water.
Add a magnetic stirrer bar and place the mixture on a heater/stirrer.
Maintain a temperature of about 45 C until the solid appears to dissolve.
Filter through Whatman No.1 filter paper.
Pour into a labelled bottle. Although the solution is deemed low hazard, it might be prudent to label the
bottle IRRITANT as the solution is strongly alkaline.
The crystal (chemical) garden

Wear eye protection and consider wearing disposable nitrile gloves.
Pour the prepared sodium silicate into a 600 ml beaker (a labelled coffee jar is also suitable), cover it
with a clock glass or sheet of glass (check edges are not sharp) and allow the liquid to settle. (After
adding crystals, it is better not to move the container so any moving should be done at this stage.)
Now add small (eg, rice-grain size) crystals of manganese(II) sulfate(VI), copper(II) sulfate(VI),
chromium(III) chloride, iron(II) sulfate(VI), iron(III) chloride, cobalt(II) chloride, tin(II) chloride,
aluminium sulfate(VI), nickel(II) sulfate(VI) or any other soluble salt you wish to try.
Leave the container undisturbed for several days. Label the container IRRITANT.
Growing lead(II) iodide crystals in gels

Dilute 16 ml of commercial sodium silicate solution to 100 ml with pure water (solution A).
Place 15 ml of 1 M ethanoic acid (see Recipe Sheet 39) in a beaker and add 0.67 g of potassium
iodide (solution B).
Add 15 ml of solution A to solution B, stirring vigorously. Pour the mixture into a boiling tube and allow
it to set (5 to 60 minutes).
Dissolve 0.66 g of lead(II) nitrate(V) in 2 ml of water and pour this onto the top of the gel. Label the
container IRRITANT.
Observe the spread of crystals of lead(II) iodide over the next few weeks.
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87 Sodium thiosulfate
Also known as hypo and it is usually supplied as hydrated crystals.
Distilled water is naturally acidic (dissolved carbon dioxide) and this may result in the slow formation of sulfur
over several hours. This is avoided by adding a pinch of sodium hydrogencarbonate or sodium sulfate(IV) to
the solution to neutralise the acidity.
A 25% solution is suitable to keep with bromine for use in case of accidents. (See Hazcard 95C.)
Formula: Na2S2O3.5H20 Molar mass: 248.17 g mol-1 Solubility: 70 g per 100 ml

0.1 M 2.48 6.20 24.82 -
0.5 M 12.41 31.02 124.08 -
1.0 M 24.82 62.04 248.17 -
Saturated (20 C) 75 188 750 -
Procedure
Measure out the indicated quantity of sodium thiosulfate-5-water.
Stir to dissolve. Do not warm the solution.
Add about 1 g of sodium hydrogencarbonate or sodium sulfate(IV) (sulfite) (HARMFUL) for every
1000 ml of solution, to help to preserve it.
Pour the solution into a labelled bottle and mix well.
A saturated solution
The saturated solution requires little water. Weigh out the sodium thiosulfate as indicated and add it to hot
(almost boiling) water with a volume in millilitres equal to about half the weight of solid in grams. Stir to
dissolve and leave to cool to room temperature. If there is no solid present in the cooled solution, add one
crystal of solid to it. If this causes further solid to form, then the solution is saturated and may be left in
contact with the solid. If not, add further solid.
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88 Stains for bacterial activity

Gram Stain To prepare the crystal violet solution, dissolve 2 g of See Hazcards 32, 36A, 40A
For details on the dye in 100 ml of industrial denatured alcohol and 85.
using this stain (Solution A) and 1 g of ammonium ethanedioate in IDA is HIGHLY FLAMMABLE.
see Guidance 100 ml of distilled/deionised water (Solution B). There should be no sources of
Leaflet GL 95 Add 25 ml of solution A to 100 ml of solution B. ignition in the vicinity
Wear eye protection during the
To prepare the Safranin counterstain, dissolve 0.5 g of preparation. Ensure the room
safranin in 100 ml of distilled/deionised water. is well ventilated. Wear
chemical-resistant gloves
To prepare Grams (Lugols) iodine, dissolve 1 g of when preparing the crystal
iodine and 2 g of potassium iodide in 300 ml of distilled violet stain.
or deionised water.
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89 Stains for cells
Aceto carmine Add 0.5 g of carmine to 55 ml of purified water in a For ethanoic acid, see
conical flask. Boil and add 45 ml of concentrated Hazcard 38. Label the stain
ethanoic acid. Plug the flask with cotton wool, boil CORROSIVE. Wear goggles.
again, cool and filter. Use a fume cupboard.
Ehrlichs Dissolve 0.64 g of haematoxylin in 32 ml of ethanol For aluminium potassium
haematoxylin heated on a water bath, and filter. Separately, dissolve sulfate(VI), see Hazcard 3B.
for animal 0.32 g of aluminium potassium sulfate(VI) in 32 ml of For ethanol; see Hazcard 40A.
histology purified water. Mix this solution with the haematoxylin For ethanoic acid, see
solution and add 32 ml of propane-1,2,3-triol (glycerol) Hazcard 38. For propane-
and 4 ml of glacial ethanoic acid. Leave exposed to 1,2,3-triol, see Hazcard 37. For
daylight for 6 weeks to ripen or add 1 ml of 0.2 M ethanoic acid, see Hazcard 38.
potassium manganate(VII) solution. For potassium manganate(VII),
see Hazcard 81.
Wear eye protection and
disposable nitrile gloves when
making up the solution.
Label the stain HIGHLY
FLAMMABLE.
Eosin for Dissolve 1 g eosin in 100 ml tap water. Used as a Wear eye protection. Low
cytoplasmic counterstain to Ehrlichs haematoxylin. For alcoholic hazard unless ethanol is used
staining solution, substitute 75% ethanol for water. as the solvent.
Feulgen's stain Dissolve 0.45 g of basic fuchsin in 87 ml of boiling For potassium metabisulfite,
for DNA water. Cool to 50 C and add 30 ml of 1 M hydrochloric see Hazcard 97. For
acid and 3 g of potassium metabisulfite. Leave in the hydrochloric acid , see
dark for 24 hours to bleach. Add 0.5 g of decolourising Hazcard 47A. Wear eye
charcoal, shake and filter into a stoppered bottle. Store protection. Use a fume
in a cool, dark place. cupboard.
No hazard label is required.
Leishman's Dissolve 0.15 g of the solid stain in 100 ml of For methanol, see Hazcard 40.
stain for blood methanol. Prepare the solution in a fume
cells cupboard wearing eye
protection and disposable
nitrile gloves.
Label the solution TOXIC and
HIGHLY FLAMMABLE.
Methyl green 1 g of methyl green pyronin is dissolved in 100 ml of Wear eye protection and
pyronin for DNA distilled/deionised water. DNA is stained green and disposable nitrile gloves.
and RNA RNA is stained red.
Methylene blue Wear eye protection, and gloves to avoid staining the For methylene blue, see
for cheek cells skin. Dissolve 0.1 to 1 g in 100 ml of water. Hazcard 32. The solution is
low hazard.
Orcein acetic The stock solution contains 2.2 g of orcein dissolved in For ethanoic acid, see
(Acetic orcein) 100 ml of glacial ethanoic acid. Dilute 10 ml of this Hazcard 38. Wear goggles and
for solution with 12 ml of water before use. This diluted chemical-resistant gloves.
chromosomes solution does not keep. Use a fume cupboard.
Label the concentrated
solution CORROSIVE.
Sudan III or IV Dissolve 0.5 g of dye in 70 ml of ethanol and 30 ml of Ethanol is HIGHLY FLAMMABLE
solutions for water, using a warm water bath, and filter. (see Hazcards 32 and 40).
lipids Label the solution HIGHLY
FLAMMABLE. Wear eye
protection.
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90 Stains for electrophoresis

Methylene blue is less sensitive than Azure A and not so convenient to use in electrophoresis but it is
cheaper and adequate for some activities.
Azure A for DNA Dissolve 0.04 g in 100 ml of 20% ethanol (ie, 0.04% Low hazard.
w/v) to apply after running the gel. Destaining is not
needed.
Colloidal See Low hazard.
Coomassie Blue www.ncbe.reading.ac.uk/NCBE/SCIENCEYEAR/recipes.html
for proteins for a recipe, or purchase from this organisation. Note
that this stain needs to be made up at least 24 hours
before use, and requires the addition of sodium
chloride solution to stabilise it.
Methylene blue Dissolve 0.05 g in 200 ml water (ie, make up to See Hazcard 32, The solution
for DNA 0.025% w/v) to apply after running the gel. Destaining is low hazard.
is needed.
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91 Stains for fungal material

Lactophenol Dissolve 10 g of phenol in 10 ml of water (do not heat). For propan-1,2,3-triol, see
Add 10 ml of propan-1,2,3-triol (glycerol) and 10 ml Hazcard 38C. For phenol, see
lactic acid. Hazcard 70.Wear goggles.
Wear disposable nitrile gloves.
Use a fume cupboard. Label
the solution TOXIC and
CORROSIVE.
Cotton blue Dilute 8 ml of 0.1% cotton blue solution in lactophenol See Hazcard 38C.
(aniline blue) for and 4 ml of 0.1% basic fuchsin solution in lactophenol Fuchsin is HARMFUL. Wear
fungi with 88 ml of lactophenol. suitable eye protection and
gloves. Label the solution
TOXIC.
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92 Stains for metabolic activity

Janus green B Dissolve 0.3 g of the dye in 100 ml of purified water. Wear eye protection and
(Diazine green) Dilute this solution ten times with water before use. disposable nitrile gloves when
for mitochondria The colour change is blue (with oxygen) to pink making up the solution. No
(anaerobic). hazard label is required.
See Guidance Leaflet PS 89 for more information.
Methylene blue For living organisms: dissolve 1 g solid in 100 ml water For ethanol, see Hazcard 40.
for cell contents and add 0.6 g sodium chloride. For dead tissue: For methyl blue see Hazcard
dissolve 0.23 g solid in 23 ml of ethanol and dilute to 32. Wear eye protection and
100 ml with water. disposable nitrile gloves.
No hazard label is required.
Neutral red Dissolve 0.1 g of neutral red in 100 ml of the Wear eye protection and
appropriate isotonic saline solution (see Recipe Sheet disposable nitrile gloves.
75). Dilute the solution ten times further with the saline
solution.
Resazurin Dissolve one tablet or 0.005 g of resazurin powder in Resazurin is IRRITANT.
solution to test 50 ml of water. (1 ml of this is added to 10 ml of a
for milk sample of milk. A colour change from blue to pink to
freshness white indicates how many bacteria are present.)
TTC, to show Dissolve 1 g of 2,3,5-triphenyl tetrazolium chloride Low hazard.
respiratory (TTC) in 100 ml of water. (A 0.5% solution is less
activity expensive and gives just as good results but takes
longer. It works well with maize seedlings.) Produces a
red colouration.
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93 Stains for plant material

Aniline Mix 89 ml of ethanol, 10 ml of 0.05 M sulfuric acid and For phenylammonium
(phenyl- 1 g of phenylammonium sulfate [aniline sulfate(VI)]. sulfate(VI): see Hazcard 4.
ammonium) Wear eye protection and
sulfate stain for disposable nitrile gloves when
lignin making up the solution.
Label the stain HIGHLY
FLAMMABLE.
FABIL for plant Prepare 3 solutions: 0.5 % solution of aniline blue in For lactophenol, see
tissues lactophenol, 0.5% solution of basic fuchsin in Hazcard 38C; for iodine, see
lactophenol and a solution containing 0.3 g of iodine Hazcard 54.
and 0.6 g of potassium iodide in 100 ml of lactophenol. Wear goggles and chemical-
When required, mix in the proportions of 4:1:5 and resistant gloves.
allow to stand overnight. Filter before use. Label the stain TOXIC.
(Cell contents stain blue, cellulose walls stain light blue
and lignin stains yellow.)
Iodine stain Use 0.01 M iodine solution. See Recipe Sheet 50.
Phloroglucinol Dissolve 5 g of phloroglucinol (benzene-1,3,5-triol) in Phloroglucinol is an IRRITANT.
for pentoses 75 ml of ethanol and 25 ml of water. Ligneous tissue Ethanol is HIGHLY FLAMMABLE
and lignin should be well-flooded and staining continued for (see Hazcards 12, 40 and 47).
about 4 minutes after which 1 drop of concentrated Label solution HIGHLY
hydrochloric acid should be added. FLAMMABLE. Wear eye
protection.
Schulzes Dissolve by warming 20 g of anhydrous zinc chloride in Zinc chloride is CORROSIVE and
solution for 8.5 ml of water and allow the mixture to cool. In a iodine is HARMFUL (see
cellulose separate container, dissolve 1 g of potassium iodide Hazcards 54 and 108). Wear
and 0.5 g of iodine in 20 ml of water. Add this solution eye protection, and chemical-
dropwise to the zinc chloride solution until iodine resistant gloves, and carry out
precipitate persists on agitation. the procedure in a fume
cupboard. Label the solution
CORROSIVE.
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94 Standard solutions for titration

A primary standard solution is one with a concentration that is accurately known. (They do not have to be
0.1 M) They are used to determine the concentration of other reagents undergoing titration.
Solids used should be of high purity (ie, more than 99.5% pure). Hydrated salts should not be used.
The solid should not alter composition during weighing (which is why sodium hydroxide is not used; it
absorbs water and carbon dioxide).
Distilled or deionised water must be used.
If possible, use a balance reading to 3 decimal places.
0.1 M sodium carbonate solution for standardising strong acid solutions
Procedure to prepare 1000 ml of solution

Heat the anhydrous sodium carbonate in an evaporating basin with a gentle flame for 30 minutes and
allow it to cool in a desiccator.
Measure 10.599 g of solid into a clean 250 ml beaker and add 150 ml of boiled pure water.
Stir the solution until the solid dissolves and pour it via a funnel into a 1000 ml volumetric flask.
Rinse the stirring rod and the beaker with water, pouring the washings into the volumetric flask.
Repeat this twice more.
Add water via the funnel into the flask so it is just up to the required mark. Add the stopper and mix the
solution well.
Titrate against a strong acid using methyl red as the indicator. The colour of the endpoint is orange.
0.1 M potassium hydrogenphthalate solution for standardising strong alkali

solutions

Measure 20.433 g of the solid into a clean 250 ml beaker and add 150 ml of boiled pure water.
solution well.
Titrate against a strong alkali using phenolphthalein as the indicator. The colour of the endpoint is pale
mauve which should last for at least 30 s.
0.1 M sodium ethanedioate solution for standardising potassium manganate(VII)

solution

Measure 13.40 g of solid into a clean 250 ml beaker. Add 150 ml of cold pure water. Stir the solution
until the solid dissolves and pour it via a funnel into a 1000 ml volumetric flask.
solution well.
Place the potassium manganate(VII) in the burette. Titrate against hot standard sodium ethanedioate
solution until there is a permanent mauve colour.
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0.0167 M potassium iodate(V) solution for standardising sodium thiosulfate solution

Dry the potassium iodate(V) at 180 C for 30 minutes before cooling and using.
Measure 3.574 g of solid into a clean 250 ml beaker and add 150 ml of cold pure water.
solution well.
To use, begin with a measured volume (10, 20 or 25 ml) of potassium iodate(V) solution. Add 10 ml of
1 M potassium iodide solution and 10 ml of 1 M sulfuric(VI) acid to produce iodine against which
sodium thiosulfate is titrated. Use a 1% starch solution as the indicator when the solution is pale
yellow. And from the results it is possible to calculate the concentration of the thiosulfate solution.
0.1 M calcium carbonate solution for standardising EDTA solution

Dry the precipitated calcium carbonate at 150 C for 1 hour.
Measure 10.00 g of cooled solid into a clean 250 ml conical flask and add enough 2 M hydrochloric
acid to dissolve it, placing a filter funnel over the flask to stop spray.
Make up this solution in a 1-litre volumetric flask.
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95 Strontium chloride
Strontium chloride hexahydrate will absorb water from the atmosphere.
Formula: SrCl2.6H2O Molar mass: 266.6 g mol-1 Solubility: 88 g per 100 ml
Procedure for preparing 100 ml of 0.1 M strontium chloride solution

Dissolve 2.67 g of strontium chloride-6-water in 70 ml of water.
NB:
If a school has only strontium carbonate, the solution can be made by adding 1.48 g of the carbonate to
100 ml of 0.1 M hydrochloric acid.
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96 Sulfur dioxide
For acid rain see the Recipe sheet 97.
This is a particularly nasty gas to prepare and use. Do make sure that the procedure is trialled with help from
a more-experienced colleague and that the fume cupboard is working satisfactorily.
General Hazards See Hazcards 47A, 78, 92 & 97. Sulfur dioxide is TOXIC by inhalation. There is no
safe Workplace Exposure Limit for sulfur dioxide. As a guide, the HSE has advised
that levels, must be less than 2.7 mg m-3 (ie, 1 ppm). Any preparation of sulfur
dioxide should be carried out in a fume cupboard.

Place between 10 and 20 g of sodium metabisulfite (HARMFUL) in a 250 cm3 conical flask.
Set up the equipment as shown below.
Pour enough 2 M hydrochloric acid (IRRITANT) to just cover the bottom of the thistle funnel tube. Place
some moist acidified potassium dichromate(VI) paper (TOXIC) above the gas jar.
Heat the conical flask gently and collect the gas by downward delivery. When the acidified potassium
dichromate(VI) paper turns green, the gas jar is full.
Add 5 M hydrochloric acid to the metabisulfite if more sulfur dioxide is required.
The leftover reagents can be poured down the fume cupboard sink with plenty of water.
It is possible to carry out the fountain experiment. A round-bottom flask is substituted for the gas jar.
Add at least 50 cm3 of

Acidified potassium
dichromate(VI) paper
Loose mineral
wool plug
Heat
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97 Sulfur dioxide solution

Sulfur dioxide is a particularly nasty gas to prepare and use. Do make sure that the procedure is trialled with
help from a more-experienced colleague and that the fume cupboard is working satisfactorily.
General Hazards See Hazcards 47A, 78, 92 & 97. Sulfur dioxide is TOXIC by inhalation. There is no
safe Workplace Exposure Limit for sulfur dioxide. As a guide, the HSE has advised
that levels must be less than 2.7 mg m-3 (ie, 1 ppm). Any preparation of sulfur
dioxide should be carried out in the fume cupboard.
Method 1
Use a fume cupboard. Make sure it is working Wear goggles.
3
Place between 10 and 20 g of sodium metabisulfite (HARMFUL) into a 250 cm conical flask.
Set up the equipment as shown below. The inverted filter funnel should be held just below the surface
of the water in the beaker.
Pour enough 2 M hydrochloric acid (IRRITANT) to just cover the bottom of the thistle funnel tube in the
flask.
Heat the conical flask gently to produce the gas, which will dissolve as it meets the water in the
beaker. The inverted funnel prevents suck back.
Add 5 M hydrochloric acid if more sulfur dioxide is required.
The leftover reagents can be poured down the fume cupboard sink with plenty of water.
Add at least 50 cm3 of

Inverted funnel
just below water
surface
Heat
Method 2
In a fume cupboard, dissolve 9.5 g of sodium metabisulfite (HARMFUL) in 100 ml of water. Add 100 ml of
0.5 M sulfuric(VI) acid and make up to 250 ml.
Acid Rain
Pupils should open containers in a fume cupboard and particular attention should be paid to known
asthmatics.
For acid rain, use 0.0001 M sulfuric(VI) acid rather than a sulphur dioxide solution.
For an acidic atmosphere, use a 0.1 M solution of sodium metabisulfite in water. This will generate enough
SO2 gas to provide an acid atmosphere.
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98 Sulfuric(VI) acid
The procedure below uses ice made from distilled or deionised water. Make sure you have a supply of this in
the freezer.
Do not make dilute solutions for the first time without seeking practical advice from a more-experienced
colleague.
Formula: H2SO4 Molar mass: 98.07 g mol-1

General Hazards See Hazcard 98A. Addition of acid to water produces a lot of heat. NEVER ADD
WATER TO THE ACID; serious accidents have occurred when this has been done.

0.01 M Ten-fold dilution of a 0.1 M solution with water -
0.1 M Ten-fold dilution of a 1 M solution with water -
0.4 M 11 22 54 -
Battery acid 115 230 575 CORROSIVE
Procedure
Wear goggles (a face shield is preferable when handling large volumes) and chemical-resistant
gloves.
Measure out the indicated volume of concentrated sulfuric(VI) acid in a measuring cylinder.
Fill the beaker or laboratory jug half to two thirds full with ice, add 200 ml of water and a stirrer bar.
Set the stirrer running on a magnetic stirrer and add the concentrated sulfuric(VI) acid slowly onto the
ice.
Keep stirring the solution until the ice melts.
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99 Testing for gases

Ammonia
Use damp red litmus Damp red litmus goes blue. Universal indicator can Wear eye protection.
paper be used as well.
Place concentrated White smoke of ammonium chloride (IRRITANT) forms. Wear eye protection.
hydrochloric acid close by
Carbon dioxide
Use lime water See Recipe Sheet 20 for calcium hydroxide. A white Wear eye protection if
precipitate of calcium carbonate forms which, in time, blowing into limewater
goes colourless as calcium hydrogencarbonate through a straw.
forms.
Carbon monoxide
Light it in a fume The gas catches fire with a blue flame forming Wear eye protection.
cupboard carbon dioxide.
Chlorine
Use damp blue litmus Damp blue litmus goes red on formation of chloric(I) Wear eye protection.
paper acid. This then bleaches the paper. Use a fume cupboard.
Dinitrogen monoxide
Add a lighted or glowing The gas supports combustion and can relight a Wear eye protection.
splint glowing splint, although it is not as active as oxygen.
Hydrogen
Add a lighted splint If the hydrogen is mixed with air there is a pop but if Wear eye protection.
the gas is pure it catches fire and there is no pop.
Hydrogen chloride
Use damp blue litmus Damp blue litmus goes red. Universal indicator can Wear eye protection.
paper be used as well.
Place concentrated White smoke of ammonium chloride forms. Wear eye protection.
ammonia solution close
by
Hydrogen sulfide
Moisten filter paper with The paper immediately turns black with the formation Wear eye protection.
0.05 M lead(II) nitrate(V) of lead sulfide. Use tongs or forceps to
solution hold the paper.
Nitrogen
There is no positive test for nitrogen.
Nitrogen dioxide
Observe the colour The only brown gas there is. Wear eye protection.
Nitrogen monoxide
Add the gas to air Brown nitrogen dioxide is formed. Wear eye protection.
Oxygen
Add a glowing splint This relights a glowing splint, sometimes with a pop. Wear eye protection.
Sulfur dioxide
Moisten filter paper with The paper goes green with the formation of Wear eye protection.
0.05 M acidified chromium(III) ions. Use a fume cupboard.
potassium dichromate(VI)
solution
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100 Testing for negative ions

Bromide ions
0.05 M silver nitrate(V) Dissolve 0.85 g of silver nitrate(V) in 100 ml of See Hazcard 6 & 87.
solution distilled/deionised water. A cream precipitate forms which Wear eye protection.
is soluble after the addition of concentrated ammonia.
Carbonate ions
2 M hydrochloric acid Adding the acid produces a lot of fizzing. The carbon See Hazcard 47A.
dioxide should be identifiable with lime water. Wear eye protection.
Chloride ions
0.05 M silver nitrate(V) Dissolve 0.85 g of silver nitrate(V) in 100 ml of water, See Hazcard 6 & 87.
solution which must be pure. A white precipitate forms which is Wear eye protection.
soluble after the addition of 2 M ammonia solution.
Ethanoate and methanoate ions

0.1 M iron(III) chloride Add 2 M ammonia solution to 0.1 M iron(III) chloride See Hazcard 6 & 55C
solution solution until a precipitate just appears. Now add more of Wear eye protection.
the original iron(III) chloride in drops with stirring until the
precipitate just dissolves. Addition of this neutral iron(III)
chloride to both ethanoates and methanoates gives a red
solution.
Iodide ions
0.05 M silver nitrate(V) Dissolve 0.85 g of silver nitrate(V) in 100 ml of See Hazcard 6 & 87.
solution distilled/deionised water. A yellow precipitate forms which Wear eye protection.
is not soluble after the addition 2 M ammonia solution.
Nitrate(III) (nitrite) ions

2 M hydrochloric acid Adding the acid produces a pale blue liquid and a brown See Hazcard 47A, 68
gas (nitrogen dioxide, TOXIC). Immediately, add water to and 93. Wear eye
quench the reaction. protection.
Nitrate(V) ions
Brown ring test Dissolve the nitrate(V) in about 1 ml of water and add Wear goggles.
0.5 g of iron(II) sulfate(VI)-7-water. Hold the test tube at See Hazcards 55B &
an angle and carefully pour about 1 ml of concentrated 98.
sulfuric(VI) acid down the side of the test tube so it forms
a layer at the bottom. A brown ring indicates nitrate(V)
ions present.
Devadas alloy Add 2 ml of suspected nitrate(V) to 5 ml of 1 M sodium Wear goggles.
hydroxide solution. Add a little Devardas alloy (contains See Hazcard 1 & 91.
aluminium, copper and zinc) and warm. Test for ammonia
gas.
Phosphate ions
0.5 M ammonium Dissolve 4 g of ammonium molybdate(VI) in 4 ml of See Hazcards 6, 8, 9A
molybdate(VI) solution concentrated ammonia and 6 ml of water. Add 12 g of & 67.
(HARMFUL) ammonium nitrate(V) and dilute to 100 ml. When testing a
sample, acidify the sample first with 0.5 ml of 1 M nitric(V)
acid and then add 2 ml of the ammonium molybdate(VI)
solution.
Sulfate(VI) ions
0.1 M barium chloride Dissolve 2.44 g in 100 ml of distilled/deionised water. A See Hazcard 10A.
solution (HARMFUL) white precipitate is formed. Sulfate(IV) ions do not give a
precipitate but it can form in time due to oxidation by
dissolved oxygen.
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101 Testing for positive ions

Metal ions in general
Reagent Recipe and observations Hazards and notes
1 Flame tests Barium ions Greenish flame See Hazcards 10A,
Calcium ions Brick red flame 19A & 47B.
Copper(II) ions Bluish flame The Hazcards also
Lead(II) ions White flame have the procedure for
Lithium Crimson red flame the tests. There is also
Potassium ions Lilac flame (best seen thro cobalt glass) the borax bead test on
Rubidium ions Blue flame Hazcard 14.
Strontium ions Crimson flame
Sodium ions Persistent yellow flame.
2 Add 0.4 M Dissolve 1.6 g of sodium hydroxide in 100 ml of water. See Hazcard 91.
sodium Precipitates form, some coloured and others colourless, The concentration of
hydroxide to a some of which dissolve in excess alkali solution. the metal solutions
small amount of Aluminium ions White precipitate which dissolves in needs to be 0.1 M.
the solution excess alkali
under test Ammonium ions No precipitate but odour of ammonia
Barium ions White precipitate
Calcium ions White precipitate
Cobalt(II) ions Blue precipitate
Copper(II) ions Blue precipitate
Iron(II) ions Green precipitate
Iron(III) ions Brown precipitate
Lead(II) ions White precipitate which dissolves in
excess alkali
Magnesium ions White precipitate
Nickel(II) ions Green precipitate
Silver ions Brown precipitate
Zinc ions White precipitate which dissolves in
excess alkali
Potassium ions No precipitate
Sodium ions No precipitate
3 0.1 M sodium, Dissolve 2.4 g of sodium sulfide-9-water or 1.0 g of See Hazcard 51. The
potassium or potassium sulfide in 100 ml of water. 4 ml of ammonium test solutions are
ammonium sulfide solution can be diluted to 100 ml with water. Some IRRITANT. Use a fume
sulphide metal ions form precipitates with the solutions. Refer to an cupboard as the odour
solution (old!) A-level text for details. is repulsive.
Aluminium ions
Alizarin solution Dissolve 0.1 g of alizarin in 100 ml of water. Addition of 2 M See Hazcard 32.
ammonia solution to an aluminium salt in the presence of
alizarin gives a pink precipitate or lake.
Ammonium ions
0.1 M sodium Dissolve 4.04 g in 100 ml of water. A yellow precipitate See Hazcard 95A. It is
hexanitrocobalt-ate(III) forms with ammonium ions. an unstable solution
solution (HARMFUL) with a maximum shelf
life of 3 weeks.
0.4 M sodium Warm with the test solution. Ammonia gas will be given Wear eye protection.
hydroxide solution off.
(IRRITANT)
Calcium ions
0.25 M ammonium Dissolve 3.5 g of ammonium ethanedioate in 100 ml of See Hazcard 36A.
ethanedioate water. Ammonium ethanedioate gives a white precipitate
of calcium ethanedioate. The precipitate can be filtered off
and dried. It decomposes under heat to give calcium
oxide. The oxide can be weighed to estimate
quantitatively the amount of calcium present in a sample.
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Cobalt(II) ions
Thiooxamide Dissolve 0.5 g in 100 ml of ethanol. A brown precipitate is See Hazcards 35 and
(rubeanic acid) produced with cobalt ions. also 40A.
(HIGHLY FLAMMABLE,
HARMFUL)
0.1 M ammonium or Dissolve 0.76 g of ammonium thiocyanate or 0.97 g of See Hazcard 9B or
potassium thiocyanate potassium thiocyanate in 50 ml of water and make up to 95C.
100 ml. A blue solution forms with cobalt(II) ions.
Copper(II) ions
2M ammonia solution Dilute 11 ml of 880 ammonia to 100 ml with water. A blue See Hazcards 6.
precipitate is formed which dissolves in excess ammonia Prepare in a fume
solution to form a deep blue solution. cupboard.
Thiooxamide Dissolve 0.5 g in 100 ml of ethanol. A black precipitate See Hazcards 35 and
(rubeanic acid) forms with copper(II) ions. also 40A.
(HIGHLY FLAMMABLE,
HARMFUL)
potassium thiocyanate potassium thiocyanate in water 50 ml of water and make 95C. The solution is
up to 100 ml. A black precipitate forms. LOW HAZARD.
Iron(II) ions
0.1 M potassium Dissolve 3.29 g in 100 ml of water. A light blue precipitate See Hazcard 79. No
hexacyanoferrate(III) forms. hazard warning is
required on the
solution.
1,10 phenanthroline Dissolve 1.49 g of 1,10 phenanthroline in 100 ml of water. See Hazcard 55B.
solution The reagent gives a red colour when mixed with iron (III) Solid Phenanthroline is
ions. TOXIC if swallowed. No
hazard warning is
required on the
solution.
Iron(III) ions
0.1 M potassium Dissolve 3.29 g in 100 ml of water. A dark blue precipitate See Hazcard 79. No
hexacyanoferrate(III) forms. hazard warning is
required on the
solution.
Ferroxyl indicator Dissolve 2 g of sodium chloride, 0.1 g potassium See Hazcards 32, 40A
(HIGHLY FLAMMABLE) hexacyanoferrate(III) and 1.0 g of phenolphthalein in and 79.
50 ml of ethanol, and mix with 100 ml of water. Very
useful for detecting the rusting process.
potassium thiocyanate potassium thiocyanate in 50 ml of water and make up to 95C. The solution is
100 ml. A dark red solution forms. [NB there is no LOW HAZARD.
colouration with iron(II) ions]
Lead(II) ions
Dithizone Dissolve 0.005 g in 100 ml of dichloromethane or See Hazcards 28, 32
methylbenzene. The reagent gives a red colouration with and 46. Label the
lead(II) ions. solution according to
which solvent is used.
Potassium iodide Prepare a 0.1 M solution. A bright yellow precipitate See Hazcard 47B.
forms.
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Nickel(II) ions
Thiooxamide Dissolve 0.5 g in 100 ml of ethanol. A violet precipitate See Hazcards 35 and
(rubeanic acid) forms. also 40A.
(HIGHLY FLAMMABLE,
HARMFUL)
Butanedione dioxime Dissolve 1 g in 100 ml of ethanol. A red precipitate forms. See Hazcard 53.
(dimethylglyoxime)
(HIGHLY FLAMMABLE &
HARMFUL)
Potassium ions
0.1 M Sodium Dissolve 4.04 g in 100 ml of water. A yellow precipitate See Hazcard 95A. It is
hexanitrocobalt-ate(III) forms. an unstable solution
(HARMFUL) with a maximum shelf
life of 3 weeks.
Zinc(II) ions
2M ammonia solution Dilute 11 ml of 880 ammonia to 100 ml with water. A white See Hazcards 6.
precipitate forms, which dissolves in excess ammonia Prepare in a fume
solution to form a colourless solution. cupboard. The solution
is LOW HAZARD.
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102 Testing for organic functional groups

Alcohols - primary, secondary & tertiary
Lucas reagent Dissolve 68 g of anhydrous zinc chloride in 43 g of concentrated See Hazcards
(CORROSIVE) (36%) hydrochloric acid. The time taken for turbidity to appear is 47A & 108A.
used to differentiate between the three classes of alcohols:
no visible reaction: primary alcohol,
solution turns cloudy in 3 - 5 minutes: secondary alcohol,
solution turns cloudy immediately, and/or phases separate:
tertiary or benzyl alcohol.
Potassium Identifies primary and secondary alcohols. To five drops of the See Hazcard 78 &
dichromate(VI) test alcohol, add 10 drops of 1 M sulfuric(VI) acid and 4 drops of 0.1 M 98A.
potassium dichromate(VI) solution. Place the solution in boiling
hot water from a kettle. The colour change is orange to green.
Potassium Identifies primary and secondary alcohols. To five drops of the See Hazcard 81 &
manganate(VII) alcohol, add 10 drops of 1 M sulfuric(VI) acid and 4 drops of 98A.
test 0.002 M potassium manganate(VII) solution. Place the solution in
boiling hot water from a kettle. The colour change is purple to
colourless.
Aldehydes and ketones

Hydrogen sulfite Dissolve 20 g of sodium metabisulfite in 100 ml of hot water. See Hazcard 92.
test White precipitate appears with the reagent is added to a carbonyl
compound.
Tollens reagent It does not work with ketones. This reagent is ammoniacal silver See Hazcard 87.
nitrate(V), it must be made immediately before use and not stored. Immediately
The breakdown products are EXPLOSIVE. Add one drop of 0.4 M dispose of liquid
sodium hydroxide solution to 1 ml of 0.1 M silver nitrate(V). Add after use down
1 M ammonia solution until the precipitate just redissolves. On the foul-water
warming with an aldehyde, a silver mirror forms. drain.
Schiffs reagent It does not work quickly with ketones. Dissolve 0.1 g of fuchsin in See Hazcards 32,
100 ml of water and add 0.9 g of sodium metabisulfite followed by 67 & 92.
2 ml of 2 M nitric(V) acid, so it is colourless. Addition of an Wear eye
aldehyde restores the colour. Be aware that sulfur dioxide, a TOXIC protection.
gas, may be evolved.
The iodoform test Works with methyl ketones (CH3CO-), methyl secondary alcohols, See Hazcards
ethanol and ethanal. To fives drops of the liquid under test, add 5 54A & 91.
drops of 0.01 M iodine, followed by enough 1 M sodium hydroxide
solution until the brown colour is removed. Warm in boiling water
bath and look for a yellow precipitate of iodoform.
Carboxylic acids
Neutral iron(III) Add 2 M ammonia solution to 0.1 M iron(III) chloride solution until See Hazcards 6 &
chloride a precipitate just appears. Now add more of the original iron(III) 55C.
chloride in drops with stirring until the precipitate just dissolves.
Add this to the liquid under test, and look for a red solution.
[Methanoic acid reacts with acidified potassium manganate(VII)
and potassium dichromate(VI) in a similar manner to alcohols.]
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Double bonds or unsaturation

Bromine water Add 0.002 M bromine water which will go clear. See Hazcard 15B.
Potassium Add an acidified 0.002 M solution of potassium manganate(VII) See Hazcards 81
manganate(VII) which will go clear. If it goes brown, it is not acidified enough. and 98.
(acidified)
Potassium Alkaline potassium manganate(VII) solution is not stable so it See Hazcards 81
manganate(VII) must be made in situ. Add enough solid anhydrous sodium and 98.
(alkaline) carbonate to make the acidified solution alkaline. Add alkaline
0.002 M solution of potassium manganate(VII) which should go
green.
OH groups in alcohols and acids

Phosphorus To about 2 ml of the liquid or solid under test, add phosphorus See Hazcard 74.
pentachloride pentachloride with a small spatula. There is a vigorous reaction
and hydrogen chloride is produced.
Phenol
Neutral iron(III) Add 2 M ammonia solution to 0.1 M iron(III) chloride solution until See Hazcards 6 &
chloride a precipitate just appears. Now add more of the original iron(III) 55C.
chloride in drops with stirring until the precipitate just dissolves.
Add this to the material under test, and look for a purple
colouration.
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103 Tin(II) chloride

Also known as stannous chloride.
Solutions stronger than 0.2 M will be difficult to prepare. This reagent has to be dissolved in concentrated
hydrochloric acid before diluting further with water. The final solution will be 1 M with respect to hydrochloric
acid.
The solution may be cloudy, so either leave it for some hours before decanting off the clear solution or filter
through fine filter paper.
Formula: SnCl2.2H20 Molar mass: 225.63 g mol-1 Solubility: 84 g per 100 ml
General Hazards See Hazcards 102 & 47A.

0.1 M 2.26 5.64 22.56 -
Procedure
Wear goggles and chemical-resistant gloves. Use a fume cupboard.
Measure out the indicated quantity of tin(II) chloride-2-water.
Add the solid to about 10% of the final volume of concentrated hydrochloric acid in a beaker.
Stir to dissolve. Do not warm the solution.
Pour the solution into a measuring cylinder or laboratory jug and dilute to the final volume with
distilled/deionised water.
Leave to clear.
Pour the solution into a labelled bottle and mix well.
CLEAPSS 2011 115

CLEAPSS Recipe Book
104 Water (sea and hard)

It is possible to obtain sea-water salt from several suppliers.
For acid rain, see Recipe Sheet 97.
A. Sea water
Dissolve the following salts in 250 ml of pure water and dilute to 1000 ml.
23.99 g of sodium chloride

0.74 g of potassium chloride
2.24 g of calcium chloride-6-water
10.89 g of magnesium chloride-6-water
4.01 g of anhydrous sodium sulfate(VI) [OR 9.10 g of sodium sulfate(VI)-10-water]
0.20 g of sodium hydrogencarbonate
0.09 g of sodium bromide
0.03 g of boric acid (if required)
0.01 g of strontium chloride (if required)
B. Permanent hard water

Measure 0.45 g of calcium sulfate(VI)-2-water and add it to 1000 ml of water in a bottle and leave it
overnight to dissolve.
C. Temporary hard water

Filter 130 ml of freshly-made calcium hydroxide solution (see Recipe Sheet 20).
Pass carbon dioxide though the solution so that calcium carbonate first precipitates and then, with
further carbon dioxide, re-dissolves to form calcium hydrogencarbonate. This may take some time.
Dilute this clear solution to 1000 ml.
116 CLEAPSS 2011

CLEAPSS Recipe Book
105 Winklers method for dissolved oxygen

This technique is better than other methods (eg, using phenosafranine) because the oxygen in the water is
fixed once the manganese(II) hydroxide is formed. However, the solutions are hazardous to prepare and
use, and should be added to the water sample as soon as possible.
General Hazards See Hazcards 60, 91 & 98. Great care must be taken in making the 8 M sodium
hydroxide solution. Gloves and eye protection (a face shield is preferable) should be
used. The preparation is very slow and must not be rushed.
Preparation of the solutions used in Winklers method

100 ml of 8 M Wear goggles and chemical-resistant gloves. Consider using a fume cupboard.
sodium hydroxide Add 8 g of sodium hydroxide pellets to 70 ml of water in a 250 ml beaker.
Once it has dissolved, cool the solution in an ice bath.
solution
When the temperature of the solution has dropped to below room temperature
add another 8 g. Stir until it has dissolved and cool again.
Repeat this procedure until a total of 32 g of sodium hydroxide has been added.
Add the solution to a 100 ml measuring cylinder and add water to the 100 ml
mark.
Stir carefully into this solution 14 g of potassium iodide.
Pour the solution into a labelled bottle and, when cool, mix well. Attach a
CORROSIVE hazard warning.
The manganese(II) Dissolve 50 g of manganese(II) sulfate(VI)-4-water or 45 g of manganese(II)
chloride-4-water in 80 ml of pure water and dilute to 100 ml.
solution
Pour it into a labelled bottle, mixing well. Add a HARMFUL hazard warning.
For the titration Prepare 0.01 M sodium thiosulfate solution (see Recipe Sheet 87) which can be
standardised against potassium iodate solution (see Recipe Sheet 94) for
accurate work.
The indicator Prepare 1% starch solution. See Recipe Sheet 49.
Winklers method for dissolved oxygen

Procedure for using these solutions
Wear goggles.
Collect 250 ml of water in a 250 ml stoppered bottle, ensuring there is no air trapped inside.
Use a 1 ml pipette (or marked teat pipette) to transfer 1 ml of the manganese(II) solution to the water
sample, injecting the liquid below the surface of the water, and taking care not to blow any more air
into the water.
Use a similar technique to transfer 1 ml of the alkaline potassium iodide solution to the water sample.
Replace the stopper on the bottle without trapping any air and agitate the bottle to mix the reagents.
Allow the contents to stand for 10 minutes.
Now the brown manganese(III) hydroxide has precipitated, the contents of the flask can be transferred
to a larger flask or bottle.
Add 1.5 ml of concentrated sulfuric(VI) acid which will dissolve the precipitate and liberate iodine.
Titrate this solution with the standardised sodium thiosulfate solution, adding 2 ml of starch solution
near the end-point. (1 mol of oxygen is equivalent to 4 mol of thiosulfate solution.)
CLEAPSS 2011 117

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106 Zinc sulfate(VI)

On standing, zinc sulfate(VI)-7-water loses water of crystallisation slowly to the air and becomes powdery.
However, it is more convenient to make solutions from the hydrated salt than the anhydrous salt which is
also available from suppliers.
The anhydrous salt readily absorbs water from the atmosphere during storage.
Formula: ZnSO4.7H2O Molar mass: 287.54 g mol-1 Solubility: 96 g per 100 ml
General Hazards Zinc sulfate(VI) is HARMFUL. See Hazcard 108B.

0.1 M 2.88 7.19 28.75 -
1.0 M 28.75 71.88 287.54 HARMFUL
Procedure
Measure out the indicated quantity of hydrated zinc sulfate(VI).
Either pour the solution from the beaker into an appropriate measuring cylinder and add water to the
required level or add water to the required level in the laboratory jug.
118 CLEAPSS 2011

CLEAPSS Recipe Book
Index
Name Recipe Name Recipe
Sheet Sheet
Acetic acid 39 Aniline blue 91
Acetic alcohol 41 Aniline sulfate(VI) 93
Acetic orcein 89 Animal tissue 74
Aceto carmine 89 preservative
Acid atmosphere 97 Anion tests 100
Acid base indicators 46 Aspirator 20
Acid rain 97 Azo dyes 9

Azure A 90
Agar 1
Alcohol tests 102 Barium chloride 10
Alcohol/water solutions 2 Barium diphenylamine- 49

4-sulfonate indicator
Alcoholic potassium 71
Barium nitrate(V) 10
hydroxide
Alcoholic solutions 2 Barium solutions 10
Aldehyde tests 102 Basic agar 1
Alginate beads 3 Belousov-Zhabotinski 63
reaction
Alizarin solution 101
Benedicts qualitative 11
Alkaline pyrogallol 84 reagent
Alum 4 Benedicts quantitative 12
Aluminium chloride 4 reagent
Bile salts 37
Aluminium etching 38
Biological buffers 18
Aluminium potassium 4
sulfate(VI) Biro ink 26
chromatography
Aluminium solutions 4
Bismuth nitrate(V) 14
Amino acid 26
chromatography Biuret reagent 15
Amino acid tests 13 Bleach 81
Ammonia gas 5 Blue-bottle reaction 63
Ammonia solution 6 Blueprints 52
Ammonia test 99 B-R reaction 63
Ammonium cerium(IV) 22 Bradys reagent 33
sulfate(VI) Briggs-Rausher reaction 63
Ammonium chloride 7 Brodies fluid 16
Ammonium 101 Bromide ion test 100
ethanedioate
Bromine water 17
Ammonium hydroxide 6
Bromocresol green 46
Ammonium iron(III) 52
citrate Bromophenol blue 46
Ammonium iron(III) 52 Bubble mixture 79
sulfate(VI)
Buffers 18
Ammonium sulfide 101
Butanedione dioxime 101
Ammonium thiocyanate 101
B-Z reaction 63
Ammonium vanadate 8
Calcium carbonate 94
Amylase 37 (standard solution)
Analgesics 26 Calcium chloride 19
chromatography Calcium hydroxide 20
Andrews arrangement 24
Calcium nitrate(V) 19
for gas preparation
CLEAPSS 2011 119

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Sheet Sheet
Carbohydrate tests 13 Cotton blue 91
Carbon dioxide 99 Cotton dying 9
Carbon dioxide gas 21 Cresol red 46
Carbon dioxide indicator 48 Crude oil alternative 32
Carbon monoxide 42 Cytological fixative 41
preparation DCPIP 13
Carbon monoxide test 99
Diammonium iron(II) 51
Carbonate ion test 100 sulfate(VI)
Carbonyl group test 33 Diazine green 92
Carboxylic acid test 102 Diazonium salts 9
Cation tests 101 2,6- dichlorophenol indo- 13
phenol
Cellular respiration 13
Dichromate(VI) titration 49
Cerium(IV) solutions 22
indicators
Cerium(IV) sulfate(VI) 22
Dimethylglyoxime 101
Cheek cells 89
Dinitrogen monoxide 42
Chemical garden 86 preparation
Chemiluminesence 23 Dinitrogen monoxide 99
China blue agar 1 test
2,3,5- Dinitrophenylhydrazine 33
Chloride ion test 100
3,5- Dinitrosalicylic acid 34
Chlorine gas 24
preparation Dipotassium hydrogen 67
Chlorine test 99 phosphate(V)
Chlorine water 25 Dipotassium hydrogen 67
phosphate(V) -3- water
Chlorophyll 26
Disappearing cross 29
chromatography
reaction
Chromatography 26
Disinfectants 81
solvents
Chrome alum 27 Disodium hydrogen 67
phosphate(V)
Chromium(III) chloride 27
Disodium hydrogen 67
Citric acid 28 phosphate(V) -7- water
Clarkes fluid 41 Dissolved oxygen 105
Clarkes soap solution 79 Dithizone 101
Clock reactions 29 DNP 33
2,4-
Cobalt(II) chloride 30 DNS 34
Cobalt(II) nitrate(V) 30 DNSA 34
Cobalt(II) thiocyanate 30 Double bond test 102
Coles reagent 13 Drosophila food base 35
Colloidal Coomassie 90 Dyes 9
blue
Complexometric 49 Dying cotton 9
indicators EDTA titration indicators 49
Coomassie blue 90 Ehrlichs haematoxylin 89
Copper electroplating 36 Eiosin indicator 49
Copper etching 38 Electrolytic agar 1
Copper(II) chloride 31 Enzymes 37
Copper(II) nitrate(V) 31 Enzymes immobilised 3
Copper(II) solutions 31 Eosin 89
Copper(II) sulfate(VI) 31 Eriochrome black 49
120 CLEAPSS 2011

CLEAPSS Recipe Book

Sheet Sheet
Etching solutions 38 Indicator agar 1
Ethanoate ion test 100 Iodide ion test 100
Ethanoic acid 39 Iodine solution 50
Ethanol/water mixtures 2 Iodine stain 93
Ethanolic potassium 71 Iodoform test 102
hydroxide
Iron etching 38
FAA 41, 74
Iron(II) chloride 51
FABIL 93
Iron(II) solutions 51
Fehling's solution 40 Iron(II) sulfate(VI) 51
Ferroin indicator 49 Iron(III) chloride 52
Ferroxyl agar 1 Iron(III) ions and iodide 29
Ferroxyl indicator 101 ion clock reaction
Feulgen's stain 89 Iron(III) solutions 52
Fixatives in biology 41 Iron(III) sulfate(VI) 52
Flame tests 101 Janus green B 13, 92
Fluorescein indicator 49 Kaiserlings 74
preservative
Formaldehyde 58
Ketone tests 102
Formalin alcohol fixative 41
Knops culture solution 66
Formalin-aceto fixative 41
Lactophenol 91
Formalin-aceto-alcohol 74
Landolt iodine reaction 29
Formic acid 59
Lead etching 38
Glucose nutrient agar 1
Lead nitrate(V) 53
Glucose quantitative 12
test Leishman's stain 89
Glucose test 11, 34 Lime water 20
Gram Stain 88 Lipase 37
Harcourt-Essen reaction 29 Lipstick 26
chromatography
Hard water 104
Lithium chloride 54
Home-made indicators 46 Litmus 46
Hydrochloric acid 43 Locating agents 26
Hydrogen 99 Lucas reagent 102
Hydrogen carbonate 48 Lugols iodine 88
indicator Luminol 23
Hydrogen chloride 42 Magnesium sulfate(VI) 55
preparation
Malt agar 1
Hydrogen chloride test 99
Manganese(II) 56
Hydrogen gas 44 sulfate(VI)
Hydrogen peroxide 45 Manometric fluid 16
Hydrogen peroxide/ 29 Mayonnaise agar 1
potassium iodide
reaction Mercury(I) nitrate(V) 57
Hydrogen sulfide 42 Mercury(II) chloride 57
preparation Metal ion 26
Hydrogen sulfide test 99 chromatography
Hydrogen-sulfite test 102 Methanal solution 58
2- hydroxy-1,2,3- 27 Methanoate ion test 100

tricarboxylic acid Methanoic acid 59
Immobilised enzymes 3 Methanol solution 2
CLEAPSS 2011 121

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Sheet Sheet
Methanolic potassium 71 Oxygen levels in water 105
hydroxide PAS reaction 13
Methyl benzoate 26 Pepsin 37
nitration
Methyl green pyronin 89 Periodic acid Schiff 13
reaction
Methyl orange 46 Permanently hard water 104
Methyl orange 9 pH buffers 18
preparation
pH probe calibration 18
Methyl red 46 buffers
Methylene blue 13, 89, 90, Phenanthroline solution 101
92
Phenol red 46
Milk agar 1
phenol- 13
Millons reagent 13
indodichlorophenol
Mohrs salt 51 Phenolphthalein 46
Molischs solution 13 Phenolphthalein 1
Murexide 49 indicator agar
Neutral Red 46, 92 Phenylammonium 93
sulfate(VI)
Neutral iron(III) chloride 102
Phenylthiocarbamide 13
Nickel electroplating 36
Phenylthiourea 13
Nickel(II) sulfate(VI) 60
Phloroglucinol 93
Ninhydrin 13
Phosphate(V) ion test 100
Nitrate(III) ion test 100
Phosphoric(V) acid 65
Nitrate(V) test 100
Phosphorus 102
Nitration of Methyl 26 pentachloride
benzoate Physiological saline 75
Nitric(V) acid 61 solutions
Nitrogen preparation 42 Plant mineral 66
Nitrogen test 99 requirement solutions
Plant tissue 74
Nitrogen dioxide 42
preservative
preparation
Polypeptide tests 13
Nitrogen dioxide test 99
Potassium chloride 68
nitrogen gas oscillating 63
reaction Potassium chromate 69
Nitrogen monoxide 42 Potassium 70
preparation dichromate(VI)
Nitrogen monoxide test 99 Potassium dihydrogen 67
phosphate(V)
N-phenylanthranilic acid 49
indicator Potassium 101
hexacyanoferrate(III)
Nutrient agar for 1
bacteria Potassium hydrogen 94
phthalate (standard
Nylon rope experiment 62 solution)
OH group test 102 Potassium hydroxide 71
Orange II preparation 9 Potassium iodate 94
Orcein acetic 90 (standard solution)
Organic functional 102 Potassium 29
group tests iodate/sodium
metabisulfite reaction
Orthophosphoric acid 65
Potassium iodide 72, 101
Oscillating reactions 63
Potassium 29
Oxygen preparation 64 iodide/potassium
Oxygen test 99 persulfate clock reaction
122 CLEAPSS 2011

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Sheet Sheet
Potassium 73 Sodium ethandioate 94
manganate(VII) (standard solution)
Potassium 67 Sodium ethanoate 83
phosphate(V) Sodium 101
Potassium sulfide 101 hexanitrocobaltate(III)
Potassium thiocyanate 101 Sodium 84
hydrogencarbonate
Precipitation indicators 49
Sodium hydroxide 85
Preservatives for animal 74
tissues Sodium hydroxide test 101
Propanone solution 2 Sodium hypochlorate 81
Protein tests 13 Sodium phosphate(V) 67
PTC 13 Sodium phosphate(V) 67
-12- water
PTU 13
Sodium silicate 86
PVA 78
Sodium sulfide 101
Pyrogallol 84
Sodium 37
Red cabbage indicator 46
tauroglycocholate
Redox indicators 49 Sodium thiosulfate 87
Reducing sugar test 11, 12 Solochrome black 49
Resazurin solution 93 Stains for cells 89
Ringers solution 75 Stains for 90
Rubeanic acid 101 electrophoresis
Stains for fungal 91
Sachs culture solutions 66
material
Safranin counterstain 88 Stains for metabolic 92
Sakaguchi test 13 activity
Saliva 37 Stains for plant material 93
Sandells solution 76 Standard solutions 94
Schiffs reagent 102 Starch agar 1
Schulzes solution 93 Starch indicator 49
Screened methyl 46 Starch malt agar 1
orange Sterilising conditions 1
Sea water 104 Strontium choride 95
Sudan III or IV solutions 89
Silicate gels 86
Sugar chromatography 26
Silver electroplating 36
Sulfate(IV) ion test 100
Silver nitrate(V) 77
Silver nitrate(V) titration 49 Sulfate(VI) ion test 100
indicators Sulfur dioxide 96
Slime 78 preparation
Sulfur dioxide test 99
Soap 79
Sodium carbonate 80 Sulfur dioxide solution 97
Sodium carbonate 94 Sulfuric(VI) acid 98

(standard solution) Technical agar 1
Sodium chlorate(I) 81 Temporary hard water 104
Sodium chloride 82
Sodium dihydrogen 67 Tests for gases 99
phosphate(V) Tests for negative ions 100
Sodium dihydrogen 67 Tests for positive ions 101
phosphate(V) -1- water
Thermochromic liquid 30
Sodium diphenylamine- 49
4-sulfonate indicator Thiooxamide 101
CLEAPSS 2011 123

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Sheet Sheet
Thiosulfate/acid 29 Unsaturation in organic 102
reaction chemistry
Thiosulfate/iodine 49 Vitamin C test 13
titration indicator
Wanklyns soap solution 79
Thymol blue 46
Washing soda 80
Thymolphthalein 46
Water 104
Tin(II) chloride 103
Tollens reagent 102 Water solutions for 66
2,3,5- Triphenyl tetrazolium 13 plants
chloride Winklers method 105
Trypsin 37 Yamada indicator 47
TTC 13 Zinc electroplating 36
Universal buffer 18 Zinc sulphate 106
Universal indicator 47
124 CLEAPSS 2011

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The

Ammonia solution (ammonium 6 3,5-Dinitrosalicylic acid 34

Citric acid 28 Mercury solutions 57

Clock reactions 29 Methanal solution 58

Potassium and sodium 67 Stains for electrophoresis 90

Agars for microbiology

Agars for other activities

2 Alcohol/water and propanone/water solutions

To prepare 100 ml of an x% (v/v) solution of ethanol in water

To prepare 1 litre of 1 M methanol solution

To prepare 1 litre of 1 M ethanol solution

To prepare 1 litre of 2 M propanone solution

Preparing immobilised enzymes/cells in alginate beads

Hardened alginate beads in calcium chloride solution

Aluminium potassium sulfate(VI)

Mass (g) of solid to be used

Aluminium chloride (hydrated)

Mass (g) of solid to be used

Preparing solutions of aluminium salts

Preparing ammonia gas

6 Ammonia solution (ammonium hydroxide)

Formula: NH3 Molar mass: 17.03 g mol-1 Solubility: infinite

General Hazards Concentrated ammonia; see Hazcards 5 & 6.

Volume of 880 ammonia to be used

Preparing ammonia solutions

General Hazards See Hazcard 9A.

Mass (g) of solid to be used

Preparing ammonium chloride solution

8 Ammonium vanadate(V) solution

Preparing 100 ml of 0.1 M ammonium vanadate solution

Preparing azo dyes as described is a two-stage process.

Stage 2 (a): Preparing Orange II (IRRITANT) and dying cotton with it

Stage 2 (b): Preparing methyl orange (TOXIC)

Mass (g) of solid to be used

Mass (g) of solid to be used

Preparing barium salts solutions

11 Benedicts qualitative reagent

Procedure to produce 1 litre of solution

Procedure for carrying out the test

12 Benedicts quantitative reagent

Preparing 1 litre of Benedicts quantitative solution (BQS)

Using the solution: volumetric procedure

A = titration volume for known glucose solution

Using the solution: colorimetric procedure

13 Biochemical indicators and tests

Tasting and genetics studies

14 Bismuth nitrate(V) solution

Formula: Bi(NO3)3.5H2O Molar mass: 395 g mol-1

General Hazards See Hazcards 67 & 73B.

Preparing 100 ml of 0.1 M of bismuth nitrate(V) solution

Qualitative Biuret Reagent

Preparing 1 litre of Qualitative Biuret reagent

Procedure and use of Biuret solution suitable for Y7 and 8

Quantitative Biuret Reagent

A. Preparing 1 litre of Brodies fluid

B. Brodies fluid substitute for short term use

C. Simple version for short-term use

Preparing 1 litre of 0.02 M solution (HARMFUL) of bromine water

Single component buffers

Temperature effects on single component buffers

Buffers for pH probe calibration

Double component buffers

Using a solid and a solution.