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
Agar
1
Alcohol/water and
propanone/water solutions
2
Alginate beads
Recipe sheet
Number
Cobalt(II) chloride solution and
thermochromic liquid
30
Copper(II) solutions
31
3
Crude oil alternative
32
Aluminium solutions
4
33
Ammonia gas
5
2,4-Dinitrophenylhydrazine
solution
Ammonia solution (ammonium
hydroxide)
6
3,5-Dinitrosalicylic acid
34
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
Benedict’s qualitative reagent
11
Fehling’s solutions
40
Benedict’s quantitative reagent
12
41
Biochemical indicators and tests
13
Fixatives used before preserving
biological specimens
Bismuth nitrate(V) solution
14
42
Biuret reagent
15
Gases less commonly used in
schools
Hydrochloric acid
43
Brodie’s fluid
16
Hydrogen gas
44
Bromine water
17
Hydrogen peroxide
45
Buffer solutions
18
Indicators (acid-base)
46
Calcium chloride and nitrate(V)
solutions
19
Indicator (universal)
47
Calcium hydroxide solution
20
Indicators (carbon dioxide)
48
Carbon dioxide
21
Indicators for redox, precipitation
and complexometric titrations
49
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
locating agents
26
Lithium chloride
54
Chromium(III) chloride and
chrome alum solutions
27
Magnesium sulfate(VI)
55
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
86
Oscillating reactions
63
Oxygen gas
64
Sodium silicate, the crystal
(chemical) garden and silicate
gels
Phosphoric(V) acid
65
Sodium thiosulfate
87
Plant mineral requirement
solutions
66
Stains for bacterial activity
88
Stains for cell contents
89
Potassium and sodium
phosphates
67
Stains for electrophoresis
90
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
biological specimens
74
Sulfuric(VI) acid
98
Testing for gases
99
Ringer’s and other saline
solutions for physiological use
75
Testing for negative ions
100
Sandell’s solution
76
Testing for positive ions
101
Silver nitrate(V)
77
Testing for organic functional
groups
102
Slime
78
Tin(II) chloride
103
Soap and bubble solutions
79
Water (sea and hard)
104
Sodium carbonate
80
105
Sodium chlorate(I) solution
81
Winkler’s method for dissolved
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
Control
measures
Procedure for
preparing
technical agar
(also called
agar-agar)
Usual sterilising
conditions
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.
Use a balance in a non-working fume cupboard, ie, not switched on, with the sash
down, to weigh out agar. Use heatproof gloves to protect from scalding when handling
freshly-sterilised molten agar.
Mix 1.5 g of agar with 10 ml of water into a paste. Slowly add more water with stirring
until the volume is 100 ml. Heat the mixture with stirring on a boiling water bath to
95 °C in the required container. This preliminary heating can be omitted if the agar is
going to be sterilised immediately, unless it is necessary to decant the agar into
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.
(If required) Autoclave the container(s) with the made-up suspension(s) for 15 minutes
at 15 psi (121 °C).
Agars for microbiology
China blue lactose
agar
Use 3.6 g of China blue lactose agar powder in 100 ml of distilled water. If this mix
proves too thin for ‘rough’ handling by students, then thicken by adding 0.5 g of agaragar (just thickener, no nutrients).
Crystal violet
agar. A selective
(against Gram
positive) medium
for soil bacteria
Glucose nutrient
agar
Malt agar for fungi
In a fume cupboard which is not switched on, add 0.005 g of crystal violet (HARMFUL,
DANGEROUS FOR THE ENVIRONMENT) to 1 litre of liquid nutrient agar solution. The
resultant solution is low hazard.
Mannitol yeast
extract agar for
growing root
nodule bacteria
Add 0.5% w/v of glucose to molten nutrient agar.
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.
Mix 10 g agar in 1 litre of water, and dissolve in a boiling water bath. Add 0.5 g
K2HPO4, 0.2 g MgSO4.7H2O, 0.2 g NaCl, 0.2 g CaCl2.6H2O,10 g mannitol, and 0.4 g
yeast extract. Dispense as required and sterilise by autoclaving before use.
Nitrogen-free
mineral salts agar
for growing
nitrogen-fixing
bacteria
Dissolve 0.05 g FeCl3.6H2O in 500 ml distilled water. Add 2 g K2HPO4, 0.25 g
MgSO4.7H2O and 10 g glucose. Check the pH and adjust, if necessary, to 8.3 using
0.1 M NaOH. Pour into a bottle containing 1 g CaCO3 and 7.5 g agar powder. Mix
and autoclave at 121 °C for 20 minutes. Before pouring plates, swirl to thoroughly
mix the CaCO3 and agar.
Nutrient agar for
bacteria
Mix 2 g of ‘Bovril’, 0.5 g of sodium chloride and 1.5 g of agar with 10 ml of water into
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
for growth of
fungi and
digestion of
starch
Mix 3 g of light malt powder (from home-brewing shops), 0.5 g of peptone (to
promote growth) in 20 ml of water. Also make a paste containing 1 g of soluble
starch in 10 ml of hot water. Add these two solutions to 1.5 g of agar with stirring and
slowly add more water with stirring until the volume is 100 ml. Stir before decanting
into smaller containers (if required) and sterilising. Autoclave the suspension at
10 psi (115 °C) for 10 minutes.
© CLEAPSS 2011
1
CLEAPSS Recipe Book
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.
Mix 0.5 g of glucose-1-phosphate and 1.5 g of agar with 10 ml of water. Slowly add
Agar for starch
more water with stirring until the volume is 100 ml. Boiling, not sterilising, should be
synthesis
sufficient.
Electrolytic agar
Add one gram of sodium sulfate(VI) or other electrolyte to the hot agar solution
before pouring.
Ferroxyl agar gel
for rusting
experiments
Indicator agar
Add 1.4 g of agar, 2 g of sodium chloride, 0.1 g of potassium hexacyanoferrate(III)
and 1 ml of phenolphthalein solution to the 100 ml of water and warm, with stirring, to
95 °C. Pour the solution into Petri dishes.
Add 1 ml of the chosen indicator solution to the agar solution before pouring.
Mayonnaise agar
for lipase activity
(1)
Dilute 4 g salad cream or mayonnaise with 5 ml water and add 1 ml 0.1 M sodium
hydroxide solution (IRRITANT). Add about 1 ml of this alkaline mixture to a solution of
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.
Alternatively, mayonnaise agar can be made up without the dye and, after
incubation, the plates can be flooded with 0.4 M copper(II) sulfate solution and left for
30 minutes before being examined. Clear areas in the blue-green matrix indicate
where lipases have broken down the fatty acids in the mayonnaise.
Stir together 2 g low-fat milk powder (Marvel is recommended as it contains very little
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.
Use 2 g agar in 100 ml boiling distilled (or deionised) water in a beaker. Add 10 ml of
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.
Mayonnaise agar
for lipase activity
(2)
Milk agar for
protease activity
Phenolphthalein
indicator agar
Starch agar for
amylase activity
Mix a paste containing 1 g of soluble starch in 10 ml of cold water. Add 1.5 g of agar,
stir well and slowly add more water with stirring until the volume is 100 ml. Heat as
for technical agar above.
2
© CLEAPSS 2011
CLEAPSS Recipe Book
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
Flash point (°C)
Density (g cm-3) at 20 °C
Hazard
0
1.00
-
10
49
0.98
20
36
0.97
30
40
29
26
0.96
0.95
FLAMMABLE
50
24
0.93
60
22
0.91
70
21
0.89
80
20
0.86
90
17
0.83
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.
Add this to a 1000 ml measuring cylinder or 1 litre measuring jug.
Add water up to the 1 litre mark.
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.
Add this to a 1000 ml measuring cylinder or 1 litre measuring jug.
Add water up to the 1 litre mark.
The solution is low hazard.
© CLEAPSS 2011
3
100
13
0.78
CLEAPSS Recipe Book
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
4
© CLEAPSS 2011
CLEAPSS Recipe Book
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)
Molar mass: 474.39 g mol-1
Formula: AlK(SO4)2.12H2O
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
required
100
0.01 M
0.1 M
0.2 M
Saturated (20 °C)
Solubility: 11 g per 100 ml
Volume (ml) of solution required
250
Ten-fold dilution of the 0.1 M solution
4.74
11.86
9.49
23.72
12
29
1000
Hazard warning
label
47.44
94.88
114
-
Aluminium chloride (hydrated)
Molar mass: 241.43 g mol-1
Formula: AlCl3.6H2O
General Hazards
See Hazcard 2A. Aluminium salts in water are acidic.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.5 M
Saturated (20 °C)
Solubility: 83 g per 100 ml
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
2.41
6.03
24.14
12.07
30.18
120.72
90
225
900
Hazard warning
label
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.
© CLEAPSS 2011
5
CLEAPSS Recipe Book
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).
Set up the equipment as shown on the right.
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.
The residual ammonia solution can be poured down the
sink in a fume cupboard with plenty of water.
6
Inverted gas jar
held by a clamp
Anti-bumping
granules
10 ml of concentrated
ammonia solution
Heat
gently
© CLEAPSS 2011
CLEAPSS Recipe Book
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.
Molar mass: 17.03 g mol-1
Formula: NH3
General Hazards
Solubility: infinite
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
Volume (ml) of solution required
Concentration
required
500
1000
2500
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
Hazard warning
label
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 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 and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
7
CLEAPSS Recipe Book
7
Ammonium chloride
Molar mass: 53.5 g mol-1
Formula: NH4Cl
General Hazards
See Hazcard 9A.
Mass (g) of solid to be used
Concentration
required
100
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Solubility: 36 g per 100 ml
2.68
5.34
40
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 1 M solution
6.69
26.75
13.38
53.50
100
400
Hazard warning
label
-
Preparing ammonium chloride solution
•
•
•
•
•
Measure out the indicated quantity of ammonium chloride.
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.
8
© CLEAPSS 2011
CLEAPSS Recipe Book
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
General Hazards
Molar mass: 117 g mol-1
Solubility: 0.52 g per 100 ml
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.
© CLEAPSS 2011
9
CLEAPSS Recipe Book
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.
10
© CLEAPSS 2011
CLEAPSS Recipe Book
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
Molar mass: 244.26 g mol-1
Formula: BaCl2.2H2O
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.5 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of 0.1 M solution
2.44
6.11
24.43
12.21
30.53
122.13
36
90
260
Solubility: 26 g per 100 ml
Hazard warning
label
HARMFUL
HARMFUL
TOXIC
Barium nitrate
Molar mass: 261.37 g mol-1
Formula: Ba(NO3)2
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
Saturated (20 °C)
2.61
9
Volume (ml) of solution required
250
1000
Ten-fold dilution of 0.1 M solution
6.53
26.14
45
90
Solubility: 9 g per 100 ml
Hazard warning
label
-
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.
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 and mix well. Add a hazard warning if appropriate.
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CLEAPSS Recipe Book
11
Benedict’s qualitative reagent
Benedict’s solution or DNSA (see Recipe sheet 34) should be used in place of Fehling’s 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.
Benedict’s solution is less satisfactory in testing for non-reducing sugars and aldehydes in organic chemistry
in which case Fehling’s or Sandell’s 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 Benedict’s 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
•
•
•
•
•
•
•
•
Wear eye protection.
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.
Stir to dissolve, warming if necessary.
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.
12
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CLEAPSS Recipe Book
12
Benedict’s 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 Benedict’s quantitative solution (BQS)
•
•
•
•
•
Wear eye protection.
Measure out 75 g of anhydrous (or 160 g of hydrated) sodium carbonate, 200 g of trisodium citrate-2water 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
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CLEAPSS Recipe Book
13
Biochemical indicators and tests
Amino acid, polypeptide and protein tests
See also Recipe Sheet 15 for the Biuret test.
Cole’s
modification
of Millon’s
reagent
Test for
soluble
proteins
Marquis’s
reagent
Test for
alkaloids
Ninhydrin
For amine
groups
Sakaguchi
test
Test for
proteins
containing
arginine
Wear goggles and gloves. Dissolve 10 g of mercury(II)
sulfate(VI) in 100 ml of 2 M sulfuric(VI) acid (solution A).
Separately, dissolve 1 g of sodium nitrate(III) (nitrite) in
100 ml of water (solution B). Before use, mix two volumes
of solution A with one of solution B. A reddish-brown
colouration or precipitate indicates the presence of soluble
proteins.
Wear goggles and gloves. Use a fume cupboard. Add 2
drops of 40% methanal solution to 3 ml of concentrated
sulfuric(VI) acid just before use. This is a spot test and
various colours appear. See
http://en.wikipedia.org/wiki/Marquis_reagent.
Wear eye protection. Dissolve 0.1 g of ninhydrin in 100 ml
of water. A purple colour, known as Ruhemann's purple, is
produced when ninhydrin reacts with primary and
secondary amines, indicating the presence of amino acids.
Can be used as a spray or a dip, but needs to be heated, in
an oven at 110 °C or with a hairdryer, for the colours to
appear.
Wear goggles. Dissolve 5 g of sodium hydroxide in 100 ml
of water (solution A). Dissolve 1 g of napthalen-1-ol in
100 ml of water (solution B). For the test, one drop of
sodium chlorate(I) solution (10-14% available chlorine, see
Recipe sheet 81) is required as well. Proteins containing
arginine appear an intense red colour.
See Hazcards
62, 93, 98A.
Solution A and
the combined
solution are
TOXIC.
See Hazcards
63 and 98.
Label the
solution
CORROSIVE and
TOXIC.
See Hazcard
66. No hazard
warning is
required on the
solution.
See Hazcards
70, 89 & 91.
Carbohydrate tests
See also 3,5-dinitrosalacylic acid (DNSA), Benedict’s solution, Sandell’s solution and Fehling’s solution.
For all
Wear goggles. Dissolve 5 g of napthalen-1-ol in 100 ml of
See Hazcards
Molisch’s
carboethanol.
40A, 70 & 98.
solution
hydrates
Label the
The solution containing a possible carbohydrate is
combined with a small amount of Molisch's reagent in a test solution HIGHLY
FLAMMABLE &
tube. After mixing, a small amount of concentrated
HARMFUL
.
sulfuric(VI) acid is slowly added down the sides of the
sloping test tube, without mixing, to form a lower layer. Look
for a purple ring at the interface of the two layers.
For
Wear goggles and chemical-resistant gloves. Dissolve 1 g
Iodic(VII) acid is
Periodic
polyCORROSIVE &
of iodic(VII) acid (periodic acid) in 100 ml of water. Used in
acid Schiff
saccharides
OXIDISING. No
conjunction with Schiff’s reagent. Changes from colourless
(PAS)
hazard warning
to purple.
reaction
is required on
the solution.
14
© CLEAPSS 2011
CLEAPSS Recipe Book
Cellular respiration
Janus
green B
Methylene
blue
TTC
Wear eye protection and disposable nitrile gloves when making up the
solution. Dissolve 0.3 g of the dye in 100 ml of purified water. Dilute this
solution ten times with water before use. Colour changes are from blue to
salmon pink.
See Guidance leaflet PS88 for more details including a practical activity.
Wear eye protection, and gloves to avoid staining the skin.
Dissolve 1 g solid in 100 ml water and add 0.6 g sodium chloride. The blue
indicator turns colourless as the dye is reduced.
Dissolve 1 g of 2,3,5-triphenyl tetrazolium chloride (TTC) in 100 ml of water.
(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.
No hazard
label is
required on
the solution.
See Hazcards
32 and 40. No
hazard label is
required on
the solution.
Low hazard.
Tasting and genetics studies
Phenylthiocarbamide
This is also called PTC, phenylthiourea or PTU. Dissolve 0.05 g in 100 ml of
water. Filter paper is soaked in the solution and then hung up to dry before
cutting into strips. See Handbook 15.13 for details on tasting investigations.
The solid is
VERY TOXIC;
see Hazcard
35. The
solution is low
hazard.
Also called 2,6-dichlorophenol indophenol, and phenol-indodichlorophenol.
Dissolve 0.1 g of dye in 100 ml of water. The standard vitamin C solution
should also be 0.1% (w/v). Add the blue dye until the colour does not
disappear.
Both are low
hazard
chemicals.
Vitamin C
DCPIP
solution
© CLEAPSS 2011
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CLEAPSS Recipe Book
14
Bismuth nitrate(V) solution
The solutions need to be kept acidic to avoid the formation of insoluble basic salts.
Molar mass: 395 g mol-1
Formula: Bi(NO3)3.5H2O
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.
• Transfer the solution to a 100 ml measuring cylinder.
• 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. It’s
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
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CLEAPSS Recipe Book
16
Brodie’s 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
A.
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.
Preparing 1 litre of Brodie’s fluid
•
•
•
B.
Wear eye protection.
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.
Brodie’s fluid substitute for short term use
•
•
•
C.
44 g of sodium bromide.
1 g of liquid detergent (eg washing-up liquid).
0.3 g of Evans blue.
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
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CLEAPSS Recipe Book
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
buffer
pH 4 buffer
pH 7 buffer
pH 9.2
buffer
pH 12.6
buffer
Dissolve 1.27 g of potassium hydrogen-ethanoate-1-ethanedioic
acid-2-water (potassium tetroxalate) in 10 ml of hot water and make
up to 100 ml with cold water.
Dissolve 1.01 g of potassium hydrogenphthalate in 10 ml of hot
water and make up to 100 ml with cold water. The solution does not
keep well because of mould growth.
Dissolve 0.77 g of ammonium ethanoate in 100 ml of cold water.
The solution does not keep well because of mould growth.
Dissolve 0.38 g of sodium tetraborate-10-water in 100 ml of water.
Use saturated limewater solution.
Low hazard
See Hazcard 36A.
Low hazard
See Hazcard 13B.
Low hazard
See Hazcard 9B.
Low hazard
See Hazcard 14.
Low hazard but wear
eye protection.
See Hazcard 18.
Temperature effects on single component buffers
Temperature / °C
pH 4 buffer
pH 9.2 buffer
5
4.01
9.39
10
4
9.33
15
4
9.27
20
4
9.23
25
4.01
9.18
20
30
4.01
9.14
40
4.03
9.07
50
4.06
9.02
60
4.09
8.97
70
4.12
8.93
80
4.16
8.89
90
4.2
8.85
© CLEAPSS 2011
CLEAPSS Recipe Book
Buffers for pH probe calibration
pH 4 buffer
pH7 buffer
pH10 buffer
NB
Dissolve 1.012 g of potassium hydrogenphthalate in 10 ml of hot
water and make up to 100 ml with cold water. The solution does not
keep well because of mould growth.
0.339 g of citric acid and 4.306 disodium hydrogenphosphate(V)12-water (or 1.707 g of anhydrous salt) dissolved in water and
made up to 100 ml with water.
0.477 g sodium tetraborate-10-water and 18.3 ml of 0.1 M sodium
hydroxide (from a burette) is made up to 100 ml with water.
See Hazcard 13B
See Hazcard 9B.
See Hazcards 14
and 91.
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
3
4
5
6
7
8
9
10
11
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.
10.21 g potassium hydrogen phthalate and 223 ml of 0.1 M
hydrochloric acid
10.21 g potassium hydrogen phthalate
10.21 g potassium hydrogen phthalate and 226 ml of freshly-made
0.1 M sodium hydroxide solution
6.81 g potassium dihydrogen phosphate and 56 ml of freshly-made
0.1 M sodium hydroxide solution
6.81 g potassium dihydrogen phosphate and 291 ml of 0.1 M sodium
hydroxide solution
6.81 g potassium dihydrogen phosphate and 467 ml of freshly-made
0.1 M sodium hydroxide solution
4.77 g sodium tetraborate-10-water and 46 ml of 0.1 M hydrochloric
acid
4.77 g sodium tetraborate-10-water and 183 ml of freshly-made 0.1 M
sodium hydroxide solution
2.1 g sodium bicarbonate and 227 ml of freshly-made 0.1 M sodium
hydroxide solution
© CLEAPSS 2011
21
Dilute each mixture to 1
litre solution with distilled or
deionised water.
CLEAPSS Recipe Book
Mixing two prepared solutions Consult the relevant Recipe Sheets for the preparation of the solutions.
All these buffers are low hazard. See relevant Hazcards for more information.
General
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
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
2.00
6.20
10.90
15.85
20.55
24.70
28.50
32.20
35.50
38.55
98.00
93.80
89.10
84.15
79.45
75.30
71.50
67.80
64.50
61.45
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
41.40
44.10
46.75
49.30
51.50
53.60
55.75
58.00
60.45
63.15
58.60
55.90
53.25
50.70
48.50
46.40
44.25
42.00
39.55
36.85
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
66.10
69.25
72.75
77.25
82.35
86.95
90.85
93.65
95.75
97.25
33.90
30.75
27.25
22.75
17.65
13.05
9.15
6.35
4.25
2.75
The ethanoic acid/sodium ethanoate buffer
system
Add the stated volume of A (0.2 M sodium
ethanoate solution) to the stated volume B
(0.2 M ethanoic acid)
pH
A (ml)
B (ml)
3.8
12.0
88.0
4.0
18.0
82.0
4.2
26.5
73.5
4.4
37.0
63.0
4.6
49.0
51.0
4.8
60.0
40.0
5.0
70.5
29.5
5.2
79.0
21.0
5.4
85.5
14.05
The ammonia/ammonium chloride buffer
system
Add the stated volume of A (0.2 M ammonia) to
the stated volume of B (0.2 M ammonium
chloride solution)
pH
A (ml)
B (ml)
8.4
12.5
87.5
8.6
18.5
81.5
8.8
26.0
74.0
9.0
36.0
64.0
9.25
50.0
50.0
9.4
58.5
41.5
9.6
69.0
31.0
9.8
78.0
22.0
10.0
85.0
15.0
The sodium hydrogencarbonate/sodium
hydroxide) system buffer system
Add the stated volume of A (0.1 M sodium
hydroxide) to 50 ml of 0.05 M sodium
hydrogencarbonate and dilute to 100 ml
(Wear eye protection.)
The phosphate(V) buffer system. (potassium
salts may also be used)
Add the stated volume of A [0.2 M sodium
dihydrogenphosphate(V)] to the stated
volume of B [0.2 M disodium
hydrogenphosphate(V)]
pH
9.6
9.8
10.0
10.2
10.4
10.6
10.8
11.0
A (ml)
5.00
6.20
10.70
13.80
16.50
19.10
21.20
22.70
pH
6.0
6.5
7.0
7.5
8.0
22
A (ml)
87.7
68.5
39.0
16.0
5.3
B (ml)
12.3
31.5
61.0
84.0
94.7
© CLEAPSS 2011
CLEAPSS Recipe Book
Universal Buffer solutions
The Universal Buffer: Recipe 1
•
•
Wear eye protection.
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
•
•
•
•
•
Wear eye protection.
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
•
•
•
Wear eye protection.
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
•
•
•
•
Wear eye protection.
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
7.0
7.5
8.0
8.5
9.0
Volume of 0.1 M
TRIS solution
100
100
100
100
100
Volume of 0.1 M
hydrochloric acid
93.2
80.6
58.4
29.4
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
•
•
•
•
•
Wear eye protection.
Dissolve 2.19 g of calcium chloride-6-water in 70 ml of water.
Transfer the solution to a 100 ml measuring cylinder.
Make up to 100 ml with water.
The solution is low hazard.
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
•
•
•
•
•
Wear eye protection.
Dissolve 2.36 g of calcium nitrate-4-water in 70 ml of water.
Transfer the solution to a 100 ml measuring cylinder.
Make up to 100 ml with water.
The solution is low hazard.
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.
Molar mass: 74.09 g mol-1
Formula: Ca(OH)2
General Hazards
Solubility: 0.15 g per 100 ml
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
•
•
•
•
•
•
•
Wear eye protection.
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 sodalime 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.
Wear eye protection.
In the 250 ml Büchner 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
•
•
•
•
•
•
•
Wear eye protection.
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.
Stir to dissolve, warming if necessary.
Pour the solution into a 1-litre volumetric flask and make it up to the mark with water.
Pour into a labelled bottle.
The solution is low hazard.
© CLEAPSS 2011
27
CLEAPSS Recipe Book
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
•
•
•
B
•
•
•
C
•
•
D
•
•
•
Solution A: Dissolve 0.4 g of luminol and 4 g of potassium hydroxide or sodium
hydroxide in 1000 ml of water. Label the container IRRITANT.
Solution B: Dilute 50 ml of fresh (10-14% available chlorine) sodium chlorate(I)
solution to 1000 ml. Label the container IRRITANT.
In a darkened room, mix equal volumes of solutions A & B together. The addition
of a pellet of potassium hydroxide or sodium hydroxide may produce more light.
Dissolve 0.2 g of luminol and 1 g of potassium hydroxide or sodium hydroxide in
1000 ml of water. To a known volume of this solution add an equal volume of
20 vol hydrogen peroxide solution.
In a darkened room, sprinkle a few crystals of potassium hexacyanoferrate(III)
onto the surface of the solution and observe the trails of light.
The addition of a pellet of potassium hydroxide or sodium hydroxide may
produce more light.
Solution A: Dissolve 0.2 g luminol, 4 g of anhydrous sodium carbonate, 24 g of
sodium hydrogencarbonate, 0.5 g of ammonium carbonate and 0.4 g of hydrated
copper(II) sulfate(VI) in 1000 ml water.
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.
Solution A: Mix together 0.2 g luminol, 11 g of anhydrous sodium carbonate,
8 g of sodium hydrogencarbonate, 0.5 g of ammonium carbonate and 0.4 g of
copper(II) sulfate(VI) in 1000 ml water. Add 25 ml of 10 vol hydrogen peroxide.
Solution B: Dissolve 0.1 g of cobalt(II) chloride and 0.1 g of sodium nitrate(III)
(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.
See Hazcards
4B, 89, 91.
Wear goggles
to prepare the
solution.
See Hazcards
4B, 50, 79, 91.
Wear goggles
to prepare the
solution.
See Hazcards
4B, 27C, 50,
95A.
See Hazcards
4B, 9A, 25,
27C, 50, 89, 93,
95A.
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.
Do not make this solution for the first time without seeking practical advice from a more-experienced
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 Büchner 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
Büchner 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.
Do not make this solution for the first time without seeking practical advice from a more-experienced
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.
Use a fume cupboard. Make sure it is working. Wear goggles.
Place at least 50 ml sodium chlorate(I) solution (CORROSIVE) in the Büchner 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)
solution
Mineral wool
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
•
•
Use a fume cupboard. Wear goggles or a face shield and chemical-resistant gloves.
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.
•
•
•
•
Use a fume cupboard. Wear goggles or a face shield and chemical-resistant gloves.
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.
© CLEAPSS 2011
31
CLEAPSS Recipe Book
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
for all of the
following solutions
Substances
separated
Amino acids
Analgesics,
eg, aspirin,
paracetamol
Anthocyanins
(plant
pigments)
Inks from
Biro pens
Chlorophyll
Lipstick
Metal ions
Nitration of
methyl
benzoate
Sugars
Solutions are better made up in a fume cupboard.
Wear eye protection.
There must be no sources of ignition in the vicinity.
Solvent: Butan-1-ol, glacial ethanoic acid, water; 6:1:2 by
volume.
Locator: Apply ninhydrin and heat in an oven at 110 °C or with a
hairdryer.
Solvent: Ethyl ethanoate, hexane, ethanoic acid; 10:9:1 by
volume.
Locator: UV light or iodine vapour. Do not look directly at UV
radiation sources.
Solvent: 100 ml 50% aqueous methanol solution with 1 ml of
ethanoic acid or
1 ml concentrated hydrochloric acid in 100 ml ethanol.
Locator: Natural colour. (The components are light sensitive so
run the chromatograms in the dark if possible and
quickly note or photograph the results.)
Solvent: Butan-1-ol, ethanol, water; 3:1:1 by volume. The
addition of a few drops of 880 ammonia is said to
produce a better chromatogram.
Locator: Natural colour.
Solvent: Propanone, petroleum spirit (100-120 °C); 1 :9 by
volume or
cyclohexane, propanone, ethoxyethane; 2:1:1 by
volume.
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.)
Solvent: 3-Methylbutan-1-ol,propanone and water; 2:1:9 by
volume.
Locator: Natural colour.
Solvent: Propanone, hydrochloric acid (conc), Distilled
water; 17:2:1 by volume.
Locator: Use conc ammonia solution followed by 0.1% dithiooxamide (rubeanic acid).
For separating the crude products obtained from the nitration of
methyl benzoate.
Solvent: Ethoxyethane, pet ether (80/100 °C); 1:9 by volume.
Locator: UV light. Do not look directly at UV radiation sources.
Solvent: Ethyl ethanoate, pyridine, water, 8:2:1 by volume.
Locator: Dab Benedict’s solution on the chromatogram and dry in
an oven at 110 °C.
32
See Hazcards 38A,
66, 84B. Label
solvent HARMFUL and
CORROSIVE.
Label the solvent
HIGHLY FLAMMABLE
and CORROSIVE. See
Hazcards 43A, 54A.
Label the solvent
HIGHLY FLAMMABLE
and TOXIC. See
Hazcards 38A, 40B.
Label the solvent
HIGHLY FLAMMABLE
and HARMFUL. See
Hazcards 6, 40A,
84B.
Label the solvents
HIGHLY FLAMMABLE.
See Hazcards 45A,
85 or 42, 45B, 85.
The solvent is LOW
See
Hazcards 84C, 85.
Label solvent HIGHLY
FLAMMABLE and
IRRITANT Label the
locator CORROSIVE.
See Hazcards 35,
47A, 85.
Label the solvent
HAZARD
HIGHLY FLAMMABLE
and HARMFUL. See
Hazcards 42, 45A.
Label the solvent
HIGHLY FLAMMABLE.
See Hazcards 4C, 8,
43A.
© 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
•
•
•
•
Wear eye protection.
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.
The solution is low hazard.
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
•
•
•
•
Wear eye protection.
Dissolve 5.0 g of chromium(III) potassium sulfate(VI)-12-water in 70 ml of cold pure water.
Make up to 100 ml with pure water.
The solution is low hazard.
© CLEAPSS 2011
33
CLEAPSS Recipe Book
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.
Molar mass: 192.12 g mol-1
Molar mass: 210.14 g mol-1
Formula: C6H8O7
Formula: C6H8O7.H2O
Solubility: 133 g per 100 ml
Solubility: 133 g per 100 ml
Mass (g) of solid to be used
Concentration
required
0.01 M
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of 0.1 M solution
1.92 (2.10)
4.80 (5.35)
19.21 (21.01)
9.60 (10.51)
24.02 (26.27)
96.06 (105.07)
19.21 (21.01)
48.03 (53.54)
192.12 (210.14)
150
450
1500
100
Hazard warning
label
IRRITANT
Procedure
• Wear eye protection.
• Measure out the indicated quantity of the solid citric acid.
• 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 and mix well. Add a hazard warning if appropriate.
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
peroxide/potassium
iodide reaction
(The HarcourtEssen reaction)
The thiosulfate/acid
reaction
(disappearing
cross)
The potassium
iodate/sodium
metabisulfite
reaction
(The Landolt iodine
clock)
The reaction
between iron(III)
ions and iodide
ions
The potassium
iodide/potassium
persulfate reaction
© CLEAPSS 2011
Solution A 1% starch solution (indicator)
Solution B* 4 ‘vol’ hydrogen peroxide
Solution C* 0.1 M hydrochloric acid
Solution D 0.01 M sodium thiosulfate
Solution E* 0.05 M potassium iodide
Add 5 drops of solution A to a suitable beaker plus 10 ml
each of solutions B to E in that order.
Solution A 0.1 M sodium thiosulfate
Solution B 1 M hydrochloric or 0.05 M sulfuric(VI) acid
Add 1 ml of the acid for every 10 ml of sodium thiosulfate.
Once the run is complete, dispose of the contents in 0.5 M
sodium carbonate solution to stop the reaction and neutralise
sulfur dioxide.
Solution A 1% starch solution (indicator)
Solution B* 0.025 M potassium iodate(V) solution (5.35 g
per 1000 ml of solution)
Solution C* 0.025 M sodium metabisulfite solution (4.75 g
per 1000 ml of solution. Prepare in a fume cupboard.)
Add 5 drops of solution A to a suitable beaker plus 10 ml
each of solutions B and C.
Solution A 1% starch solution (indicator)
Solution B 0.01 M sodium thiosulfate
Solution C* 0.025 M iron(III) chloride using 0.1 M
hydrochloric acid to dilute a more concentrated iron(III)
solution
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.
Solution A Dissolve 0.25 g of sodium thiosulfate in 100 ml
of 1% starch solution
Solution B* Dissolve 13.50 g of potassium persulfate in
1000 ml of solution
Solution C* 0.1 M potassium iodide
Mix 5 ml of solution A with 50 ml each of solutions B and C in
that order.
35
See Hazcards 47A
& B, 67, 95C.
Wear eye
protection.
See Hazcards 47A,
95C & 98.
Wear eye
protection. Do not
use temperatures
above 50 °C.
See Hazcards 80,
92.
Wear eye
protection.
See Hazcards 41,
47B, 55, 67, 95C.
Wear eye
protection.
See Hazcards 47B,
95B, 95C.
Wear eye
protection.
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.
Make up to 100 ml with pure 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
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of 0.1 M solution
6.24
24.97
2.50
31.21
124.84
12.49
62.42
249.68
24.97
100
400
40
Formula: CuCl2.2H2O
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Molar mass: 170.48 g mol-1
Volume (ml) of solution required
250
1000
Ten-fold dilution of 0.1 M solution
4.26
17.05
1.71
21.31
85.24
8.52
42.62
170.48
17.05
200
800
80
Formula: Cu(NO3)2.3H2O
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Molar mass: 249.68 g mol-1
Molar mass: 241.6 g mol-1
Volume (ml) of solution required
250
1000
Ten-fold dilution of 0.1 M solution
6.03
24.15
2.42
30.19
120.75
12.08
60.34
241.50
24.15
350
1400
140
Solubility: 32 g per 100 ml
Hazard warning
label
HARMFUL
HARMFUL
Solubility: 76 g per 100 ml
Hazard warning
label
(HARMFUL)
HARMFUL
Solubility: 138 g per 100 ml
Hazard warning
label
(HARMFUL)
HARMFUL
Procedure
•
•
•
•
•
•
Wear eye protection.
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.
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 and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
37
CLEAPSS Recipe Book
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 Newton’s 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
hold the test tubes
Heat
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 Brady’s 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 Brady’s 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).
Molar mass: 198.14 g mol-1
Formula: (NO2)2C6H3NHNH2
Procedure for making about 100 ml of Brady’s reagent
Traditional method
New method
• Wear eye protection and gloves.
Wear eye protection, gloves and work in a fume
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 Brady’s 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 Brady’s 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
CLEAPSS Recipe Book
34
3,5-Dinitrosalicylic acid
Also called DNSA or DNS.
3,5-Dinitrosalicylic acid is an alternative to Benedict’s 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 Benedict’s 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, (KNaC4H4O6·4H2O). 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
•
•
•
•
•
•
•
•
Wear eye protection.
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.
© CLEAPSS 2011
41
CLEAPSS Recipe Book
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
Copper
electroplating
Nickel
electroplating
Silver
electroplating
Zinc electroplating
Recipe for 100 ml of solution
Use 1 M copper(II) sulfate(VI) solution
Dissolve 5 g of ammonium nickel(II)
sulfate(VI) in 80 ml of water and dilute to
100 ml.
Dissolve 1.6 g of silver nitrate(V) and 32 g of
potassium iodide in 100 ml of water. Add 3
drops of concentrated sulfuric(VI) acid. Use a
carbon rod as the anode.
Dissolve 33 g of zinc sulfate(VI)-7-water in
50 ml of water and dilute to 100 ml. Add five
drops of 2 M sulfuric(VI) acid and 2 “spatulas”
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
Notes
See Hazcard 27C. Label the
solution HARMFUL.
See Hazcard 65B. Label the
solution HARMFUL.
See Hazcards 87 and 98A. Label
the solution IRRITANT.
See Hazcards 14, 98A and 108B.
It would be advisable to label the
solution HARMFUL.
© CLEAPSS 2011
CLEAPSS Recipe Book
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
CLEAPSS Recipe Book
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
Aluminium
etching
Copper etching
Iron etching 1
(Fry’s reagent)
Iron etching 2
(Nital solution)
Lead etching
Recipe for 100 ml of solution
Use 2 M sodium hydroxide solution at 60-70 °C.
Notes
See Hazcard 91, Wear goggles.
Dissolve 20 g of iron(III) chloride-6-water in
80 ml of water and 20 ml of concentrated
hydrochloric acid (36% w/v).
Dissolve 59 g of copper(II) choride-2-water in
40 ml of water and add 60 ml of concentrated
hydrochloric acid (36% w/v).
Add 2 ml of concentrated nitric(V) acid to 98 ml
of ethanol. Under no circumstances use more
than 2 ml of nitric(V) acid in making up 100 ml
of solution.
Wear goggles and chemical resistant gloves.
Before use, mix together 50 ml of 5 M nitric(V)
acid and 50 ml of 15% (w/v) ammonium
molybdate(VI) solution. Apply with a swab for
30 s before rinsing.
See Hazcards 47A and 55C.
Label the solution IRRITANT.
44
See Hazcards 27A and 47A. Label
the solution CORROSIVE.
See Hazcards 40A and 67. Label
the solution HIGHLY FLAMMABLE &
HARMFUL. Do not store.
See Hazcards 9A and 67. Label
the solution CORROSIVE.
© CLEAPSS 2011
CLEAPSS Recipe Book
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.
Molar mass: 60.05 g mol-1
Formula: CH3COOH
General Hazards
See Hazcard 38A. Ethanoic acid is CORROSIVE. It has a very sharp odour.
Volume (ml) of concentrated acid to be used
Concentration
required
0.01 M
0.1 M
0.5 M
1M
2M
500
14
29
57
Volume (ml) of solution required
1000
Ten-fold dilution of 0.1 M solution
Ten-fold dilution of 1 M solution
29
57
114
2500
71
143
286
Hazard warning
label
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.
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 and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
45
CLEAPSS Recipe Book
40
Fehling’s solutions
Benedict’s 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.
Sandell’s 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
(Fehling’s 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 Fehling’s solution
Fehling’s solution A
•
•
•
•
Wear eye protection.
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.
Fehling’s 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
CLEAPSS Recipe Book
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
for fixing animal
tissue
Dilute 10 ml of 40% methanal solution to
100 ml. Leave specimen in this solution
for 16-48 hours before preserving.
Formalin-alcohol
fixative for plant
tissue
Mix together 90 ml of ethanol with 10 ml
of 40% methanal solution. Leave
specimen in this solution for 12 hours
before preserving.
Formalin-acetoalcohol (FAA)
fixative for plant
tissue
Mix together 5 ml of 40% methanal and
5 ml of glacial ethanoic acid in 90 ml of
70% ethanol. Leave specimen in this
solution for 12 hours before preserving.
Acetic alcohol
(Clarke’s fluid) (a
cytological fixative
especially for
chromosomes)
Mix 25 ml of glacial ethanoic acid with
75 ml of absolute alcohol (Industrial
denatured alcohol is normally
adequate).
© CLEAPSS 2011
47
For methanal, see Hazcard 63. Label
the final solution HARMFUL. Use a
fume cupboard, wear eye protection
and disposable nitrile gloves.
For ethanol; see Hazcard 40A. For
methanal, see Hazcard 63. Label the
solution HIGHLY FLAMMABLE and
HARMFUL. Use a fume cupboard,
wear eye protection and disposable
nitrile gloves.
For ethanol; see Hazcard 40A. For
methanal, see Hazcard 63. For
ethanoic acid, see Hazcard 38.
Label the solution HIGHLY FLAMMABLE
& HARMFUL. Use a fume cupboard,
wear eye protection and disposable
nitrile gloves.
For ethanol; see Hazcard 40A. For
ethanoic acid, see Hazcard 38.
Label the solution HIGHLY FLAMMABLE
and CORROSIVE. Use a fume
cupboard, wear eye protection and
disposable nitrile gloves.
CLEAPSS Recipe Book
42
Gases less commonly used in schools
Gas
Carbon
monoxide
Dinitrogen
monoxide
Hazard
information
See Hazcard 21
See Hazcard 68
Hydrogen
chloride
See Hazcard 49
Hydrogen
sulfide
Nitrogen
See Hazcard 51
Nitrogen
dioxide
See Hazcard 68
Nitrogen
monoxide
See Hazcard 68
See Hazcard 93
Minimum amount of reagents to prepare 1000 ml of gas
Concentrated sulfuric(VI) acid is slowly added to 2.9 g of solid sodium
methanoate. Collect over water.
5.8 g of hydroxyammonium chloride in 30 ml of water is added to a warm
solution containing 81 g of ammonium iron(III) sulfate(VI) in 160 ml of
water. Collect over warm water.
2.1 ml of concentrated sulfuric(VI) acid is slowly added to excess solid
sodium chloride. Collect by downward delivery (a tube into the bottom of
an upright gas jar).
42 ml of 2 M hydrochloric acid is added to excess iron(II) sulfide. Collect
over hot water.
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.
8 ml of concentrated nitric(V) acid (70%) is added to an excess of copper
turnings. Collect by downward delivery (a tube into the bottom of an
upright gas jar).
30 ml of half-concentrated nitric(V) acid (35%) is added to an excess of
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.
Density: 1.18 g cm-3
Formula: HCl
General Hazards
Concentration
required
0.01 M
0.1 M
0.4 M
0.5 M
1M
2M
Molar mass: 36.46 g mol-1
See Hazcard 47A. Hydrochloric acid is CORROSIVE. It has a very sharp odour.
500
17
21
42
84
Volume (ml) of solution required
1000
Ten-fold dilution of 0.1 M solution
Ten-fold dilution of 1 M solution
34
42
84
167
2500
84
105
209
417
Hazard warning
label
IRRITANT
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.
• Add the liquid to about two thirds of the final volume of water in a beaker or laboratory jug.
• Stir the mixture well.
• 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 and mix well. Add a hazard warning if appropriate.
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CLEAPSS Recipe Book
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.
The reaction needs to be speeded up at the
start, to flush out air quickly.
Add about 5 ml of 1 M copper sulfate(VI)
(HARMFUL) down the thistle funnel. This reacts
with zinc, coating it with copper which acts as
a catalyst.
Add 2 M sulfuric(VI) acid (CORROSIVE) down
the thistle funnel.
Collect the gas in the 250 ml measuring
cylinder. Once the measuring cylinder is full,
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
larger than the hydrogen generator.
• Once the measuring cylinder is full, disconnect
at A, and attach other equipment but before
using the hydrogen, leave time to flush the
air out of this newly-attached equipment.
The gas collected in the measuring cylinder
can be discarded.
50
1 to 5 ml of 1M
copper(II) sulfate(VI) plus
2M sulfuric acid
250 ml measuring
cylinder
250 ml
plastic bottle
Hydrogen
generator
Zinc
A
500ml measuring
cylinder;
to ensure
all air is flushed
through
U-tube
Calcium chloride
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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.
Molar mass: 34.02 g mol-1
Formula: H2O2
See Hazcard 50. ‘100 vol’ hydrogen peroxide is HARMFUL.
General Hazards
Concentration
required
0.08 M
0.1 M
0.34 M
1M
1.7 M
3.4 M
1 vol
1.2 vol
4 vol
12 vol
20 vol
40 vol
0.29%
0.34%
1.14%
3.3%
5.7%
11.4%
250
Volume (ml) of solution required
1000
2500
Twenty-fold dilution of the 20 ‘vol’ solution
Ten-fold dilution of the 1 M solution
Five-fold dilution of the 20 ‘vol’ solution
29
115
288
50
200
500
100
400
1000
Hazard
warning
label
IRRITANT
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.
• 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 and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
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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
(g)
Volume of
IDA
Acid
Alkali
pH range
Bromophenol blue
Methyl orange
Bromocresol green
Methyl red
Bromothymol blue
Phenol red
Neutral Red
Cresol red
Thymol blue
Phenolphthalein
Thymolphthalein
0.4
0.4
0.4
0.2
0.4
0.4
0.4
0.2
0.4
1.0
2.0
200
200
200
300
200
200
200
200
200
600
1000
Yellow
Red
Yellow
Red
Yellow
Yellow
Red
Yellow
Yellow
Colourless
Colourless
Blue
Yellow
Blue
Yellow
Blue
Red
Yellow
Purple
Violet
Mauve
Blue
3.0 - 4.5
3.0 - 4.5
4.0 - 5.4
4.0 - 6.0
6.0 - 7.5
6.6 - 8.0
6.8 - 8.0
7.2 - 8.8
8.0 - 9.6
8.0 - 10.0
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
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Indicator (universal)
General Hazards
See Hazcard 32. The hazards of many dyes and indicators are not well known.
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
© CLEAPSS 2011
4.5
5
5.5
6
6.5
7
7.5
53
8
8.5
9
9.5
10
10.5 11
CLEAPSS Recipe Book
48
Indicators (carbon dioxide)
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).
Hydrogencarbonate (bicarbonate) indicator: Preparation of the stock solution:
•
•
•
•
•
Wear eye protection.
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).
•
•
•
Wear eye protection.
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.
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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
0.70 g of iron(II) sulfate(VI). (Ferroin indicator).
Wear eye protection. 1:10phenanthroline is TOXIC. The solution is
low hazard.
Dissolve 0.1 g of N-phenylanthranilic acid in 5 ml of
0.1 M sodium hydroxide. Dilute to 100 ml with water.
See Hazcard 78.
Dichromate(VI) titrations
Dissolve 0.1 g of N-phenylanthranilic acid in 5 ml of
0.1 M sodium hydroxide. Dilute to 100 ml with water.
See Hazcard 78.
Dissolve 0.2 g of sodium or barium diphenylamine-4-sulfonate
[diphenylamine-4-sulfonic acid (sodium salt)] in 100 ml of water.
See Hazcard 10B. Sodium
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
stir it vigorously to form a paste.
Boil 100 ml of pure water and add it to the paste with stirring and
allow to cool.
This ‘solution’ (it is really a suspension)
does not keep. Dispose of it if it starts to
go cloudier or ‘lumpy’.
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
30 ml of water.
See Hazcard 32 & 40A. Label this
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 Volhard’s method.) Dissolve 10 g of ammonium iron(III)
sulfate(VI).12-water in 90 ml of water and add enough 2 M
nitric acid to clear the solution and prevent hydrolysis. Make up to
100 ml. Use 1 ml per titration.
See Hazcard 55C. Colour change: white
ppt to orange red colouration.
EDTA titrations
Dissolve 1 g of the Eriochrome black T (Solochrome black) in
100 ml of ethanol or grind 1 g of the solid dye with 100 g of
sodium chloride. Use about 0.2 g of this solid mixture for each
titration.
See Hazcard 32 & 40A. Label this
solution HARMFUL. (Colour change is red
to blue.)
Grind 1 g of murexide with 100 g of sodium chloride. Use 0.2 g for
each titration.
See Hazcard 32 & 40A. Label this
solution HARMFUL. (Colour change is red
to blue.)
© CLEAPSS 2011
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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.
Molar mass: 253.80 g mol-1
Formula: I2
General Hazards
See Hazcard 54. The organic solvents used are also hazardous and the relevant
Hazcard should be consulted.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
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
Hazard
warning
label
-
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.
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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
Volume (ml) of solution required
Concentration
required
0.01 M
0.1 M
1M
Saturated (20 °C)
Solubility: 48 g per 100 ml
100
250
1000
Ten-fold dilution of a 0.1 M solution with 0.1 M sulfuric(VI) acid
2.78
6.95
27.80
27.80
69.50
278.01
50
125
500
Hazard warning
label
IRRITANT
IRRITANT
IRRITANT
Iron(II) chloride-4-water
Do not use the anhydrous salt.
Formula: FeCl2.4H2O
Concentration
required
0.01 M
0.1 M
1M
Saturated (20 °C)
Molar mass: 198.8 g mol-1
Solubility: 68 g per 100 ml
Volume (ml) of solution required
100
250
1000
Ten-fold dilution of a 0.1 M solution with 0.1 M hydrochloric acid
1.99
4.97
19.88
19.88
49.7
198.80
70
175
700
Hazard warning
label
IRRITANT
IRRITANT
IRRITANT
Procedure
• Wear eye protection.
• 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.
• Stir to dissolve (do not warm the solution).
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
either 1 M sulfuric(VI) acid for the sulfate(VI) or 1 M hydrochloric acid for the chloride to the required
level.
• Pour into a labelled bottle and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
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CLEAPSS Recipe Book
Diammonium iron(II) sulfate(VI)
Also known as ammonium ferrous sulfate(VI) or Mohr’s salt.
Formula: (NH4)2SO4.FeSO4.6H2O
General Hazards
Concentration
required
0.01 M
0.1 M
0.5 M
Saturated (20 °C)
Molar mass: 392.14 g mol-1
Solubility: 36 g per 100 ml
1 M sulfuric(VI) acid; Hazcard 98A. Iron(II) salts; Hazcard 55B.
Volume (ml) of solution required
100
250
1000
Ten-fold dilution of a 0.1 M solution with 0.1 M sulfuric(VI) acid
3.92
9.80
39.21
19.61
49.02
196.07
40
100
400
Hazard warning
label
IRRITANT
IRRITANT
IRRITANT
IRRITANT
Procedure
• Wear eye protection.
• 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.
• Stir to dissolve (do not warm the solution).
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
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.
Molar mass: 270.30 g mol-1
Formula: FeCl3.6H2O
General Hazards
Concentration
required
0.01 M
0.1 M
1M
Saturated (20 °C)
Solubility: 92 g per 100 ml
See Hazcard 55C. Do make sure you are using then hydrated salt and not the
anhydrous salt.
Volume (ml) of solution required
100
250
1000
Ten-fold dilution of a 0.1 M solution with 0.2 M hydrochloric acid
2.70
6.76
27.03
27.03
67.58
270.30
95
238
950
Hazard warning
label
IRRITANT
IRRITANT
IRRITANT
Procedure
• Wear eye protection.
• 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.
• Stir to dissolve (do not warm the solution).
• 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.
• Pour into a labelled bottle and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
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CLEAPSS Recipe Book
Iron(III) sulfate(VI)
Molar mass: 562 g mol-1
Formula: Fe2(SO4)2.9.H2O
General Hazards
See Hazcards 55C and 98. 1 M sulfuric(VI) acid. Label the final solution IRRITANT
because of the acid present.
Volume (ml) of solution required
Concentration
required
0.01 M
0.1 M
0.5 M
1M
100
250
1000
Ten-fold dilution of a 0.1 M solution with 0.1 M sulfuric(VI) acid
5.6
14.1
56.2
28.1
70.3
281
56.2
140.5
562
Hazard warning
label
IRRITANT
IRRITANT
IRRITANT
IRRITANT
Ammonium iron(III) sulfate(VI)
Molar mass: 482.19 g mol-1
Formula: NH4Fe(SO4)2.12.H2O
General Hazards
Concentration
required
0.01 M
0.1 M
0.5 M
Saturated (20 °C)
Solubility: 124 g per 100 ml
See Hazcards 55C and 98. 1 M sulfuric(VI) acid. Label the final solution IRRITANT
because of the acid present.
Volume (ml) of solution required
100
250
1000
Ten-fold dilution of a 0.1 M solution with 0.1 M sulfuric(VI) acid
4.82
12.05
48.22
24.11
96.4
241.10
125
313
1250
Hazard warning
label
IRRITANT
IRRITANT
IRRITANT
IRRITANT
Procedure
• Wear eye protection.
• 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.
• Stir to dissolve (do not warm the solution).
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
1 M sulfuric(VI) acid to the required level.
• Pour into a labelled bottle and mix well. Add a hazard warning if appropriate.
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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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.
Molar mass: 331.21 g mol-1
Formula: Pb(NO3)2
General Hazards
Concentration
required
0.005 M
0.01 M
0.05 M
0.1 M
1M
Saturated (20 °C)
Solubility: 56 g per 100 ml
See Hazcards 57A & B.
Volume (ml) of solution required
100
250
1000
Twenty-fold dilution of a 0.1 M solution with water
or ten-fold dilution of 0.05 M solution
Ten-fold dilution of a 0.1 M solution with water
1.66
16.55
3.31
8.28
33.12
33.12
82.80
331.21
56
140
560
Hazard warning
label
TOXIC
TOXIC
TOXIC
TOXIC
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).
• 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 and mix well. Add a hazard warning if appropriate.
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Lithium chloride
Molar mass: 42.39 g mol-1
Formula: LiCl
General Hazards
Solubility: 84 g per 100 ml
Lithium salts are HARMFUL if swallowed and irritants. See Hazcards 47A and 58.
Mass (g) of solid to be used
Concentration
required
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
100
250
2.12
4.24
90
Ten-fold dilution of the 1 M solution
5.30
10.60
225
1000
21.20
42.39
900
Hazard warning
label
HARMFUL
Procedure
• Measure out the indicated quantity of lithium chloride.
• 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. Add a hazard warning if appropriate.
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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.
Molar mass: 246.47 g mol-1
Formula: MgSO4.7H2O
General Hazards
See Hazcard 59B.
Mass (g) of solid to be used
Concentration
required
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Solubility: 71 g per 100 ml
100
Volume (ml) of solution required
250
1000
Hazard warning
label
2.47
12.32
24.65
75
6.16
30.81
61.62
190
24.65
123.24
246.47
750
-
Procedure
• Measure out the indicated quantity of hydrated magnesium sulfate(VI).
• 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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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.
Molar mass: 223.07 g mol-1
Molar mass: 169.02 g mol-1
Formula: MnSO4.4H2O
Formula: MnSO4.H2O
General Hazards
Manganese(II) sulfate(VI) is HARMFUL. See Hazcard 60.
Mass (g) of solid to be used
Concentration
required
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Solubility: 95 g per 100 ml
Solubility: 40 g per 100 ml
Volume (ml) of solution required
100
250
1000
2.23 (1.69)
11.15 (8.45)
22.31 (16.90)
100 (45)
5.58 (4.23)
27.89 (21.13)
55.77 (42.26)
250 (115)
22.31 (16.90)
111.54 (84.51)
223.07 (169.02)
1000 (450)
Hazard warning
label
HARMFUL
HARMFUL
Procedure
• Measure out the indicated quantity of hydrated manganese(II) sulfate(VI).
• 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. 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.
• Transfer the solution to a 100 ml measuring cylinder.
• 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
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.
• Transfer the solution to a 100 ml measuring cylinder.
• Make up to 100 ml with water.
• Label the solution TOXIC.
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65
Solubility: 7.4 g per 100 ml
CLEAPSS Recipe Book
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.
Molar mass: 30.03 g mol-1
Formula: HCHO
General Hazards
See Hazcard 63. It can cause burns, conjunctivitis or sensitisation by skin contact.
Volume of liquid required
Concentration
required
0.3%
1%
3%
4%
0.1 M
0.3 M
1M
1.3 M
Volume (ml) of solution required
500
1000
2500
Ten-fold dilution of a 0.1 M solution with water
13
25
63
38
75
190
50
100
250
Hazard warning
label
IRRITANT
HARMFUL
HARMFUL
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 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 and mix well. Add the appropriate hazard warning.
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Methanoic acid
Also known as formic acid.
It is usually supplied as a 90% (w/w) solution.
The concentration of this solution is 23.9 mol dm-3.
Density: 1.22 g cm-3
Formula: HCOOH
General Hazards
Concentration
required
0.01 M
0.1 M
1M
2M
Molar mass: 46.02 g mol-1
See Hazcard 38A. Methanoic acid is CORROSIVE. It has a sharp odour.
250
10.5
21
Volume (ml) of solution required
500
1000
Ten-fold dilution of the 0.1 M solution
Ten-fold dilution of 1 M solution
21
42
42
84
2500
105
210
Hazard
warning label
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.
• Add the liquid to about two thirds of the final volume of water in a beaker or laboratory jug.
• Stir the mixture well.
• 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 and mix well. Add a hazard warning if appropriate.
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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
General Hazards
Molar mass: 262.86 g mol-1
Solubility: 65 g per 100 ml
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
• Wear eye protection.
• Dissolve 2.63 g of nickel(II) sulfate(VI)-6-water in 70 ml of water.
• Make up to 100 ml with pure water.
• The solution should be labelled TOXIC.
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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.
Molar mass: 63.01 g mol-1
Formula: HNO3
General Hazards
Concentration
required
0.01 M
0.1 M
0.4 M
1M
5M
See Hazcard 67. If in contact with the skin, this becomes yellow and layers of skin
peel off.
Volume (ml) of solution required
500
1000
2500
Ten-fold dilution of a 0.1 M solution with water
Ten-fold dilution of a 1 M solution with water
13
25
63
32
63
158
158
317
792
Hazard warning
label
IRRITANT
IRRITANT
CORROSIVE
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 from the beaker into an appropriate measuring cylinder or laboratory jug and add
water to the required level.
• 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.
Wear eye protection for the experiment.
• In a 500 ml flask, dissolve 10 g of glucose in 300 ml of 0.4 M sodium hydroxide
solution. Add 6 drops of a 0.2% aqueous methylene blue solution. Fit a rubber
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.
•
See Hazcards 32 &
91. The solution
should be labelled
IRRITANT.
The Belousov-Zhabotinski (B-Z) reaction
All reagents in the Belousov-Zhabotinski (B-Z) reactions must be free of chloride ions,
which inhibit the reaction.
• Wear eye protection.
• 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.
See Hazcards 6B,
80 & 98A.
The Briggs-Rausher (B-R) reaction
All reagents in the Briggs-Rausher (B-R) reaction must be free of chloride ions, which
inhibit the reaction.
• Solution A: 10 vol hydrogen peroxide solution.
• Solution B: 6% (w/v) potassium iodate(V) solution in 0.1 M sulfuric(VI) acid.
• Solution C: 1% starch solution.
• 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.
See Hazcards 6B,
50 & 80. The
diluted solutions do
not require a
hazard warning.
D. The nitrogen gas oscillating reaction
Use a fume cupboard (nitrogen dioxide, VERY TOXIC, is produced).
Wear eye protection.
• 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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See Hazcards 68 &
93.
CLEAPSS Recipe Book
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.
Wear eye protection.
In the 250 ml Büchner 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
Büchner
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).
The concentration of this solution is 14.7 mol dm-3.
Density: 1.7 g cm-3
Formula: H3PO4
General Hazards
Concentration
required
0.01 M
0.1 M
0.5 M
1M
Molar mass: 98 g mol-1
See Hazcard 72.
Volume (ml) of solution required
500
1000
2500
Ten-fold dilution of the 0.01 M solution
3.5
7.0
17.0
17
34
85
34
68
170
Hazard warning
label
IRRITANT
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.
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
water to the required level.
• Pour the solution into a labelled bottle and mix well. Add a hazard warning if appropriate.
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66
Plant mineral requirement solutions
A
Sach’s culture solution (complete recipe)
Dissolve the following salts in 1000 ml of pure water:
•
•
•
•
•
•
B
0.25 g of calcium sulfate(VI)-2-water
0.25 g of calcium phosphate(V)-2-water
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
Sach’s culture solution with mineral deficiencies
Deficient in calcium:
Deficient in iron:
Deficient in magnesium:
Deficient in nitrogen:
Deficient in phosphorus:
Deficient in potassium:
Deficient in sulfur:
C
[CaH4(PO4)2.2H2O]
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.
Omit iron(III) chloride-6-water in recipe A.
0.17 g of potassium sulfate(VI) replaces magnesium sulfate(VI) in recipe A.
0.52 g of potassium chloride replaces potassium nitrate(V) in recipe A.
0.16 g of calcium nitrate(V)-4-water replaces calcium phosphate(V) in
recipe A.
0.59 g of sodium nitrate(V) replaces potassium nitrate(V) in recipe A.
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.
Knop’s 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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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
phosphate(V)
Sodium dihydrogen
phosphate(V)
Sodium dihydrogen
phosphate(V)-1-water
KH2PO4
0.2 M
NaH2PO4
0.2 M
NaH2PO4.H2O
0.2 M
Dipotassium hydrogen
phosphate(V)
Dipotassium hydrogen
phosphate(V)-3-water
Disodium hydrogen
phosphate(V)
Disodium hydrogen
phosphate(V)-7-water
K2HPO4
0.2 M
K2HPO4.3H2O
0.2 M
Na2HPO4
0.2 M
Na2HPO4.7H20
0.2 M
Potassium
phosphate(V)
Sodium phosphate(V)
K3PO4
0.2 M
Na3PO4
0.2 M
Sodium phosphate(V)12-water
Na3PO4.12H2O
0.2 M
© CLEAPSS 2011
Dissolve 27.218 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 23.996 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 27.598 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 34.836 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 45.644 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 28.392 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 53.614 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 42.454 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 32.788 g of the solid in 500 ml of water
and add water to 1000 ml.
Dissolve 76.024 g of the solid in 500 ml of water
and add water to 1000 ml.
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68
Potassium chloride
Molar mass: 74.55 g mol-1
Formula: KCl
Solubility: 11 g per 100 ml
Mass (g) of solid to be used
Concentration
required
100
0.1 M
1.0 M
3.0 M (used for
filing pH probes)
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 1 M solution
7.46
18.64
74.55
22.34
55.91
223.65
40
100
400
Hazard warning
label
-
Procedure
• Measure out the indicated quantity of potassium chloride.
• 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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69
Potassium chromate(VI)
Molar mass: 194.19 g mol-1
Formula: K2CrO4
General Hazards
See Hazcard 78.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
1.0 M
Saturated (20 °C)
Solubility: 64 g per 100 ml
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
1.94
4.86
19.42
19.42
48.58
194.19
68
158
630
Hazard warning
label
TOXIC
TOXIC
VERY TOXIC
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).
• 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. 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’.
Molar mass: 294.18 g mol-1
Formula: K2Cr2O7
General Hazards
See Hazcard 78.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
Saturated (20 °C)
Solubility: 12 g per 100 ml
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution with 0.1 M sulfuric(VI) acid
2.94
7.36
29.42
12
30
120
Hazard warning
label
TOXIC
TOXIC
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 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.
• Stir to dissolve, warming if necessary.
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
0.1 M sulfuric(VI) acid to the required level.
• Pour into a labelled bottle. Add the hazard warning.
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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.
Molar mass: 56.11 g mol-1
Formula: KOH
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.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.4 M
1.0 M
5.0 M
Solubility: 112 g per 100 ml
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
Ten-fold dilution of the 1 M solution
2.24 g
5.61 g
22.44 g
5.61 g
14.03 g
56.11 g
28.06 g
70.14 g
280.55 g
Hazard warning
label
IRRITANT
IRRITANT
CORROSIVE
CORROSIVE
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 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 the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
water to the required level.
• Pour into a labelled plastic bottle. Add a hazard warning if appropriate.
A 10% (w/v) alcoholic solution of potassium hydroxide
Solutions absorb carbon dioxide on standing. Solutions lower than 0.1 M are most significantly affected and
should, therefore, be freshly made.
•
•
•
•
•
•
•
Wear chemical-resistant gloves and goggles. Use a fume cupboard if the solid is a fine powder.
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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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
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
1.66
4.15
16.60
16.60
41.50
166.00
150
375
1500
Solubility: 144 g per 100 ml
Hazard warning
label
-
Procedure
• Wear eye protection.
• Measure out the indicated quantity of potassium iodide.
• 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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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.
Molar mass: 158.03 g mol-1
Formula: KmnO4
General Hazards
Solubility: 6 g per 100 ml
See Hazcard 81.
Mass (g) of solid to be used
Concentration Conc of acid
required
for dilution
0.002 M
0.1 M
0.02 M
0.1 M
Saturated
0.1 M
1M
1M
Volume (ml) of solution required
500
1000
2500
Ten-fold dilution of the 0.02 M solution
with 0.1 M sulphuric(VI) acid
1.58
3.16
7.90
7.90
15.80
39.51
32
64
160
Hazard
warning label
IRRITANT
IRRITANT
Procedure
• Wear eye protection.
• 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.
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
dilute sulfuric(VI) acid to the required level.
• Pour into a labelled bottle.
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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
tissue (Recipe 1)
Preserving animal
tissue (Recipe 2)
Add 10 ml of odourless preservative
(eg, Phenoxetol or Opresol) to 50 ml
of propane-1,2-diol. Dilute this
mixture to 100 ml with purified water.
(Another version of this recipe
includes and additional 1 g of sodium
chloride.)
Mix together 10 g of 4-methoxybenzaldehyde (p-anisaldehyde), 50 ml of
Teepol and 50 ml of propane-1,2-diol
and make up this mixture to 1000 ml
with purified water.
Preserving plant tissue
Use the formalin-aceto-alcohol (FAA)
recipe on the fixative recipe sheet 41.
To preserve the red or green colour
add copper(II) sulfate(VI)-5-water
crystals; the amount is not too
critical.
Kaiserling’s
preservative
Add 36 g of sodium ethanoate, 5 ml
of a 5% camphor solution in ethanol
and 72 ml of propane-1,2,3-triol
(glycerol). Add this mixture to 120 ml
of purified water.
82
Wear eye protection. See
Hazcard 37.
Animal tissue should not be
preserved for more than 1 month in
these solutions unless it is fixed
first. Label the solution IRRITANT.
Wear eye protection. See
Hazcard 37.
Animal tissue should not be
preserved for more than 1 month in
these solutions unless it is fixed
first. Label the solution IRRITANT.
4-Methoxybenzaldehyde is
HARMFUL if swallowed. Label the
solution HARMFUL.
Use a fume cupboard. Wear eye
protection.
For ethanol; see Hazcard 40A.
For methanal, see Hazcard 63.
For ethanoic acid, see Hazcard 38.
For copper(II) sulfate(VI), see
Hazcard 27C. Label the solution
HIGHLY FLAMMABLE, IRRITANT and
HARMFUL.
Wear eye protection.
Camphor is HARMFUL. For propane1,2,3-triol, see Hazcard 37. For
sodium ethanoate see Hazcard
38A. for ethanol, see Hazcard 40A.
Label the solution HARMFUL and
HIGHLY FLAMMABLE.
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Ringer’s and other saline solutions for physiological use
Saline solutions are suitable for temporary microscopial preparations and short experiments.
For more prolonged investigations, a suitable Ringer’s solution should be used.
Ringer’s 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
For amphibian tissues
For mammalian tissues
For mammalian blood
© CLEAPSS 2011
Dissolve 7.5 g of sodium chloride in 1000 ml of water.
Dissolve 6.4 g of sodium chloride in 1000 ml of water.
Dissolve 9 g of sodium chloride in 1000 ml of water.
Dissolve 6 g of sodium chloride in 1000 ml of water.
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Sandell’s solution
Sandell’s solution is a safer alternative than Fehling’s testing for sugars.
Sandell’s 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 Fehling’s 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 Benedict’s 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 Sandell’s 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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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.
Tollen’s 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.
Molar mass: 169.87 g mol-1
Formula: AgNO3
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.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1
0.2
0.5
Saturated (20 °C)
Solubility: 216 g per 100 ml
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M with water
1.70
4.25
16.99
3.40
8.50
33.98
8.49
21.23
84.94
220
550
2200
Hazard warning
label
IRRITANT
CORROSIVE
CORROSIVE
Procedure
• Wear eye protection.
• Measure out the indicated quantity of silver nitrate(V).
• 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. Add a hazard warning if appropriate.
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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 below 85,000 g mol-1
PVA with a molar mass above 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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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 Clarke’s or Wanklyn’s 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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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.
Molar mass: 105.99 g mol-1
Molar mass: 286.14 g mol-1
Formula: Na2CO3
Formula: Na2CO3.10H2O
General Hazards
Solubility: 22 g per 100 ml
Solubility: 50 g per 100 ml
See Hazcard 95A.
Anhydrous sodium carbonate
Mass (g) of solid to be used
Concentration
required
250
0.05 M
0.1 M
0.5 M
1M
Saturated
Volume (ml) of solution required
500
1000
Two-fold dilution of 0.1 M solution
2.65
5.30
10.60
13.25
26.50
53.00
26.50
53.00
105.99
55
110
220
Hazard warning
label
IRRITANT
Hydrated sodium carbonate
Mass (g) of solid to be used
Concentration
required
250
0.05 M
0.1 M
0.5 M
1M
Saturated
Volume (ml) of solution required
500
1000
Two-fold dilution of 0.1 M solution
7.15
14.31
28.61
35.77
71.54
143.07
71.54
143.07
286.14
125
250
500
Hazard warning
label
IRRITANT
Procedure
• Wear eye protection.
• Measure out the indicated quantity of anhydrous or hydrated sodium carbonate.
• 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. Add a hazard warning if appropriate.
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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.
Molar mass: 74.45 g mol-1
Formula: NaClO
General Hazards
See Hazcard 89.
Concentration required
1%
10000 ppm
0.15 M
0.25%
2500 ppm
0.038 M
0.1%
1000 ppm
0.015 M
0.01%
100 ppm
0.0015 M
Preparation
See below for procedure.
No need for further dilution.
Mix, by volume, 1 part of the 1%
solution with 3 parts of water.
Mix by volume, 1 part of the 1%
solution with 9 parts of water.
Dilute the 0.1% solution further
by mixing by volume, 1 part of
the 0.1% solution with 9 parts of
water.
Use
Disinfectant for blood spills or
dirty conditions.
Disinfectant for micro-biological
discard pots.
Disinfectant for general use, eg,
sterilising solution.
Disinfectant for sterilising
mouthpieces, swabbing skin.
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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Sodium chloride
Molar mass: 58.44 g mol-1
Formula: NaCl
Mass (g) of solid to be used
Concentration
required
100
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
2.92
5.84
37
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 1 M solution
7.31
29.22
14.61
58.44
93
370
Solubility: 36 g per 100 ml
Hazard warning
label
-
Procedure
• Measure out the indicated quantity of sodium chloride.
• 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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Sodium ethanoate
Sodium ethanoate is sold as the anhydrous salt or as the trihydrate.
Anhydrous sodium ethanoate
Molar mass: 82.03 g mol-1
Formula: NaCH3COO
Mass (g) of solid to be used
Concentration
required
100
0.2 M
1.0 M
Saturated (20 °C)
8.20
50
Volume (ml) of solution required
250
1000
5-fold dilution of the 1 M solution
20.51
82.03
125
500
Solubility: 46 g per 100 ml
Hazard warning
label
-
Hydrated sodium ethanoate
Molar mass: 136.08 g mol-1
Formula: NaCH3COO.3H2O
Mass (g) of solid to be used
Concentration
required
100
0.2 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
5-fold dilution of the 1 M solution
13.61
34.02
136.08
80
200
800
Solubility: 76 g per 100 ml
Hazard warning
label
-
Procedure
• Measure out the indicated quantity of sodium ethanoate.
• 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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Sodium hydrogencarbonate
Molar mass: 84.01 g mol-1
Formula: NaHCO3
Mass (g) of solid to be used
Concentration
required
100
0.1 M
0.5
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 1 M solution
4.20
11.50
42.00
8.40
21.00
84.01
12
30
120
Solubility: 10 g per 100 ml
Hazard warning
label
-
Procedure
• Measure out the indicated quantity of sodium hydrogencarbonate.
• Add the solid to about two thirds of the final volume of water in a beaker or laboratory jug.
• Stir to dissolve. Do not warm as decomposition may take place.
• 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.
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.
General Hazards
See Hazcard 12.
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 water’s 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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Sodium hydroxide
Solutions absorb carbon dioxide on standing. Solutions lower than 0.1 M are most significantly affected and
should, therefore, be freshly made.
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
General Hazards
Molar mass: 40.00 g mol-1
Solubility: 108 g per 100 ml
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.
Mass (g) of solid to be used
Volume (ml) of solution required
Concentration
required
250
1000
2500
0.01 M
Ten-fold dilution of the 0.1 M solution
0.1 M
Ten-fold dilution of the 1 M solution
0.4 M
4.00
16.00
40.00
1.0 M
10.00
40.00
100.00
5.0 M
50.00
200.00
500.00
Hazard warning
label
IRRITANT
IRRITANT
CORROSIVE
CORROSIVE
Procedure
• Wear chemical-resistant gloves and goggles. Use a fume cupboard if the solid is a fine powder.
• 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.)
• 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. Add a hazard warning if appropriate.
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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
• Wear eye protection.
• 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
• Wear eye protection.
• 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.
94
© CLEAPSS 2011
CLEAPSS Recipe Book
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.)
Molar mass: 248.17 g mol-1
Formula: Na2S2O3.5H20
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
0.5 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
2.48
6.20
24.82
12.41
31.02
124.08
24.82
62.04
248.17
75
188
750
Solubility: 70 g per 100 ml
Hazard warning
label
-
Procedure
• Measure out the indicated quantity of sodium thiosulfate-5-water.
• Add the solid to about two thirds of the final volume of water in a beaker or laboratory jug.
• Stir to dissolve. Do not warm the solution.
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
water to the required level.
• 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.
© CLEAPSS 2011
95
CLEAPSS Recipe Book
88
Stains for bacterial activity
Gram Stain
For details on
using this stain
see Guidance
Leaflet GL 95
To prepare the crystal violet solution, dissolve 2 g of
the dye in 100 ml of industrial denatured alcohol
(Solution A) and 1 g of ammonium ethanedioate in
100 ml of distilled/deionised water (Solution B).
Add 25 ml of solution A to 100 ml of solution B.
To prepare the Safranin counterstain, dissolve 0.5 g of
safranin in 100 ml of distilled/deionised water.
To prepare Gram’s (Lugol’s) iodine, dissolve 1 g of
iodine and 2 g of potassium iodide in 300 ml of distilled
or deionised water.
96
See Hazcards 32, 36A, 40A
and 85.
IDA is HIGHLY FLAMMABLE.
There should be no sources of
ignition in the vicinity
Wear eye protection during the
preparation. Ensure the room
is well ventilated. Wear
chemical-resistant gloves
when preparing the crystal
violet stain.
© CLEAPSS 2011
CLEAPSS Recipe Book
89
Stains for cells
Aceto carmine
Ehrlich’s
haematoxylin
for animal
histology
Add 0.5 g of carmine to 55 ml of purified water in a
conical flask. Boil and add 45 ml of concentrated
ethanoic acid. Plug the flask with cotton wool, boil
again, cool and filter.
Dissolve 0.64 g of haematoxylin in 32 ml of ethanol
heated on a water bath, and filter. Separately, dissolve
0.32 g of aluminium potassium sulfate(VI) in 32 ml of
purified water. Mix this solution with the haematoxylin
solution and add 32 ml of propane-1,2,3-triol (glycerol)
and 4 ml of glacial ethanoic acid. Leave exposed to
daylight for 6 weeks to ‘ripen’ or add 1 ml of 0.2 M
potassium manganate(VII) solution.
Eosin for
cytoplasmic
staining
Dissolve 1 g eosin in 100 ml tap water. Used as a
counterstain to Ehrlich’s haematoxylin. For alcoholic
solution, substitute 75% ethanol for water.
Feulgen's stain
for DNA
Dissolve 0.45 g of basic fuchsin in 87 ml of boiling
water. Cool to 50 °C and add 30 ml of 1 M hydrochloric
acid and 3 g of potassium metabisulfite. Leave in the
dark for 24 hours to bleach. Add 0.5 g of decolourising
charcoal, shake and filter into a stoppered bottle. Store
in a cool, dark place.
Leishman's
stain for blood
cells
Dissolve 0.15 g of the solid stain in 100 ml of
methanol.
Methyl green
pyronin for DNA
and RNA
Methylene blue
for cheek cells
1 g of methyl green pyronin is dissolved in 100 ml of
distilled/deionised water. DNA is stained green and
RNA is stained red.
Wear eye protection, and gloves to avoid staining the
skin. Dissolve 0.1 to 1 g in 100 ml of water.
Orcein acetic
(Acetic orcein)
for
chromosomes
The stock solution contains 2.2 g of orcein dissolved in
100 ml of glacial ethanoic acid. Dilute 10 ml of this
solution with 12 ml of water before use. This diluted
solution does not keep.
Sudan III or IV
solutions for
lipids
Dissolve 0.5 g of dye in 70 ml of ethanol and 30 ml of
water, using a warm water bath, and filter.
© CLEAPSS 2011
97
For ethanoic acid, see
Hazcard 38. Label the stain
CORROSIVE. Wear goggles.
Use a fume cupboard.
For aluminium potassium
sulfate(VI), see Hazcard 3B.
For ethanol; see Hazcard 40A.
For ethanoic acid, see
Hazcard 38. For propane1,2,3-triol, see Hazcard 37. For
ethanoic acid, see Hazcard 38.
For potassium manganate(VII),
see Hazcard 81.
Wear eye protection and
disposable nitrile gloves when
making up the solution.
Label the stain HIGHLY
FLAMMABLE.
Wear eye protection. Low
hazard unless ethanol is used
as the solvent.
For potassium metabisulfite,
see Hazcard 97. For
hydrochloric acid , see
Hazcard 47A. Wear eye
protection. Use a fume
cupboard.
No hazard label is required.
For methanol, see Hazcard 40.
Prepare the solution in a fume
cupboard wearing eye
protection and disposable
nitrile gloves.
Label the solution TOXIC and
HIGHLY FLAMMABLE.
Wear eye protection and
disposable nitrile gloves.
For methylene blue, see
Hazcard 32. The solution is
low hazard.
For ethanoic acid, see
Hazcard 38. Wear goggles and
chemical-resistant gloves.
Use a fume cupboard.
Label the concentrated
solution CORROSIVE.
Ethanol is HIGHLY FLAMMABLE
(see Hazcards 32 and 40).
Label the solution HIGHLY
FLAMMABLE. Wear eye
protection.
CLEAPSS Recipe Book
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
Colloidal
Coomassie Blue
for proteins
Methylene blue
for DNA
Dissolve 0.04 g in 100 ml of 20% ethanol (ie, 0.04%
w/v) to apply after running the gel. Destaining is not
needed.
See
Low hazard.
Low hazard.
www.ncbe.reading.ac.uk/NCBE/SCIENCEYEAR/recipes.html
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.
Dissolve 0.05 g in 200 ml water (ie, make up to
0.025% w/v) to apply after running the gel. Destaining
is needed.
98
See Hazcard 32, The solution
is low hazard.
© CLEAPSS 2011
CLEAPSS Recipe Book
91
Stains for fungal material
Lactophenol
Dissolve 10 g of phenol in 10 ml of water (do not heat).
Add 10 ml of propan-1,2,3-triol (glycerol) and 10 ml
lactic acid.
Cotton blue
(aniline blue) for
fungi
Dilute 8 ml of 0.1% cotton blue solution in lactophenol
and 4 ml of 0.1% basic fuchsin solution in lactophenol
with 88 ml of lactophenol.
© CLEAPSS 2011
99
For propan-1,2,3-triol, see
Hazcard 38C. For phenol, see
Hazcard 70.Wear goggles.
Wear disposable nitrile gloves.
Use a fume cupboard. Label
the solution TOXIC and
CORROSIVE.
See Hazcard 38C.
Fuchsin is HARMFUL. Wear
suitable eye protection and
gloves. Label the solution
TOXIC.
CLEAPSS Recipe Book
92
Stains for metabolic activity
Janus green B
(Diazine green)
for mitochondria
Methylene blue
for cell contents
Neutral red
Resazurin
solution to test
for milk
freshness
TTC, to show
respiratory
activity
Dissolve 0.3 g of the dye in 100 ml of purified water.
Dilute this solution ten times with water before use.
The colour change is blue (with oxygen) to pink
(anaerobic).
See Guidance Leaflet PS 89 for more information.
For living organisms: dissolve 1 g solid in 100 ml water
and add 0.6 g sodium chloride. For dead tissue:
dissolve 0.23 g solid in 23 ml of ethanol and dilute to
100 ml with water.
Dissolve 0.1 g of neutral red in 100 ml of the
appropriate isotonic saline solution (see Recipe Sheet
75). Dilute the solution ten times further with the saline
solution.
Dissolve one tablet or 0.005 g of resazurin powder in
50 ml of water. (1 ml of this is added to 10 ml of a
sample of milk. A colour change from blue to pink to
white indicates how many bacteria are present.)
Dissolve 1 g of 2,3,5-triphenyl tetrazolium chloride
(TTC) in 100 ml of water. (A 0.5% solution is less
expensive and gives just as good results but takes
longer. It works well with maize seedlings.) Produces a
red colouration.
100
Wear eye protection and
disposable nitrile gloves when
making up the solution. No
hazard label is required.
For ethanol, see Hazcard 40.
For methyl blue see Hazcard
32. Wear eye protection and
disposable nitrile gloves.
No hazard label is required.
Wear eye protection and
disposable nitrile gloves.
Resazurin is IRRITANT.
Low hazard.
© CLEAPSS 2011
CLEAPSS Recipe Book
93 Stains for plant material
Aniline
(phenylammonium)
sulfate stain for
lignin
Mix 89 ml of ethanol, 10 ml of 0.05 M sulfuric acid and
1 g of phenylammonium sulfate [aniline sulfate(VI)].
FABIL for plant
tissues
Prepare 3 solutions: 0.5 % solution of aniline blue in
lactophenol, 0.5% solution of basic fuchsin in
lactophenol and a solution containing 0.3 g of iodine
and 0.6 g of potassium iodide in 100 ml of lactophenol.
When required, mix in the proportions of 4:1:5 and
allow to stand overnight. Filter before use.
(Cell contents stain blue, cellulose walls stain light blue
and lignin stains yellow.)
Use 0.01 M iodine solution.
Dissolve 5 g of phloroglucinol (benzene-1,3,5-triol) in
75 ml of ethanol and 25 ml of water. Ligneous tissue
should be well-flooded and staining continued for
about 4 minutes after which 1 drop of concentrated
hydrochloric acid should be added.
Iodine stain
Phloroglucinol
for pentoses
and lignin
Schulze’s
solution for
cellulose
© CLEAPSS 2011
Dissolve by warming 20 g of anhydrous zinc chloride in
8.5 ml of water and allow the mixture to cool. In a
separate container, dissolve 1 g of potassium iodide
and 0.5 g of iodine in 20 ml of water. Add this solution
dropwise to the zinc chloride solution until iodine
precipitate persists on agitation.
101
For phenylammonium
sulfate(VI): see Hazcard 4.
Wear eye protection and
disposable nitrile gloves when
making up the solution.
Label the stain HIGHLY
FLAMMABLE.
For lactophenol, see
Hazcard 38C; for iodine, see
Hazcard 54.
Wear goggles and chemicalresistant gloves.
Label the stain TOXIC.
See Recipe Sheet 50.
Phloroglucinol is an IRRITANT.
Ethanol is HIGHLY FLAMMABLE
(see Hazcards 12, 40 and 47).
Label solution HIGHLY
FLAMMABLE. Wear eye
protection.
Zinc chloride is CORROSIVE and
iodine is HARMFUL (see
Hazcards 54 and 108). Wear
eye protection, and chemicalresistant gloves, and carry out
the procedure in a fume
cupboard. Label the solution
CORROSIVE.
CLEAPSS Recipe Book
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
Procedure to prepare 1000 ml of solution
• Measure 20.433 g of the 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 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
Procedure to prepare 1000 ml of 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.
• 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.
• Place the potassium manganate(VII) in the burette. Titrate against hot standard sodium ethanedioate
solution until there is a permanent mauve colour.
102
© CLEAPSS 2011
CLEAPSS Recipe Book
0.0167 M potassium iodate(V) solution for standardising sodium thiosulfate solution
Procedure to prepare 1000 ml of 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.
• 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.
• 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
Procedure to prepare 1000 ml of 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.
© CLEAPSS 2011
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CLEAPSS Recipe Book
95
Strontium chloride
Strontium chloride hexahydrate will absorb water from the atmosphere.
Formula: SrCl2.6H2O
General Hazards
Molar mass: 266.6 g mol-1
Solubility: 88 g per 100 ml
See Hazcard 19A.
Procedure for preparing 100 ml of 0.1 M strontium chloride solution
• Wear eye protection.
• Dissolve 2.67 g of strontium chloride-6-water in 70 ml of water.
• Make up to 100 ml with pure water.
• The solution is low hazard.
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.
104
© CLEAPSS 2011
CLEAPSS Recipe Book
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.
Use a fume cupboard. Make sure it is working. Wear goggles.
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
2 M hydrochloric acid
Acidified potassium
dichromate(VI) paper
Loose mineral
wool plug
Heat
© CLEAPSS 2011
105
CLEAPSS Recipe Book
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
2 M hydrochloric acid
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.
106
© CLEAPSS 2011
CLEAPSS Recipe Book
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
General Hazards
Concentration
required
0.01 M
0.1 M
0.4 M
1M
Battery acid
5M
Molar mass: 98.07 g mol-1
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.
Volume (ml) of solution required
500
1000
2500
Ten-fold dilution of a 0.1 M solution with water
Ten-fold dilution of a 1 M solution with water
11
22
54
27
54
136
115
230
575
135
270
680
Hazard warning
label
IRRITANT
CORROSIVE
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.
• Pour the solution from the beaker into an appropriate measuring cylinder or laboratory jug and add
water to the required level.
• Pour the solution into a labelled bottle and mix well. Add a hazard warning if appropriate.
© CLEAPSS 2011
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CLEAPSS Recipe Book
99
Testing for gases
Ammonia
Use damp red litmus
paper
Place concentrated
hydrochloric acid close by
Damp red litmus goes blue. Universal indicator can
be used as well.
White smoke of ammonium chloride (IRRITANT) forms.
Wear eye protection.
See Recipe Sheet 20 for calcium hydroxide. A white
precipitate of calcium carbonate forms which, in time,
goes colourless as calcium hydrogencarbonate
forms.
Wear eye protection if
blowing into limewater
through a straw.
The gas catches fire with a blue flame forming
carbon dioxide.
Wear eye protection.
Damp blue litmus goes red on formation of chloric(I)
acid. This then bleaches the paper.
Wear eye protection.
Use a fume cupboard.
The gas supports combustion and can relight a
glowing splint, although it is not as active as oxygen.
Wear eye protection.
If the hydrogen is mixed with air there is a ‘pop’ but if
the gas is pure it catches fire and there is no ‘pop’.
Wear eye protection.
Damp blue litmus goes red. Universal indicator can
be used as well.
White smoke of ammonium chloride forms.
Wear eye protection.
The paper immediately turns black with the formation
of lead sulfide.
Wear eye protection.
Use tongs or forceps to
hold the paper.
Wear eye protection.
Carbon dioxide
Use lime water
Carbon monoxide
Light it in a fume
cupboard
Chlorine
Use damp blue litmus
paper
Dinitrogen monoxide
Add a lighted or glowing
splint
Hydrogen
Add a lighted splint
Hydrogen chloride
Use damp blue litmus
paper
Place concentrated
ammonia solution close
by
Wear eye protection.
Hydrogen sulfide
Moisten filter paper with
0.05 M lead(II) nitrate(V)
solution
Nitrogen
There is no positive test for nitrogen.
Nitrogen dioxide
Observe the colour
The only brown gas there is.
Wear eye protection.
Brown nitrogen dioxide is formed.
Wear eye protection.
This relights a glowing splint, sometimes with a ‘pop’.
Wear eye protection.
The paper goes green with the formation of
chromium(III) ions.
Wear eye protection.
Use a fume cupboard.
Nitrogen monoxide
Add the gas to air
Oxygen
Add a glowing splint
Sulfur dioxide
Moisten filter paper with
0.05 M acidified
potassium dichromate(VI)
solution
108
© CLEAPSS 2011
CLEAPSS Recipe Book
100 Testing for negative ions
Bromide ions
0.05 M silver nitrate(V)
solution
Dissolve 0.85 g of silver nitrate(V) in 100 ml of
distilled/deionised water. A cream precipitate forms which
is soluble after the addition of concentrated ammonia.
See Hazcard 6 & 87.
Wear eye protection.
Adding the acid produces a lot of fizzing. The carbon
dioxide should be identifiable with lime water.
See Hazcard 47A.
Wear eye protection.
Dissolve 0.85 g of silver nitrate(V) in 100 ml of water,
which must be pure. A white precipitate forms which is
soluble after the addition of 2 M ammonia solution.
See Hazcard 6 & 87.
Wear eye protection.
Carbonate ions
2 M hydrochloric acid
Chloride ions
0.05 M silver nitrate(V)
solution
Ethanoate and methanoate ions
0.1 M iron(III) chloride
solution
Add 2 M ammonia solution to 0.1 M iron(III) chloride
solution until a precipitate just appears. Now add more of
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.
See Hazcard 6 & 55C
Wear eye protection.
Dissolve 0.85 g of silver nitrate(V) in 100 ml of
distilled/deionised water. A yellow precipitate forms which
is not soluble after the addition 2 M ammonia solution.
See Hazcard 6 & 87.
Wear eye protection.
Iodide ions
0.05 M silver nitrate(V)
solution
Nitrate(III) (nitrite) ions
2 M hydrochloric acid
Adding the acid produces a pale blue liquid and a brown
gas (nitrogen dioxide, TOXIC). Immediately, add water to
quench the reaction.
See Hazcard 47A, 68
and 93. Wear eye
protection.
Dissolve the nitrate(V) in about 1 ml of water and add
0.5 g of iron(II) sulfate(VI)-7-water. Hold the test tube at
an angle and carefully pour about 1 ml of concentrated
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.
Add 2 ml of suspected nitrate(V) to 5 ml of 1 M sodium
hydroxide solution. Add a little Devarda’s alloy (contains
aluminium, copper and zinc) and warm. Test for ammonia
gas.
Wear goggles.
See Hazcards 55B &
98.
Dissolve 4 g of ammonium molybdate(VI) in 4 ml of
concentrated ammonia and 6 ml of water. Add 12 g of
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.
See Hazcards 6, 8, 9A
& 67.
Dissolve 2.44 g in 100 ml of distilled/deionised water. A
white precipitate is formed. Sulfate(IV) ions do not give a
precipitate but it can form in time due to oxidation by
dissolved oxygen.
See Hazcard 10A.
Nitrate(V) ions
Brown ring test
Devada’s alloy
Wear goggles.
See Hazcard 1 & 91.
Phosphate ions
0.5 M ammonium
molybdate(VI) solution
(HARMFUL)
Sulfate(VI) ions
0.1 M barium chloride
solution (HARMFUL)
© CLEAPSS 2011
109
CLEAPSS Recipe Book
101 Testing for positive ions
Metal ions in general
Reagent
1 Flame tests
2 Add 0.4 M
sodium
hydroxide to a
small amount of
the solution
under test
3 0.1 M sodium,
potassium or
ammonium
sulphide
solution
Recipe and observations
Barium ions
Greenish flame
Calcium ions
Brick red flame
Copper(II) ions
Bluish flame
Lead(II) ions
White flame
Lithium
Crimson red flame
Potassium ions
Lilac flame (best seen thro’ cobalt glass)
Rubidium ions
Blue flame
Strontium ions
Crimson flame
Sodium ions
Persistent yellow flame.
Dissolve 1.6 g of sodium hydroxide in 100 ml of water.
Precipitates form, some coloured and others colourless,
some of which dissolve in excess alkali solution.
Aluminium ions
White precipitate which dissolves in
excess alkali
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
Dissolve 2.4 g of sodium sulfide-9-water or 1.0 g of
potassium sulfide in 100 ml of water. 4 ml of ammonium
sulfide solution can be diluted to 100 ml with water. Some
metal ions form precipitates with the solutions. Refer to an
(old!) A-level text for details.
Hazards and notes
See Hazcards 10A,
19A & 47B.
The Hazcards also
have the procedure for
the tests. There is also
the borax bead test on
Hazcard 14.
See Hazcard 91.
The concentration of
the metal solutions
needs to be 0.1 M.
See Hazcard 51. The
test solutions are
IRRITANT. Use a fume
cupboard as the odour
is repulsive.
Aluminium ions
Alizarin solution
Dissolve 0.1 g of alizarin in 100 ml of water. Addition of 2 M
ammonia solution to an aluminium salt in the presence of
alizarin gives a pink precipitate or ‘lake’.
See Hazcard 32.
Ammonium ions
0.1 M sodium
hexanitrocobalt-ate(III)
solution (HARMFUL)
Dissolve 4.04 g in 100 ml of water. A yellow precipitate
forms with ammonium ions.
0.4 M sodium
hydroxide solution
(IRRITANT)
Warm with the test solution. Ammonia gas will be given
off.
See Hazcard 95A. It is
an unstable solution
with a maximum shelf
life of 3 weeks.
Wear eye protection.
Calcium ions
0.25 M ammonium
ethanedioate
Dissolve 3.5 g of ammonium ethanedioate in 100 ml of
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.
110
See Hazcard 36A.
© CLEAPSS 2011
CLEAPSS Recipe Book
Cobalt(II) ions
Thiooxamide
(rubeanic acid)
(HIGHLY FLAMMABLE,
HARMFUL)
0.1 M ammonium or
potassium thiocyanate
Dissolve 0.5 g in 100 ml of ethanol. A brown precipitate is
produced with cobalt ions.
See Hazcards 35 and
also 40A.
Dissolve 0.76 g of ammonium thiocyanate or 0.97 g of
potassium thiocyanate in 50 ml of water and make up to
100 ml. A blue solution forms with cobalt(II) ions.
See Hazcard 9B or
95C.
Dilute 11 ml of 880 ammonia to 100 ml with water. A blue
precipitate is formed which dissolves in excess ammonia
solution to form a deep blue solution.
Dissolve 0.5 g in 100 ml of ethanol. A black precipitate
forms with copper(II) ions.
See Hazcards 6.
Prepare in a fume
cupboard.
See Hazcards 35 and
also 40A.
Dissolve 0.76 g of ammonium thiocyanate or 0.97 g of
potassium thiocyanate in water 50 ml of water and make
up to 100 ml. A black precipitate forms.
See Hazcard 9B or
95C. The solution is
LOW HAZARD.
0.1 M potassium
hexacyanoferrate(III)
Dissolve 3.29 g in 100 ml of water. A light blue precipitate
forms.
1,10 phenanthroline
solution
Dissolve 1.49 g of 1,10 phenanthroline in 100 ml of water.
The reagent gives a red colour when mixed with iron (III)
ions.
See Hazcard 79. No
hazard warning is
required on the
solution.
See Hazcard 55B.
Solid Phenanthroline is
TOXIC if swallowed. No
hazard warning is
required on the
solution.
Copper(II) ions
2M ammonia solution
Thiooxamide
(rubeanic acid)
(HIGHLY FLAMMABLE,
HARMFUL)
0.1 M ammonium or
potassium thiocyanate
Iron(II) ions
Iron(III) ions
0.1 M potassium
hexacyanoferrate(III)
Dissolve 3.29 g in 100 ml of water. A dark blue precipitate
forms.
Ferroxyl indicator
(HIGHLY FLAMMABLE)
Dissolve 2 g of sodium chloride, 0.1 g potassium
hexacyanoferrate(III) and 1.0 g of phenolphthalein in
50 ml of ethanol, and mix with 100 ml of water. Very
useful for detecting the rusting process.
Dissolve 0.76 g of ammonium thiocyanate or 0.97 g of
potassium thiocyanate in 50 ml of water and make up to
100 ml. A dark red solution forms. [NB there is no
colouration with iron(II) ions]
0.1 M ammonium or
potassium thiocyanate
See Hazcard 79. No
hazard warning is
required on the
solution.
See Hazcards 32, 40A
and 79.
See Hazcard 9B or
95C. The solution is
LOW HAZARD.
Lead(II) ions
Dithizone
Dissolve 0.005 g in 100 ml of dichloromethane or
methylbenzene. The reagent gives a red colouration with
lead(II) ions.
Potassium iodide
Prepare a 0.1 M solution. A bright yellow precipitate
forms.
© CLEAPSS 2011
111
See Hazcards 28, 32
and 46. Label the
solution according to
which solvent is used.
See Hazcard 47B.
CLEAPSS Recipe Book
Nickel(II) ions
Thiooxamide
(rubeanic acid)
(HIGHLY FLAMMABLE,
HARMFUL)
Butanedione dioxime
(dimethylglyoxime)
(HIGHLY FLAMMABLE &
HARMFUL)
Dissolve 0.5 g in 100 ml of ethanol. A violet precipitate
forms.
See Hazcards 35 and
also 40A.
Dissolve 1 g in 100 ml of ethanol. A red precipitate forms.
See Hazcard 53.
Dissolve 4.04 g in 100 ml of water. A yellow precipitate
forms.
See Hazcard 95A. It is
an unstable solution
with a maximum shelf
life of 3 weeks.
Dilute 11 ml of 880 ammonia to 100 ml with water. A white
precipitate forms, which dissolves in excess ammonia
solution to form a colourless solution.
See Hazcards 6.
Prepare in a fume
cupboard. The solution
is LOW HAZARD.
Potassium ions
0.1 M Sodium
hexanitrocobalt-ate(III)
(HARMFUL)
Zinc(II) ions
2M ammonia solution
112
© CLEAPSS 2011
CLEAPSS Recipe Book
102 Testing for organic functional groups
Alcohols - primary, secondary & tertiary
Lucas reagent
(CORROSIVE)
Potassium
dichromate(VI) test
Potassium
manganate(VII)
test
Dissolve 68 g of anhydrous zinc chloride in 43 g of concentrated
(36%) hydrochloric acid. The time taken for turbidity to appear is
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.
Identifies primary and secondary alcohols. To five drops of the
alcohol, add 10 drops of 1 M sulfuric(VI) acid and 4 drops of 0.1 M
potassium dichromate(VI) solution. Place the solution in boiling
hot water from a kettle. The colour change is orange to green.
Identifies primary and secondary alcohols. To five drops of the
alcohol, add 10 drops of 1 M sulfuric(VI) acid and 4 drops of
0.002 M potassium manganate(VII) solution. Place the solution in
boiling hot water from a kettle. The colour change is purple to
colourless.
See Hazcards
47A & 108A.
See Hazcard 78 &
98A.
See Hazcard 81 &
98A.
Aldehydes and ketones
Hydrogen sulfite
test
Tollen’s reagent
Schiff’s reagent
The iodoform test
Dissolve 20 g of sodium metabisulfite in 100 ml of hot water.
White precipitate appears with the reagent is added to a carbonyl
compound.
It does not work with ketones. This reagent is ammoniacal silver
nitrate(V), it must be made immediately before use and not stored.
The breakdown products are EXPLOSIVE. Add one drop of 0.4 M
sodium hydroxide solution to 1 ml of 0.1 M silver nitrate(V). Add
1 M ammonia solution until the precipitate just redissolves. On
warming with an aldehyde, a silver mirror forms.
It does not work quickly with ketones. Dissolve 0.1 g of fuchsin in
100 ml of water and add 0.9 g of sodium metabisulfite followed by
2 ml of 2 M nitric(V) acid, so it is colourless. Addition of an
aldehyde restores the colour. Be aware that sulfur dioxide, a TOXIC
gas, may be evolved.
Works with methyl ketones (CH3CO-), methyl secondary alcohols,
ethanol and ethanal. To fives drops of the liquid under test, add 5
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.
See Hazcard 92.
See Hazcard 87.
Immediately
dispose of liquid
after use down
the foul-water
drain.
See Hazcards 32,
67 & 92.
Wear eye
protection.
See Hazcards
54A & 91.
Carboxylic acids
‘Neutral’ iron(III)
chloride
© CLEAPSS 2011
Add 2 M ammonia solution to 0.1 M iron(III) chloride solution until
a precipitate just appears. Now add more of the original iron(III)
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.]
113
See Hazcards 6 &
55C.
CLEAPSS Recipe Book
Double bonds or unsaturation
Bromine water
Potassium
manganate(VII)
(acidified)
Potassium
manganate(VII)
(alkaline)
Add 0.002 M bromine water which will go clear.
Add an acidified 0.002 M solution of potassium manganate(VII)
which will go clear. If it goes brown, it is not acidified enough.
See Hazcard 15B.
See Hazcards 81
and 98.
Alkaline potassium manganate(VII) solution is not stable so it
must be made in situ. Add enough solid anhydrous sodium
carbonate to make the acidified solution alkaline. Add alkaline
0.002 M solution of potassium manganate(VII) which should go
green.
See Hazcards 81
and 98.
OH groups in alcohols and acids
Phosphorus
pentachloride
To about 2 ml of the liquid or solid under test, add phosphorus
pentachloride with a small spatula. There is a vigorous reaction
and hydrogen chloride is produced.
See Hazcard 74.
Add 2 M ammonia solution to 0.1 M iron(III) chloride solution until
a precipitate just appears. Now add more of the original iron(III)
chloride in drops with stirring until the precipitate just dissolves.
Add this to the material under test, and look for a purple
colouration.
See Hazcards 6 &
55C.
Phenol
‘Neutral’ iron(III)
chloride
114
© CLEAPSS 2011
CLEAPSS Recipe Book
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.
Molar mass: 225.63 g mol-1
Formula: SnCl2.2H20
General Hazards
See Hazcards 102 & 47A.
Mass (g) of solid to be used
Concentration
required
100
0.01 M
0.1 M
Solubility: 84 g per 100 ml
Volume (ml) of solution required
250
1000
Ten-fold dilution of the 0.1 M solution
2.26
5.64
22.56
Hazard warning
label
-
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.
•
•
•
•
•
•
•
•
•
B.
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)
Permanent hard water
•
C.
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.
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 Winkler’s 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 Winkler’s method
• Wear goggles and chemical-resistant gloves. Consider using a fume cupboard.
100 ml of 8 M
• Add 8 g of sodium hydroxide pellets to 70 ml of water in a 250 ml beaker.
sodium hydroxide
• 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.
• Dissolve 50 g of manganese(II) sulfate(VI)-4-water or 45 g of manganese(II)
The 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
•
The indicator
•
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.
Prepare 1% starch solution. See Recipe Sheet 49.
Winkler’s 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
CLEAPSS Recipe Book
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.
Molar mass: 287.54 g mol-1
Formula: ZnSO4.7H2O
General Hazards
Solubility: 96 g per 100 ml
Zinc sulfate(VI) is HARMFUL. See Hazcard 108B.
Mass (g) of solid to be used
Concentration
required
0.1 M
1.0 M
Saturated (20 °C)
Volume (ml) of solution required
100
250
1000
2.88
7.19
28.75
28.75
71.88
287.54
100
250
1000
Hazard warning
label
HARMFUL
HARMFUL
Procedure
• Measure out the indicated quantity of hydrated zinc sulfate(VI).
• 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.
• 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.
• Pour into a labelled bottle.
118
© CLEAPSS 2011
CLEAPSS Recipe Book
Index
Name
Name
Recipe
Sheet
Recipe
Sheet
Acetic acid
39
Aniline blue
91
Acetic alcohol
41
Aniline sulfate(VI)
93
Acetic orcein
89
74
Aceto carmine
89
Acid atmosphere
97
Animal tissue
preservative
Anion tests
100
Acid base indicators
46
Aspirator
20
Acid rain
97
Azo dyes
9
Agar
1
Azure A
90
Alcohol tests
102
Alcohol/water solutions
2
Alcoholic potassium
hydroxide
Alcoholic solutions
71
2
Aldehyde tests
102
Alginate beads
3
Alizarin solution
101
Alkaline pyrogallol
84
Alum
4
Aluminium chloride
4
Aluminium etching
38
Aluminium potassium
sulfate(VI)
4
Aluminium solutions
4
Amino acid
chromatography
Amino acid tests
26
Barium chloride
10
Barium diphenylamine4-sulfonate indicator
49
Barium nitrate(V)
10
Barium solutions
10
Basic agar
1
Belousov-Zhabotinski
reaction
Benedict’s qualitative
reagent
63
Benedict’s quantitative
reagent
Bile salts
12
Biological buffers
18
Biro ink
chromatography
Bismuth nitrate(V)
26
11
37
14
Biuret reagent
15
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)
sulfate(VI)
22
Brady’s reagent
33
Briggs-Rausher reaction
63
Ammonium chloride
7
Brodie’s fluid
16
Bromide ion test
100
Ammonium
ethanedioate
Ammonium hydroxide
101
Ammonium iron(III)
citrate
Ammonium iron(III)
sulfate(VI)
Ammonium sulfide
52
101
Ammonium thiocyanate
101
6
52
Ammonium vanadate
8
Amylase
37
Analgesics
chromatography
Andrews arrangement
for gas preparation
26
© CLEAPSS 2011
24
119
Bromine water
17
Bromocresol green
46
Bromophenol blue
46
Bubble mixture
79
Buffers
18
Butanedione dioxime
101
B-Z reaction
63
Calcium carbonate
(standard solution)
Calcium chloride
94
19
Calcium hydroxide
20
Calcium nitrate(V)
19
CLEAPSS Recipe Book
Name
Name
Recipe
Sheet
Recipe
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
preparation
Carbon monoxide test
42
Cytological fixative
41
99
DCPIP
13
Carbonate ion test
100
51
Carbonyl group test
33
Diammonium iron(II)
sulfate(VI)
Diazine green
Carboxylic acid test
102
Diazonium salts
9
Cation tests
101
13
Cellular respiration
13
dichlorophenol indophenol
Cerium(IV) solutions
22
49
Cerium(IV) sulfate(VI)
22
Dichromate(VI) titration
indicators
Cheek cells
89
Dimethylglyoxime
101
Chemical garden
86
42
Chemiluminesence
23
China blue agar
1
Chloride ion test
100
Dinitrogen monoxide
preparation
Dinitrogen monoxide
test
Dinitrophenylhydrazine
33
Chlorine gas
preparation
Chlorine test
24
Dinitrosalicylic acid
34
Dipotassium hydrogen
phosphate(V)
67
Chlorine water
25
67
Chlorophyll
chromatography
Chromatography
solvents
Chrome alum
26
Dipotassium hydrogen
phosphate(V) -3- water
Disappearing cross
reaction
Disinfectants
29
27
67
Chromium(III) chloride
27
Disodium hydrogen
phosphate(V)
Citric acid
28
Disodium hydrogen
phosphate(V) -7- water
67
2,6-
2,3,53,5-
99
26
92
99
81
Clarke’s fluid
41
Dissolved oxygen
105
Clarke’s soap solution
79
Dithizone
101
Clock reactions
29
DNP
33
Cobalt(II) chloride
30
Cobalt(II) nitrate(V)
30
DNS
34
Cobalt(II) thiocyanate
30
DNSA
34
Cole’s reagent
13
Double bond test
102
Colloidal Coomassie
blue
Complexometric
indicators
Coomassie blue
90
Drosophila food base
35
Dyes
9
Dying cotton
9
EDTA titration indicators
49
90
Ehrlich’s 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
2,4-
49
120
© CLEAPSS 2011
CLEAPSS Recipe Book
Name
Recipe
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
hydroxide
71
Iodoform test
102
Iron etching
38
Iron(II) chloride
51
Iron(II) solutions
51
Iron(II) sulfate(VI)
51
FAA
2-
Name
Recipe
Sheet
41, 74
FABIL
93
Fehling's solution
40
Ferroin indicator
49
Ferroxyl agar
1
Iron(III) chloride
52
29
Ferroxyl indicator
101
Iron(III) ions and iodide
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
Fluorescein indicator
49
Formaldehyde
58
Formalin alcohol fixative
41
Kaiserling’s
preservative
Ketone tests
Formalin-aceto fixative
41
Formalin-aceto-alcohol
74
Formic acid
59
Glucose nutrient agar
1
Glucose quantitative
test
Glucose test
12
11, 34
13, 92
74
102
Knop’s culture solution
66
Lactophenol
91
Landolt iodine reaction
29
Lead etching
38
Lead nitrate(V)
53
Leishman's stain
89
Lime water
20
Gram Stain
88
Lipase
37
Harcourt-Essen reaction
29
26
Hard water
104
Lipstick
chromatography
Lithium chloride
Home-made indicators
46
Litmus
46
Hydrochloric acid
43
Locating agents
26
Hydrogen
99
Lucas reagent
102
Hydrogen carbonate
indicator
Hydrogen chloride
preparation
Hydrogen chloride test
48
Lugol’s iodine
88
Luminol
23
Magnesium sulfate(VI)
55
99
Malt agar
1
Hydrogen gas
44
56
Hydrogen peroxide
45
Manganese(II)
sulfate(VI)
Manometric fluid
Hydrogen peroxide/
potassium iodide
reaction
Hydrogen sulfide
preparation
Hydrogen sulfide test
29
Mayonnaise agar
1
Mercury(I) nitrate(V)
57
42
Mercury(II) chloride
57
Hydrogen-sulfite test
102
Metal ion
chromatography
Methanal solution
26
99
58
hydroxy-1,2,3tricarboxylic acid
27
Methanoate ion test
100
Methanoic acid
59
Immobilised enzymes
3
Methanol solution
2
© CLEAPSS 2011
42
121
54
16
CLEAPSS Recipe Book
Name
Methanolic potassium
hydroxide
71
Methyl benzoate
nitration
Methyl green pyronin
26
89
Methyl orange
46
Methyl orange
preparation
Methyl red
9
Methylene blue
Milk agar
46
13, 89, 90,
92
1
Millon’s reagent
13
Mohr’s salt
51
Molisch’s solution
13
Murexide
49
Neutral Red
46, 92
Neutral iron(III) chloride
102
Nickel electroplating
36
Nickel(II) sulfate(VI)
60
Ninhydrin
13
Nitrate(III) ion test
100
Nitrate(V) test
100
Nitration of Methyl
benzoate
Nitric(V) acid
26
Nitrogen preparation
42
Nitrogen test
99
Nitrogen dioxide
preparation
Nitrogen dioxide test
42
61
99
nitrogen gas oscillating
reaction
63
Nitrogen monoxide
preparation
Nitrogen monoxide test
42
99
N-phenylanthranilic acid
indicator
49
Nutrient agar for
bacteria
Nylon rope experiment
1
62
OH group test
102
Orange II preparation
Name
Recipe
Sheet
9
Orcein acetic
90
Organic functional
group tests
Orthophosphoric acid
102
Oscillating reactions
63
Oxygen preparation
64
Oxygen test
99
65
Oxygen levels in water
105
PAS reaction
13
Pepsin
37
Periodic acid Schiff
reaction
Permanently hard water
pH buffers
13
104
18
pH probe calibration
buffers
Phenanthroline solution
101
Phenol red
46
phenolindodichlorophenol
Phenolphthalein
13
18
46
Phenolphthalein
indicator agar
Phenylammonium
sulfate(VI)
Phenylthiocarbamide
13
Phenylthiourea
13
Phloroglucinol
93
Phosphate(V) ion test
100
Phosphoric(V) acid
65
Phosphorus
pentachloride
Physiological saline
solutions
Plant mineral
requirement solutions
102
Plant tissue
preservative
Polypeptide tests
74
Potassium chloride
68
Potassium chromate
69
Potassium
dichromate(VI)
Potassium dihydrogen
phosphate(V)
70
Potassium
hexacyanoferrate(III)
Potassium hydrogen
phthalate (standard
solution)
Potassium hydroxide
101
Potassium iodate
(standard solution)
Potassium
iodate/sodium
metabisulfite reaction
Potassium iodide
94
Potassium
iodide/potassium
persulfate clock reaction
122
Recipe
Sheet
1
93
75
66
13
67
94
71
29
72, 101
29
© CLEAPSS 2011
CLEAPSS Recipe Book
Name
94
Sodium
hexanitrocobaltate(III)
Sodium
hydrogencarbonate
Sodium hydroxide
101
Sodium hydroxide test
101
2
Sodium hypochlorate
81
13
Sodium phosphate(V)
67
13
Sodium phosphate(V)
-12- water
67
101
Potassium thiocyanate
101
Precipitation indicators
49
Preservatives for animal
tissues
74
Propanone solution
Protein tests
73
67
PTU
13
PVA
78
Pyrogallol
84
Red cabbage indicator
46
Redox indicators
Reducing sugar test
Recipe
Sheet
Sodium ethandioate
(standard solution)
Sodium ethanoate
Potassium
manganate(VII)
Potassium
phosphate(V)
Potassium sulfide
PTC
Name
Recipe
Sheet
83
84
85
Sodium silicate
86
Sodium sulfide
101
37
49
Sodium
tauroglycocholate
Sodium thiosulfate
11, 12
87
Solochrome black
49
Resazurin solution
93
Stains for cells
89
Ringer’s solution
75
101
Sach’s culture solutions
66
Safranin counterstain
88
Sakaguchi test
13
Saliva
37
Stains for
electrophoresis
Stains for fungal
material
Stains for metabolic
activity
Stains for plant material
90
Rubeanic acid
93
Sandell’s solution
76
Standard solutions
94
Schiff’s reagent
102
Starch agar
1
Schulze’s solution
93
Starch indicator
49
Screened methyl
orange
Sea water
46
Starch malt agar
1
104
Silicate gels
86
Silver electroplating
36
Silver nitrate(V)
77
Silver nitrate(V) titration
indicators
91
92
Sterilising conditions
1
Strontium choride
95
Sudan III or IV solutions
89
Sugar chromatography
26
Sulfate(IV) ion test
100
49
Sulfate(VI) ion test
100
78
Soap
79
Sulfur dioxide
preparation
Sulfur dioxide test
96
Slime
Sodium carbonate
80
Sulfur dioxide solution
97
94
Sulfuric(VI) acid
98
Technical agar
1
Sodium carbonate
(standard solution)
Sodium chlorate(I)
81
Sodium chloride
82
Sodium dihydrogen
phosphate(V)
67
Sodium dihydrogen
phosphate(V) -1- water
67
Sodium diphenylamine4-sulfonate indicator
49
© CLEAPSS 2011
123
99
Temporary hard water
104
Tests for gases
99
Tests for negative ions
100
Tests for positive ions
101
Thermochromic liquid
30
Thiooxamide
101
CLEAPSS Recipe Book
Name
Thiosulfate/acid
reaction
Thiosulfate/iodine
titration indicator
2,3,5-
Name
Recipe
Sheet
Recipe
Sheet
29
Unsaturation in organic
chemistry
102
49
Vitamin C test
13
Wanklyn’s soap solution
79
Washing soda
80
Water
104
Water solutions for
plants
Winkler’s method
66
105
Yamada indicator
47
Zinc electroplating
36
Zinc sulphate
106
Thymol blue
46
Thymolphthalein
46
Tin(II) chloride
Tollen’s reagent
103
102
Triphenyl tetrazolium
chloride
Trypsin
13
TTC
13
Universal buffer
18
Universal indicator
47
37
124
© CLEAPSS 2011