Qualitative Analysis Reagents
Preparation of laboratory reagents and solutions used in qualitative analysis. Bench reagents consist of a stack of acids and bases on each bench top. Side bench and Special reagents
are placed away from the work bench.
A lab will only put out those reagents and solutions that are necessary for the determinations being carried out on that day.
LIST OF SIDE BENCH REAGENTS
If reagent not found in this list check the alphabetical listing below.
Dissolve 231 g of the salt in 1 liter of water. (3M)
The commercial salt is a mixture of bicarbonate
and carbamate. Dissolve 192 g of the salt in a mixture of 140ml conc.
ammonia solution and water to make up 1 liter of solution. (2M)
dissolve 169 g of the salt in 1 liter of solution (3M).
dissolve 80 g of the salt in 1 liter of water. (1M)
dissolve 71 g of the crystaline salt in 1 liter of water.(0.5M)
dissolve 132 g of the salt in 1 liter of water.(1M)
use the yellow commercial product
Dissolve38 g of the salt in 1 liter of water (0.5M)
dissolve 61 g of the salt in 1liter of water.(0.25M)
a saturated aqueous solution is prepared by shaking 35g or 11ml liquid bromine with water. Add more bromine if necessary to a slight excess.
dissolve 55 g of the hydrated salt in 1 liter of water (0.25M)
shake 3 g of the salt with 1 liter of water, filter and decant the saturatrd solution after several hours (0.015M)
saturate 250ml of water with chlorine.the chlorine may be prepared by dropping conc. HCl upon KmnO4. Preserve in a dark coloured bottle.(6.8g/l)
dissolve 44 g of the salt in 1 liter of water. (0.15M)
dissolve 125 g of the salt in 1 liter of water containing 3ml of conc. Sulphuric acid.(0.5M)
dissolve 135 g of the hydrated salt in 1 liter of water containing 20ml of conc. HCl (0.5M)
dissolve 140 g of the salt in 1 liter of water containing 7 ml of conc. Sulphuric acid (0.5M)
H2S generated from a Kipps apparatus ( ~42g/l)
dissolve 12.7 g of iodine in a solution of 20g of pure KI in 30 ml of water, and dilute to 1 liter with water.(0.05m)
dissolve 95 g of the salt in 1 liter of water (0.25M)
dissolve 62 g of the salt in 1 liter of water (0.25M)
Magneson (4-(4-nitrophenylazo) resorcinol
0.001% in 1M NaOH.
dissolve 27 g of the salt in 1 liter of water (0.1M)
dissolve 49g of the salt in 1 liter of water (0.25M)
dissolve 55 g of the salt in 1 liter of water (0.25M)
dissolve 53 g of the salt in 1 liter of water (0.125M)
dissolve 83 g of the salt in 1 liter of water (0.5M)
dissolve 3.2g of the salt in hot water, dilute to 1 liter, and filter through glass wool (0.01M)
dissolve 49g of the salt in 1 liter of water (0.25M)
dissolve 17g of the salt in 1 liter of water (0.1M)
dissolve 8g of the salt in 1 liter of water. this is nearly a saturated solution (0.025M)
dissolve 408g of the crystaline salt in 1 liter of water (3M)
dissolve 430g of the crystaline salt in 1 liter of water (1.5M)
Disodium hydrogen phosphate
dissolve 120g of the salt in 1 liter of water 0.34M)
dissolve with heat 56g of the salt in 100ml conc. HCl and dilute to 1 liter. Keep a few pieces of granulated zinc in the stored solution to avoid oxidation. (0.25M)
10g in 100ml of water (10%)
dissolve 1g in 100ml of water (1%)
dissolve 150g of the salt in 1 liter of water (0.5M)
LIST OF SPECIAL REAGENTS
Use a 0.1% aqueous solution.
Make a saturated solution in ethanol.
Sodium alizarin sulphate
Use a 0.1% aqueous solution.
Dissolve 0.2 g in 195 ml of sodium bisulphite solution (3 in20) and 5 ml of anhydrous sodium sulphite solution (1 in 5) and filter if necessary. Stopper and store in a cool dark place. Use within 10 days.
Ammoniacal silver nitrate.
Add conc ammonia to bench silver nitrate (0.1M) until the initially formed ppt. Just disappears.
Ammonium citrate, lead free
Dissolve 40g of citric acid in 100 ml of water and make alkaline to phenol red with ammonium hydroxide. Remove lead by shaking with small portions of dithizone extraction solution in chloroform until the dithizone solution retains its original green color.Discard the extraction solution.
Dissolve 9g of ammonium thiocyanate and 8g of mercuric chloride in 100ml of watwer.
Dissolve 2.5g of NH4VO3 in 500 ml of boiling water, cool, and add 20 ml of nitric acid. Dilute to 1 liter and store in polythene bottle.
Ammonium molybdate reagent
Method A: Dissolve 45 gms. of the commercial salt or 40 gms. of pure molybdenum trioxidein a mixture of 70 ml.of concentrated ammonia solution and 140 ml. of water; when solution is complete, add it very slowly and with vigorous stirring to a mixture of 250ml.of concentrated nitric acid and 500ml.of water, and dilute to 1 litre.Allow to stand 1 to 2 days and decant and use the clear solution.
Method B: Dissolve 45 gms. of pure commercial ammonium molybdate in mixture of 40ml. concentrated ammonia solution and 60 ml. of water, add 120gms. of ammonium nitrate and dilute to a litre with water.
Note: The alkaline solution of ammonium molybdate keeps better than the nitric acid solution; there is little tendency for the separation of solid. Before using the alkaline solution, it is important that the test solution contains a slight excess of nitric acid.
Ammoniacal silver nitrate
Add ammonia dropwise to a 1 in 20 solution of silver nitrate until the ppt. that first forms is almost , but not entirely, dissolved. Filter and store in dark bottle.Forms explosive compounds on standing! Prepare fresh.
To 2.5 g of sulfanilic acid add 15 ml water and 3 ml ammonia and mix. If necessary add more ammonia until the the acid dissolves. Adjust the pH to 4.5 with dilute HCl and dilute to 25 ml.
To 0.2 g of amylase crystal, add 100ml water, shake well and filter. Prepare fresh.
Dissolve 0.5g in 100 ml ethanol
Dissolve about 0.1 g in 100 ml sulphuric acid. Prepare fresh.
Mix one part by volume of conc. nitric acid with three (3) parts by volume of conc. hydrochloric acid in a pyrex beaker and allow to stand until a bright red color develops.
Dissolve 13.3gm.of crystalised neutral copper acetate in 200ml.of 1% acetic acid solution. This reagent does not keep well.
Bradys reagent (2,4 DNPH)
Dissolve 40g of 2, 4 dinitrophenylhydrazine in 80ml conc. Sulphuric acid. Cool and add 900ml methanol and 100ml water.
Benedicts solution (qualitative)
Dissolve 86.5gm.of sodium citrate and 50gm. of anhy. sodium carbonate in about 350ml. of water. Filter if necessary. Add a solution of 8.6gm. of copper sulphate in 50ml. of water with constant stirring. Dilute to 500ml. The resulting solution should be perfectly clear; if not, filter through a fluted filter paper.
Benedicts solution (quatitative)
Dissolve 200g sodium citrate, 75g sodium carbonate and 125g potassium thiocynate in about 600 ml water. Dissolve separately, 18g copper (11) sulphate in 100 ml water. When the solutions have cooled, mix them together with stirring. Now add 5 ml of a 5% potassium ferrocyanide to the solution, and make up to 1 liter.
Dissolve 50 g in 10 ml glacial acetic acid, dilute to 100 ml with water and mix. caution: Toxic!).
Dissolve 1g in 10ml acetic acid and dilute to 100 ml with water.
Dissolve 0.100 g in 50ml purified absolute ethanol.
Take enough urea to cover the bottom of a test tube. Heat very gently , until the liquid which forms resolidifies. This white solid is biuret. Dissolve the biuret in about 2ml. water, and use this solution for the biuret test.
Soln. A : 0.1m sodium hydroxide.
SoLn. B : 0.01M copper (11) sulphate solution.
Add soln. A first to sample, then add B. Pink or purple color confirms protein.
Biuret reagent (quantitative)
Dissolve in order, 3g copper (11) sulphate, 5g potassium iodide, 9g potassium sodium tartrate, and 8g sodium hydroxide in about 600 ml water. Make up the dissolved solids to 1 liter solution.
Boil a 2 to 1 mixture of conc. sulphuric acid and water to to remove nitrates, cool , and dissolve into it 0.6g of brucine sulphate and dilute to 1 liter.
Weigh an unopened bottle of antimony trichloride. Open the bottle and empty the contents into a wide mouth glassstoppered amber bottle containing about 100 ml of chloroform. By difference, obtain the weight of antimony trichloride and then add sufficient chloroform to supply 100 ml for each 25 g. Dissolve by warming or shaking for several hours and filter through sodium sulphate into a dry clean amber bottle with ground stopper.Store at room temperature and keep in dark when not in use. Rinse all glassware coming into contact with this reagent wit chloroform or a mixture of ethanol and ether, since the antimony oxychloride which forms is insoluble in water.
Dissolve 0.25 g in 100 ml of water.
Cadion 2B reagent
Dissolve 0.25 g in 100 ml of water.
Dissolve 2.7 g in 10 mls. of water.
Weigh out 10 g of sodium dichromate crystals, make it into a slurry with a few mls of watwer, then dissolve in 250mls conc. sulphuric acid with stirring and cooling (ice-bath), to give a thick syrupy dark brown mixture.(very corrosive!)
Use a 0.005% in conc. sulphuric acid.
Dissolve 1g of 4,5-dihydroxy-2,7-naphthalenedisulfonic acid in 100 ml water.
Dissolve 1g in 100 ml hot water containing a few drops of nitric acid, cool, and add 2g of KI.
Dissolve with warming, 40 g of uranyl acetate in a mixture of30 ml acetic acid and 470 ml water.Similarly, prepare a solution containing 200 g of cobaltous acetate in a mixture of 30 ml acetic acid and 470 ml water. Mix the two solutions while still warm, cool to room temperature to separate xs salts from the solution and filter.
Dissolve 5 g in 100ml solution.(5%) Does not keep. Add about 1g of ammonium carbonate to stock to enhance stability.
Dissolve 5g of a-benzoin oxime in 100ml ethanol
Dissolve 5 g of yellow mercuric oxide (HgO) in a mixture of 40 ml of water,and while stirring slowly add 20 ml of sulphuric acid, then add another 40 ml of water, and stir until complety dissolved <0.5N)
Dissolve 1gm. of the solid in 100ml. of 95% ethyl alcohol.
Measure and pour into a 250 mL beaker 89 mL of distilled water. Stir on a magnetic stirrer.
Carefully add 15 mL of concentrated sulfuric acid.
Add and dissolve 1.0 g n,n-dimethyl-p-phenylenediamine sulfate.
Add 5 g of Florisil and stir the mixture until all is absorbed.
Allow the adsorbant to settle and decant the supernatant solution.
Add 200 ml sulphuric acid to 700 ml water, cool, and add 1g of N,N-dimethyl-p-phenylenediamine (p-aminodimethylaniline) and dilute ti 1 liter.
Dinitro-p-diphenylcarbazide Dissolve 0.1g in 100 ml ethanol
Use a saturated ethanolic solution; or dissolve 0.125 g in a mixture of 25ml acetone 25 ml water; or dissolve 0.2g in 10 ml acetic acid and dilute to 100 ml with methanol
Dissolve an approximately 1% solution in ethanol.
Dissolve 0.2g in 20 ml of 0.1M sodium carbonate solution, boil, cool, then filter.
Dissolve 0.1g in 0.5ml ethanol,or in 0.1M HCl
Dissolve 0.2g in 100 ml of 1% NaOH
Dissolve 30 mg (milligram) of dithizone in 1 liter chloroform, add 5 ml alcohol, and store in refrigerator.
Solution 1: dissolve 0.85g of basic bismuth nitrate in 10 ml acetic acid and 40 ml water.
Solution 2: dissolve 8 g of potassium iodide in 20 ml water.
Mix 5 ml of Solution 1; 5 ml of Solution 2; 20 ml of acetic acid; and 100 ml of water before use.
Add copper sulphate to a solution of ethylenediamine until the color becomes dark-blue violet
Dissolve 50 g in a mixture of 25 ml water and 25 ml ethanol, add 3 drops hydrochloric acid, stirr vigorously, and filter if necessary to obtain a clear solution.
Solution A; Dissolve 34.6gms. of pure copper sulphate in distilled water and dilute to 500ml. ( blur )
Solution B: Dissolve 173gms. of sodium potassium tartrate and 30gms.of pure sodium hydroxide in water and dilute to 500ml. Alternately, dissolve 121gms. of pure sodium hydroxide and 93.1gms.of pure tartaric acid in water, then dilute the solution to 500ml.( colourless ).
Mix equal volumes of solutions A and B immediately before use, and then use as the reagent.
Ferric thiocyanate reagent
Dissolve 1.5 g of ferric chloride and 2.0 g of potassium thiocyanate in 100 ml water.
Dissolve 0.2 g in 100 ml water
Make a saturated solution in 50% ethanol.
Dilute the commercial 40% solution (1 part) with water (7 parts)
Dissolve 0.15gm. of fuchsin in 100ml. water.
Dissolve 10 g in 100 ml ethanol.
Dissolve 2 g in 100 ml water.
Use the commercial 10 volume (3%) or 20 volume 6%) solution.
Dissolve 10 g in 100 ml water.
Use a 5% solution in ethanol.
Gently warm 1gm. of indigo with 12ml. of concentrated sulphuric acid, allow to stand for 48hrs. and pour into 240ml. of water. Filter if necessary.
Dissolve 0.15 g in 100 ml ethanol
Dissolve 20g KI and 10g iodine crystals in 100ml water.
Mix 25g of chromium trioxide (chromic anhydride CrO3) with conc. sulphuric acid to a paste, then dilute with water to
Karl Fisher Reagent
Dissolve 762 g of iodine in 2,420 ml of pyridine in a 10 liter glass stoppered bottle, and add 6 liters of methanol. To prepare the active stock, add 3 liter of the foregoing stock to a 4 liter bottle, cool in ice bath. Add carefully 135 ml of liquid sulfur dioxide, collected in a calibrated cold trap,and stopper the bottle. Shake the mixture until homogeneous, and set aside for one or two days before use.
Dissolve 100gms. of magnesium chloride and 100gms.of ammonium chloride in water, add 50ml. of concentrated ammonia solution and dilute to 1 litre with distilled water.
Magnesium nitrate reagent
Dissolve 130gms. of magnesium nitrate and 200gms. of ammonium nitrate in water, add 15-20mls.concentrated ammonia solution and dilute to 1 litre.
Use a 0.001% in 1M sodium hydroxide.
A 1% solution of malachite green oxalate in glacial acetic acid.
Dissolve 90 g manganese sulphate in 200 ml water, 175 ml phosphoric acid and 350 ml of diluted sulphuric acid (1 in2). Add water to make up 1 liter.
Dissolve 1.358 g of mercuric chloride in 60 ml water. Dissolve 5 g of potassium iodide in 10 ml water. Mix the two solutions and add water to make 100 ml.
Dissolve 2 g in 10 ml water. To 10 ml of this solution add 90 ml of water and 20 g of sodium bisulfite.
Warm one globule of Mercury with concentrated nitric acid and dilute the solution with twice its volume of water.
20% soln. in naphthol. Dissolve 20g of 1-naphthol in 100ml ethanol.
Add 0.4 g of murexide to 40 g of powdered potassium sulphate, and grind in glass mortar to a homogeneous mixture.
Dissolve 0.1 g of 2,7-dihydroxynaphthalene in 1 liter sulphuric acid and allow the solution to stand in the dark until the yellow color has diappeared (at least 18 hrs.)
Dissolve 1 g of 1-naphthol in 25 ml methanol. Prepare fresh.
A 1% solution in benzol.
Boil 0.3 g in 70 ml water, filter or decant, and mix with 30 ml of glacial acetic acid.
Dissolve 10gms. of potassium iodide in 10mls. of ammonia-free water, adding saturated mercuric chloride solution (60gms. / litre) in small quantities at a time with shaking, until a slight permanent precipitate is formed, then adding 80ml. of 9M sodium hydroxide solution and diluting to 200ml.Allow to stand overnight and decant the clear liquid.
Nesslers reagent has been described as a solution which is about 0.09M in potassium mercuri-iodide and 2.5M in potassium hydroxide.
An alternative method is to dissolve23gms. of mercuric iodide and 16gms. of potassium iodide in ammonia-free water and make up the volume to 100ml; add 100ml. of 6M sodium hydroxide. Allow to stand for 24hrs. and decant the solution from any precipitate that may have formed, the solution should be kept in the dark.
Another method that reacts promptly and consistantly is to dissolve 143 g of sodium hydroxide in 700 ml water. Disolve 50 g of red mercuric iodide and 40 g of potassium iodide in 200 ml water. Pour the iodide solution into the hydroxide solution, and dilute with water to 1 liter. Allow to settle, and use the supernatant liquid.
A 0.2% solution of ninhydrin (triketohydrindene hydrate, C9H4O3.H2O) in water. Prepare fresh.
Dissolve 5g nitron in5 ml acetic acid and make up to 100 ml with water.
Dissolve 10 g in 100 ml of either 1:1 acetic acid , ethyl alcohol or acetone.
Dissolve 1 g in 100 ml water.
Dissolve 0.1 g of the monohydrate in 100 ml water.
Dissolve 0.15 g orthophenanthroline (C12H8N2.H2O) in 10 ml solution of ferrous sulphate, prepared by dissolving 1.48 g of ferrous sulphate in 100 ml water. THe ferrous sulphate solution must be prepared immediately before dissolving the orthophenanthroline.
Dissolve 5 g in water, filter and dilute to 100 ml.
Dissolve the equivalent of 1 g of anhydrous trinitrophenol in 100 ml of hot water. Cool, and filter if necessary.
Dissolve 10 g of KCN in sufficient water to make 20 ml. Dilute to 100 ml. Shake with dithizone solution to remove lead.
Dissolve 70 g of sodium molybdate (Na2MoO42H2O) in 150 ml water (Slution A). Dissolve 60 g of acetic acid in a mixture of 85 ml nitric acid and 150 ml of water and cool (Solution B) Gradually add Solution A to Solution B, with stirring, to produce Solution C. Dissolve 5 g of synthetic quinoline in a mixture of 35 ml nitric acid and 100 ml water (Solution D) Gradually add Solution D to Solution C, mix well, and allow to stand overnight. Filter the mixture, add 280 ml of acetone to the filtrate, dilute to 1 liter with water, and mix. Store in polythene bottle.(Caution: Flammable).
Neutralize 1g of the acid with NaOH and dilute ti 100 ml
Dissolve 0.02g in 100 ml ethanol, or dissolve 0.05g in 100 ml of 0.01M sodium hydroxide.
Dissolve 0.01 g in 100ml water, or dissolve 0.05 g rhodamine B and 15g KCL in a solution of 15 mls conc. HCl and 85 mls water
(dithio-oxamide) - 0.05% in ethanol., a saturated ethanolic solution.
A 20% solution in ethanol.
Method 1: Dissolve 0.2gm. of pure p rosaniline hydrochloride in 20ml. of a cold, freshly prepared, saturated aqueous solution of sulphur dioxide; allow the solution to stand for a few hours until it becomes colourless or pale yellow. Dilute the solution to 200ml. and keep it in a tightly stoppered bottle.The solution keeps well, and should not be exposed to light or air. Store in the dark.
Method 2: Add 2gm. of sodium bisulphite to a solution of 0.2gm.of p- rosaniline hydrochloride and 2ml.of concentrated hydrochloric acid in 200ml. of water.
Dissolve 10 g ofsilicotungstic acid (Si02.12WO3.26H2O) in water and neutralize with 10% sodium hydroxide (pH 6)
Silver ammonium nitrate
Dissolve 1 g of silver nitrate in 20 ml of water.Add ammonia dropwise, withconstant stirring, until the ppt. is almost but not entirely dissolved. Filter and store in dark container.
Dissolve 1 g in 200 ml of freshly distilled pyridine.
A 5% solution of sodium azide in water.
Dissolve 0.6 g sodium borohydride and 0.5g of sodium hydroxide with stirring and dilute to 100 ml with water.
Sodium cobaltinitrite solution
Dissolve 17gms. of the pure salt in 250
Alternately, the solution may be prepared as follows: Dissolve 7.5 gms. cobalt nitrate in 30ml. of water; dissolve 60gms. of sodium nitrite in 30ml. of water, mix the two solutions with vigorous stirring and add 15ml. of glacial acetic acid, stir, dilute to 250ml, allow to stand and filter.Make up new solution every 2-3 weeks.
Dissolve 1 g in water and dilute to 1 liter.
Dissolve 10 g of sodium metal in 120 ml of ethanol using the following method: remove surplus oil from sodium with filter paper, dry again on filter paper, and cut the weighed metal into small pieces about the size of a pea. pour the ethanol into a 500 ml flask cooled on ice bath, and add one or two pieces at a time until dissolved.
Sodium hypochlorite solution
The commercial product contains about 10-14 per cent w / v of available chlorine. Dilute with an equal volume of water.
Sodium nitroprusside solution
Prepare a solution as required by dissolving a crystal in 5 ml. of water.
Sodium rhodizonate reagent
Dissolve 0.5 g in 100 ml water
Triturate 0.5gm. of soluble starch with a little cold water into a thin paste and add 25ml. of boiling water.Boil until a clear solution is obtained (5-mins.). This solution should be freshly prepared as required. Amore stable starch solution is obtained by adding 0.5gm. of potassium iodide and 2-3 drops of chloroform.
A more satisfactory starch solution for use as an indicator is prepared as follows:* Mix 5.0 gm.of powdered sodium starch glycollate with 1-2 ml. ethyl alcohol, add 100ml. of cold water and boil for a few minutes with stirring.This 5% stock solution is stable for many months; it is diluted to 0.1% strength when required for use.
Dissolve 1 g in 100 ml of warm 30% acetic acid
Dissolve 1 g of tannic acid (tannin) in 1 ml ethanol, and add water to make 10 ml. Prepare fresh.
Tartrate solution, alkaline
Dissolve 34.6 g of sodium potassium tartrate (rochelle salt) and 10 g of sodium hydroxide in water, dilute to 100 ml, let stand for two days, and filter through glass wool.
10g in 100ml of water (10%)
Dissolve 1g in 100ml of water (1%)
(also called thiazole yellow, clayton yellow)
Dissolve 5 g in water, filter and dilute to 100 ml.
Cool separately in small beakers surrounded by crushed ice 10 ml of 20% hydrochloric acid and 10 ml of clear, colorless titanium tetrachloride.Add the tetrachloride dropwise to the chilled acid.Stand at ice temperature until all the solid disolves, then dilute to 1 liter with 20% hydrochloric acid.
Add sodium hydroxide soln. to silver nitrate soln. to form a ppt. then add dilute ammonia soln. until ppt.
20% solution: dissolve 0.20 g of Triton-X-100 (polyethelene glycol ether of isooctylphenol) in water, and dilute to 100ml.
Uranyl magnesium acetate
Make up an aqueous saturated solution of the salt.
Make up a 1% solution in ethanol
Zirconyl nitrate reagent
( for fluoride test )
Dissolve 0.1gm. of zirconyl nitrate in 20ml. concentrated hydrochloric acid and dilute with water to 100ml.
Zirconyl nitrate reagent
( for phosphate separations )
Heat 10gms. of commercially pure zirconium nitrate and 100ml. of 1M nitric acid to the boiling point with constant stirring.Leave to stand for about 24hrs.and decant the clear solution.
Add about 10 g of granulated zinc to to 20 ml mercury, to produce a liquid amalgam on cooling, and heat to 150 degrees with stirringuntil the zinc is dissolved.
Zinc amalgated (Jones Reductor)
The zinc is amalgated by immersing it in a solution of mercuric chloride in hydrochloric acid. A quantity of 250 g of 20 mesh zinc is covered with water in a 1 liter flask, and a solution of 11 g of mercuric chloride in 100 ml of hydrochloric acid is poured into the flask. The system is slowly mixed and shaken for about 2 minutes. The solution is poured off, and the amalgam is washed thoroughly with hot tap water, then distilled water.
Zinc uranyl acetate
Dissolve 10gms. of uranyl acetate in 6gms. of 30% acetic acid, warming if necessary and dilute with water to 50ml. (soln.A) In a separate vessel, stir 30gms. of zinc acetate with 3gms.of 30% acetic acid and dilute with water to 50ml. (soln.B) Mix the two solutions A and b, and add a small quantity of sodium chloride. Allow to stand for 24 hrs. and filter from the precipitated sodium zinc uranyl acetate.
Alternatively, a reagent of equivalent concentration may be prepared by dissolving uranyl zinc acetate in the appropriate volume of water or 1M acetic acid
Mix 1 ml of pyridine with 4 ml of a 10% aqueous solution of copper sulphate and 5 ml of water.