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APPENDIX 5 CHEMICAL TESTS

5.1 GENERAL IDENTIFICATION TESTS

Acetates

          A. Acetates, when warmed with a 50 per cent v/v solution of sulfuric acid, yield acetic acid, which is recognizable by its odour.

          B. Acetates, when warmed with sulfuric acid and a small quantity of ethanol, yield ethyl acetate, which is recognizable by its odour.

          C. Neutral solutions of acetates produce a redbrown colour with iron(III) chloride TS and a red-brown precipitate when boiled. The precipitate dissolves and the colour of the solution changes to yellow by the addition of hydrochloric acid.

Acetyl Groups

          In a test-tube (about 180 mm × 18 mm) place about 15 mg of the test substance or the prescribed quantity and 3 drops of phosphoric acid. Close the tube with a stopper through which passes a small test-tube (about 100 mm × 10 mm) containing water to act as a condenser. On the outside of the smaller tube hang a drop of a 5 per cent w/v solution of lanthanum nitrate.

          Place the apparatus in a water-bath for 5 minutes and remove the smaller tube. Mix the drop with 1 drop of 0.01 M iodine on a tile. Add at the edge 1 drop of 2 M ammonia. After 1 to 2 minutes a blue colour develops at the junction of the two drops which intensifies and persists for a short time.

          For substances hydrolyzable only with difficulty, heat the mixture slowly to boiling point over an open flame instead of using a water-bath.

Alkaloids

          Dissolve a few mg of alkaloids, or the prescribed quantity, in 5 ml of water, add dilute hydrochloric acid until an acid reaction occurs. Then add 1 ml of acetic potassium iodobismuthate TS: an orange or orange-red precipitate is produced immediately.

Aluminium Salts

          A. Solutions of aluminium salts yield with ammonia TS a gelatinous, white precipitate which does not dissolve upon the addition of excess ammonia TS. Sodium hydroxide TS or sodium sulfide TS produces the same precipitate which dissolves in an excess of either of these reagents.

          B. To a solution of aluminium salts, add ammonia TS until a faint precipitate is produced, and then add 5 drops of a freshly prepared 0.05 per cent w/v solution of quinalizarin in a 1 per cent w/v solution of sodium hydroxide. Heat to boiling, cool, and acidify with an excess of 5 M acetic acid: a reddish violet colour is produced.

Amines, Primary Aromatic

          Acidify the prescribed solution with 2 M hydrochloric acid, or dissolve 100 mg in 2 ml of 2 M hydrochloric acid,and add 4 drops of sodium nitrite TS. After 1 to 2 minutes add the solution to 1 ml of 2-naphthol TS: an intense orange or red colour, usually together with a precipitate of the same colour, is produced.

Amino Acids

          To 5 ml of a 0.1 per cent w/v solution of the test substance add 1 ml of ninhydrin TS. After heating for 3 minutes, add 20 ml of water and allow to stand for 15 minutes: a blue-purple colour develops.

Ammonium Salts

          Ammonium salts are decomposed by the addition of an excess of sodium hydroxide TS, with the evolution of ammonia, recognizable by its odour and by its alkaline effect upon moistened red litmus paper exposed to the vapour. Warming the solution accelerates the decomposition.

Antimony Compounds

          Dissolve with gentle heating 10 mg of the substance being examined in a solution of 500 mg of potassium sodium tartrate in 10 ml of water and allow to cool. To 2 ml of this solution or to 2 ml of the prescribed solution add sodium sulfide TS1 dropwise: an orange-red precipitate is produced which dissolves on addition of 2 M sodium hydroxide.

Arsenic Compounds

          Heat 5 ml of the prescribed solution on a water-bath with 5 ml of hypophosphorous TS: a brown precipitate is produced.

Barbiturates, Non-nitrogen Substituted

          Dissolve 5 mg of the substance being examined in 3 ml of methanol, add 2 drops of a solution containing 10 per cent w/v of cobalt(II) nitrate and 10 per cent w/v of calcium chloride, mix and add, with shaking, 2 drops of 2 M sodium hydroxide: a violet-blue colour and precipitate are produced.

Barium

          With 1 M sulfuric acid, solutions of barium salts yield a white precipitate. This precipitate is insoluble in hydrochloric acid and in nitric acid. Barium salts impart a yellowish green colour to a nonluminous flame that appears blue when viewed through green glass.

Benzoates

          A. To 1 ml of a 10 per cent w/v neutral solution of benzoates add 0.5 ml of iron(III) chloride TS: a dull yellow precipitate, soluble in ether, is produced.

          B. Moisten 200 mg of benzoates, treated if necessary as prescribed, with 4 to 6 drops of sulfuric acid and gently warm the bottom of the tube: a white sublimate is deposited on the inner walls of the tube.

          C. Dissolve 500 mg of benzoates in 10 ml of water, or use 10 ml of the prescribed solution and add 0.5 ml of hydrochloric acid: the precipitate obtained, after crystallization from water and drying in vacuum, melts between 120º and 124º (Appendix 4.3).

Bicarbonates

          A. Solutions of bicarbonates, on boiling, liberate carbon dioxide which produces a white precipitate in calcium hydroxide TS.

          B. Solutions of bicarbonates yield no precipitate with magnesium sulfate TS at room temperature, but on boiling the mixture, a white precipitate is produced.

Bismuth Salts

          A. To 500 mg of bismuth salts add 10 ml of 2 M hydrochloric acid or use 10 ml of the prescribed solution. Boil for 1 minute, cool, filter if necessary and to 1 ml of the resulting solution add 20 ml of water: a white or slightly yellow precipitate is produced which turns brown on addition of 1 or 2 drops of sodium sulfide TS.

          B. To 40 to 50 mg of bismuth salts add 10 ml of 2 M nitric acid or use 10 ml of the prescribed solution. Boil for 1 minute, allow to cool, filter if necessary and to 5 ml of the resulting solution add 2 ml of a 10 per cent w/v solution of thiourea: an orange-yellow colour or an orange precipitate is produced. Add 4 ml of a 2.5 per cent w/v solution of sodium fluoride: the solution is not decolorized within 30 minutes.

Bisulfites

          See Sulfites.

Borate

          A. To 1 ml of a borate solution, acidified with hydrochloric acid to litmus, add 3 or 4 drops of iodine TS and 3 or 4 drops of a 2 per cent v/v solution of polyvinyl alcohol: an intense blue colour is produced.

          B. When a borate is treated with sulfuric acid, methanol is added, and the mixture is ignited, it burns with a green-bordered flame.

Bromides

          A. Solutions of bromides yield, with silver nitrate TS, a yellowish white precipitate, somewhat soluble in strong ammonia solution but slightly soluble in ammonia TS and in nitric acid.

           B. Solutions of bromides, upon the addition of chlorine TS, dropwise, liberate bromine, which is dissolved by shaking with chloroform, colouring the chloroform red to reddish brown.

Calcium Salts

          A. To 0.2 ml of a neutral solution containing the equivalent of about 0.04 mg of calcium or to 0.2 ml of the prescribed solution, add 0.5 ml of a 0.2 per cent w/v solution of glyoxal bis(2-hydroxyanil) in ethanol, 4 drops of 2 M sodium hydroxide, and 4 drops of 1 M sodium carbonate. Extract with 1 to 2 ml of chloroform and add 1 to 2 ml of water: the chloroform layer is red.

          B. Dissolve 20 mg of calcium salts or the prescribed quantity in 5 ml of 5 M acetic acid. Add 0.5 ml of potassium hexacyanoferrate(II) TS: the solution remains clear. Add 50 mg of ammonium chloride: a white crystalline precipitate forms.

          C. To a solution of calcium salts add a few drops of a solution of ammonium oxalate: a white precipitate is obtained that is only sparingly soluble in 6 M acetic acid but is soluble in hydrochloric acid.

          D. Moisten calcium salts with hydrochloric acid and introduce on a platinum wire into an open flame: a brick-red colour develops.

Carbonates

          A. Carbonates effervesce with dilute acids, liberating carbon dioxide: the gas is colourless and produces a white precipitate in calcium hydroxide TS (common with bicarbonates).

          B. Solutions of carbonates produce, at room temperature, a white precipitate with magnesium sulfate TS (distinction from bicarbonates).

Chlorides

          A. Solutions of chlorides yield with silver nitrate TS a white, curdy precipitate, which is insoluble in nitric acid, but is soluble in ammonia TS.

          B. Dry chlorides, when mixed with an equal weight of manganese(IV) oxide, moistened with sulfuric acid, and gently heated, evolve chlorine which is recognizable by its odour and by the production of a blue colour with moistened starch-iodide paper. Carry out the reaction preferably under a hood.

Citrates

          A. Upon the addition of a few mg of a citrate to a mixture of 15 ml of pyridine and 5 ml of acetic anhydride: a carmine-red colour is produced.

          B. Neutral solutions of citrates, when treated with calcium chloride TS, do not yield a precipitate; on boiling they yield a white precipitate which is soluble in acetic acid.

          C. Solutions of citrates, when boiled with an excess of mercury(II) sulfate TS and filtered if necessary, yield a solution which, after being boiled and treated with a few drops of potassium permanganate TS, decolorizes the reagent and yields a white precipitate.

Copper Salts

          A. Solutions of copper(II) compounds, acidified with hydrochloric acid, deposit a red film of metallic copper upon a bright, untarnished surface of metallic iron.

          B. An excess of ammonia TS, added to a solution of a copper(II) salt, produces first a bluish precipitate and then a deep blue-coloured solution.

          C. With potassium hexacyanoferrate(II) TS, solutions of copper(II) salts yield a reddish brown precipitate, insoluble in diluted acids.

Esters

          To 30 mg of the test substance or to the prescribed quantity add 0.5 ml of a 7 per cent w/v solution of hydroxylamine hydrochloride in methanol and 0.5 ml of a 10 per cent w/v solution of potassium hydroxide in ethanol. Heat to boiling, cool, acidify with 2 M hydrochloric acid, and add 4 drops of a 1 per cent w/v solution of iron(III) chloride: a bluish red or red colour is produced.

Iodides

          A. Solutions of iodides yield, with silver nitrate TS, a yellow, curdy precipitate, practically insoluble in ammonia TS and in nitric acid.

          B. Solutions of iodides, upon the addition of chlorine TS, dropwise, liberate iodine, which colours the solution yellow to red. When the solution is shaken with chloroform, the latter is coloured violet.

Iron Salts

          Iron(II) and iron(III) compounds in solution yield a black precipitate with ammonium sulfide TS. This precipitate is dissolved by cold dilute hydrochloric acid with the evolution of hydrogen sulfide.

IRON(II) SALTS

          A. Solutions of iron(II) salts yield a dark blue precipitate with potassium hexacyanoferrate(III) TS. This precipitate is insoluble in dilute hydrochloric acid, but is decomposed by sodium hydroxide TS.

          B. Solutions of iron(II) salts yield with sodium hydroxide TS a greenish white precipitate, the colour rapidly changing to green and then to brown when shaken.

IRON(III) SALTS

          A. Acid solutions of iron(III) salts yield a dark blue precipitate with potassium hexacyanoferrate(II) TS.

          B. With an excess of sodium hydroxide TS, solutions of iron(III) salts yield a reddish brown precipitate.

          C. Solutions of iron(III) salts produce with ammonium thiocyanate TS: a deep red colour which is not destroyed by dilute mineral acids.

Lactates

          A. Lactates in solution, heated with dilute sulfuric acid and potassium permanganate TS, evolve acetaldehyde recognizable by its odour.

          B. Lactates, treated with iodine and potassium iodide TS in the presence of a slight excess of sodium hydroxide TS, give a yellowish precipitate of iodoform which, after extraction with ether and spontaneous evaporation, is characterized by its crystalline form and its odour: it melts at 118º to 124º (Appendix 4.3).

          C. Lactates, shaken with a few drops of water and 2 ml of sulfuric acid and treated with 2 drops of guaiacol TS, slowly give a red colour.

Lead and Lead Compounds

          A. To 100 mg of the substance being examined dissolved in 1 ml of 5 M acetic acid or to 1 ml of the prescribed solution add 2 ml of potassium chromate TS: a yellow precipitate is produced which is soluble in 2 ml of 10 M sodium hydroxide.

          B. To 50 mg of the substance being examined dissolved in 1 ml of 5 M acetic acid or to 1 ml of the prescribed solution add 10 ml of water and 4 drops of potassium iodide TS: a yellow precipitate is produced. Heat to boiling for 1 to 2 minutes and allow to cool: the precipitate reappears as glistening, yellow plates.

Lignin

          A. Lignified cell walls are coloured bright red by soaking them in phloroglucinol TS and adding 2 to 4 drops of hydrochloric acid.

          B. Lignified tissues are coloured yellow by aniline hydrochloride TS.

Magnesium

          A. Dissolve 10 to 15 mg of magnesium salts in 2 ml of water or use 2 ml of the prescribed solution. Add 1 ml of ammonia TS: a white precipitate forms that is redissolved by adding 1 ml of 2 M ammonium chloride. Add 1 ml of 0.25 M disodium hydrogenphosphate: a white crystalline precipitate forms.

          B. To 0.5 ml of a neutral or slightly acidic solution of magnesium salts, add 4 drops of a 0.1 per cent w/v solution of titan yellow and 0.5 ml of 0.1 M sodium hydroxide: a bright red turbidity forms, which gradually settles to give a bright red precipitate.

Manganese Salts

          A. Solutions of manganese(II) salts yield, with ammonium sulfide TS, an orange-pink precipitate, which dissolves in acetic acid.

          B. To 100 mg of manganese(II) salts, add 2 g of lead(IV) oxide and 5 ml of nitric acid, boil gently for a few minutes, add 100 ml of water and filter: a purple solution is produced.

Mercury and Mercury Compounds

          MERCURY Solutions of mercury salts, free from an excess of nitric acid, when applied to bright copper foil, yield a deposit that, upon rubbing, becomes bright and silvery in appearance. With hydrogen sulfide, solutions of mercury compounds yield a black precipitate that is insoluble in ammonium sulfide TS and in boiling 2 M nitric acid.

          MERCURY(I) SALTS Mercury(I) compounds are decomposed by 1 M sodium hydroxide, producing a black colour. Solutions of mercury(I) salts yield with hydrochloric acid a white precipitate that is blackened by ammonium TS. With potassium iodide TS, a yellow precipitate, that may become green upon standing, is formed.

          MERCURY(II) SALTS Solutions of mercury(II) salts yield a yellow precipitate with 1 M sodium hydroxide. They yield also, in neutral solutions with potassium iodide TS, a scarlet precipitate that is very soluble in an excess of the reagent.

Nitrates

          A. Mix a solution of nitrates with an equal volume of sulfuric acid, cool the mixture, and superimpose a solution of iron(II) sulfate: a brown colour is produced at the junction of the two liquids.

          B. Nitrates heated with sulfuric acid and copper liberate brownish red fumes.

          C. Nitrates do not decolourize acidified potassium permanganate TS (distinction from nitrites).

Penicillins

          To 2 mg of a penicillin, add 2 mg of chromotropic acid sodium salt and 2 ml of sulfuric acid. Immerse in an oilbath at 150º: the solution, when shaken and examined every 30 seconds, exhibits the colours stated in the table below.

Permanganates

          Solutions of permanganates acidified with sulfuric acid are decolorized by hydrogen peroxide TS and by sodium metabisulfite TS, in the peroxide cold, and by oxalic acid TS, in hot solution.

Peroxides

​          Solutions of peroxides slightly acidified with sulfuric acid yield a deep blue colour upon the addition of potassium dichromate TS. On shaking the mixture with an equal volume of ether and allowing the liquids to separate, the blue colour is found in the ether layer.

Phosphates (Orthophosphates)

​          A. Solutions of phosphates, when neutralized to about pH 7 treated with silver nitate TS, yield a light yellow precipitate which is readily soluble in ammonia TS and in dilute nitric acid:

​          B. Solutions of phosphates in dilute nitric acid, when treated with and equal volume of ammonium molybdate TS and warmed, yield a bright canary-yellow precipitate.

Potassium Salts

​          A. Ignite a small quantity of potassium salts, dissolve the residue in water, filter and make alkaline with sodium hydroxide TS. Treat the solution so obtained with sodium tetraphenylborate TS: a white precipitate is produced.

​          B. To 1 ml of a 5 per cent w/v solution of potassium salts, add 1 ml of 2 M acetic acid and 1 ml of a freshly prepared 10 per cent w/v solution of sodium hexanitrocobaltate(III): a yellow or orange-yellow precipitate forms immediately.

​          C. Potassium compounds impart a violet colour to a nonluminous flame, but the presence of small quantities of sodium masks the colour unless being viewed through a cobalt-blue glass.

Salicylates

​          A. To 1 ml of a 10 per cent w/v neutral solution of salicylates, add 0.5 ml of iron(III) chloride TS: a violet colour is produced which persists after the addition of 2 drops of 5 M acetic acid.

​          B. Dissolve 500 mg of salicylates in 10 ml of water or use 10 ml of the prescribed solution. Add 0.5 ml of hydrochloric acid: the precipitate obtained, after recrystallization from hot water and drying in vacuum, melts between 156º and 161º (Appendix 4.3).

Silicates

​          In a lead or platinum crucible mix to a thin slurry using a copper wire the prescribed quantity of the test substance with 10 mg of sodium fluoride and 4 drops of sulfuric acid. Cover the crucible with a thin, transparent plate of plastic from which a drop of water is suspended and warm gently: a white ring is produced around the drop of water within a short time.

Silver Salts

​          A. Dissolve 10 mg of silver salts in 10 ml of water or use 10 ml of the prescribed solution. Add 6 drops of 7 M hydrochloric acid: a curdy, white precipitate is produced that is soluble in 3 ml of ammonia TS. Add potassium iodide TS: a yellow precipitate is produced. 

​          B. A solution of a silver salt to which ammonia TS and a small quantity of formaldehyde solution are added deposits, upon warming, a mirror of metallic silver on the inner walls of the tube.

Sodium Salts

​          A. Ignite a small quantity of sodium compounds and dissolve the residue in dilute acetic acid, filter, if necessary, and treat with zinc uranyl(IV) acetate TS: a yellow crystalline precipitate is formed.

​          B. Sodium compounds impart an intense yellow colour to a nonluminous flame.

Sulfates

​          A. Solutions of sulfates yield with barium chloride TS a white precipitate, which is insoluble in hydrochloric acid and in nitric acid.

​          B. Solutions of sulfates, when treated with lead(II) acetate TS, yield a white precipitate, which is soluble in an 8 per cent w/v solution of ammonium acetate and in sodium hydroxide TS.

Sulfites

​          When treated with 3 M hydrochloric acid, sulfites and bisulfites yield sulfur dioxide, which blackens filter paper moistened with mercury(II) nitrate TS.

Tartrates

​          A. Add a few mg of a tartrate to a mixture of 15 ml of pyridine and 5 ml of acetic anhydride: an emeraldgreen colour is produced.

​          B. To 5 ml of a 0.4 per cent w/v solution of the substance being examined, add 1 drop of a 1 per cent w/v solution of iron(II) sulfate and 1 drop of hydrogen peroxide TS (10 volumes): a transient yellow colour is produced. When 2 M sodium hydroxide is added dropwise, an intense blue colour is produced.

​          C. Mix a few ml of sulfuric acid with a few drops of a 2 per cent w/v solution of resorcinol and a few drops of a 10 per cent w/v solution of potassium bromide, and add 2 or 3 drops of the solution being examined. Warm the mixture on a water-bath for 5 to 10 minutes: an intense blue colour is produced that changes to red when the solution is cooled and poured into water.

Thiocyanates

​          With iron(III) chloride TS solutions of thiocyanates yield a red colour that is not destroyed by moderately concentrated mineral acids.

Thiosulfates

​          With hydrochloric acid solutions of thiosulfates yield a white precipitate that soon turns yellow, and sulfur dioxide, which blackens filter paper moistened with mercury(II) nitrate TS. The addition of iron(III) chloride TS to solutions of thiosulfates produces a dark violet colour that quickly disappears.

Xanthines

​          Mix a few mg of xanthines or the prescribed quantity with 0.1 ml of hydrogen peroxide TS (100 volumes) and 6 drops of 2 M hydrochloric acid. Heat to dryness on a water bath until a yellowish red residue is produced and add 2 drops of 2 M ammonia: the colour of the residue changes to violet-red.

Zinc Salts

​          A. Solutions of zinc salts, prepared if necessary by the addition of hydrochloric acid, when treated with. solutions of alkali hydroxides, yield a white precipitate which is soluble in an excess of the solution of alkali hydroxide; the solution remains clear on the addition of ammonium chloride TS but yields a white, flocculent precipitate on the addition of sodium sulfide TS.

​          B. Solutions of zinc salts, when treated with potassium hexacyanoferrate(II) TS, yield a white precipitate which is insoluble in dilute hydrochloric acid.

​          C. Solutions of zinc salts, when acidified with dilute sulfuric acid and treated with 1 drop of a 0.1 per cent w/v solution of copper(II) sulfate and 2 ml of ammonium mercuri-thiocyanate TS, yield a violet precipitate.