Todd R.G. (General Editor), Clarke E.G.C. (Editor) Isolation and Identification of drugs in pharmaceuticals, body fluids and post-mortem material, Ed. The Pharmaceutical Press, Londres, Inglaterra, Págs 123-134.

Colour Tests

H. M. Stevens

Many substances give distinct colours when brought into contact with various chemical reagents. Sometimes the colour reaction with a particular reagent is not confined to a single compound but is produced by most of the compounds in a given class or even by substances not in that class. This type of test is called a 'general test’ and can only be used as a preliminary guide to the nature of the substance to be identified.

The second type of colour test is the 'special test’, where a colour produced with a certain reagent is specific for the compound under investigation. The special tests help considerably in identifying substances but absolute and final identification is usually only possible if some physical characteristic such as an infra-red spectrum can be obtained from the material. Special tests are described in detail in the relevant monographs in Part 2 and will not be dealt with further at this stage.

Many colour tests, both 'general’ and 'special’, can be carried out as 'micro’ tests by using the techniques introduced by Clarke and Williams (1955). These techniques, which are described at the end of this chapter under the heading Microtests (p. 133), enable the identification of compounds to be effected on the microgram scale.

The compounds cited below have in the main been restricted to those for which monographs appear in Part 2; it must be realised, however, that many other organic compounds will give similar responses to these tests.

GENERAL TESTS

In the following paragraphs the 'general’ tests are described, the various substances which respond being listed under the heading of the colour(s) obtained. These colours are set down in order of the spectrum from violet/purple through green and red to brown and black.

The tests have been classified according to the main component of the reagent and subdivided on the basis of the subsidiary components.

I. Sulphuric Acid

A drop of the cold concentrated acid is brought into contact with a smear (if a liquid) or a few fragments (if a solid) of the test material on a white tile. In many instances this test may be carried out on the microscale.

Purple: Lymecycline, oxytetracycline, rolitetracyclinc, telracycline.

Blue: Chlortetracycline, demethylchlortetracycline,thioridazine.

Green: Strophanthin-K---- sulphuric acid: water (4:1) must be used.

Yellow: Bromodiphenhydramine, cyclizine, diphenhydramine, diphenyl-pyraline, ethyl biscoumacetate, griseofulvin, oestradiol, orphen-adrine, pipradrol, prednisone.

Orange: Amitriptyline, chlorprothixene, cortisone, ethinyloestradiol, hydrocortisone, methacycline, spironolactone (50% sulphuric acid must be used; when heated the solution turns red).

Red: Caramiphen (if heated), hexamine (if heated), methylprednisolone (after 1 minute), ouabain, phenothiazines (but not trifluoperazine), prednisolone (after 1 minute).

Brown: Methylpentynol.

Ia Sulphuric Acid---- with nitrous acid; Liebermann's reagent

This reagent (see Appendix 1) is prepared by dissolving 1 g of potassium nitrite in 10 ml of sulphuric acid. The reagent was originally developed for giving intense colours with phenols. The test is carried out as for Sulphuric Acid (I).

Violet:o-Aminophenol, methocarbamol, paracetamol, phenacetin, resorcinol.

Blue:p-Aminophenol, diphenylamine, mefenamic acid, phenol (changing to red), yohimbin.

Green:Alphanaphthol, betanaphthol, chlorotrianisene (changing to pink on the addition of water), colchicine, hydrastine.

Yellow:Benzathine penicillin, benzoic acid, benzylpenicillin, cocaine, o-, m-, and p-hydroxybenzoic acid, physostigmine, tropacocaine.

Orange:Compounds generally containing a mono-substituted benzene group not attached directly to a carbonyl group-acetanilide, amphetamine, aniline, atropine, caramiphen, ephedrine, glutethimide, hyoscine, methoin, methylphenobarbitone, pethidine, phenacemide, phenobarbitone. phenylmethylbarbituricacid, phenytoin, sulphinpyrazone, warfarin.

Red:Acepromazine (brownish-pink), brucine, oxytetracycline.

Brown:m-Aminophenol, chlortetracycline, cotarnine, cresols, ethinamate, methylpentynol, noscapine, tetracycline, veratrine.

Black:Codeine, diamorphine, emetine, morphine, narceine, papaverine.

I bSulphuric Acid (60%)---- containing potassium dichromate (0-5%)

This test is carried out as for Sulphuric Acid (I) unless any modifications are mentioned.

Purple: Strychnine (changing to red), yohimbine (fades rapidly to slate blue).

Blue: Aniline, desipramine, imipramine, thioridazine.

Red: Brucine, griseofulvin (5 mg is dissolved in 1 ml of sulphuric acid and 5 mg of powdered potassium dichromate added).

Brown: Warfarin.

Note. The above tests are carried out in the cold. However, when alcohols and other reducing agents are warmed with a solution of potassium dichromate in dilute sulphuric acid (acidified potassium dichromate solution, p. 804) the potassium dichromate is reduced to the green, chromium salt.

I c Sulphuric Acid---- with chromotropic acid (see also under Screening Tests, p. 6)

Red: Formaldehyde. A few drops of a solution are added to a dilute solution of chromotropic acid in sulphuric acid.

I d Sulphuric Acid---- with resorcinol

Violet: Formaldehyde. A little resorcinol is shaken with the test solution and some sulphuric acid is run down the inside of the tube to form a layer under the mixed solution---- a reddish-violet ring appears at the junction of the 2 layers.

Green: Saccharin. A little is heated with an excess of resorcinol in sulphuric acid to just below the boiling point of the mixture---- green. When the solution is cooled, diluted, and made alkaline with sodium hydroxide a green fluorescence is produced.

Red: Tartaric acid. A little is warmed with resorcinol and a few drops of sulphuric acid.

I e Sulphuric Acid---- with vanillin

Violet: Menthol. About 10 mg is dissolved in 1 ml of sulphuric acid and 1 ml of a 1% solution of vanillin in sulphuric acid is added---- orange-yellow; the addition of 1 ml of water gives a violet colour which distinguishes menthol from thymol.

I f

Sulphuric Acid---- with pyrocatechol

Violet:

Metaldehyde. One drop of sulphuric acid is added to a small amount of solid followed by a trace of pyrocatechol---- violet-red.

I g

Sulphuric Acid---- with betanaphthol

Green:

Tartaric acid. The substance is heated with an equal bulk of betanaphthol and a few drops of sulphuric acid.

Yellow:

Malic acid (the test is carried out as above).

Red:

Oestradiol. About 0-05 mg is heated at 100° with I ml of a 2-5% solution of betanaphthol in sulphuric acid for 2 minutes. The mixture is cooled and 1 ml of water is added---- orange-yellow which changes to red when the solution is heated at 100° for 90 seconds.

I h

Sulphuric Acid---- with phenol

Green:

Sulphonal. A little of the substance is heated with a few drops of sulphuric acid containing a trace of phenol.

Note. The colour tests using sulphuric acid in conjunction with formaldehyde (Marquis test), ammonium vanadate (Mandelin's test), and ammonium molybdate (Fröhde's test) are described under Microtests, p. 133.

II

Nitric Acid

One of the most important tests using this reagent is Vitali's test (p. 133). Drugs which give a colour when treated with concentrated nitric acid, in the same way as with sulphuric acid in test I, p. 123, include:

Blue:

Desipramine, imipramine, thioridazine (with 50% nitric acid).

Yellow:

Phenols and phenolic substances (warming with the acid is sometimes necessary).

Red:

Antazoline, bialamicol (100 mg is dissolved in 2 ml of water and 2 ml of concentrated nitric acid added), brucine, hexylresorcinol (1 ml of 70% nitric acid is added to 1 ml of a saturated aqueous solution).

II a

Nitric Acid---- with eerie ammonium sulphate

Violet:

Primaquine. 10 mg is dissolved in 5 ml of water and 1 ml of a 5% solution of ceric ammonium sulphate in dilute nitric acid is added.

II b

Nitric Acid---- with mercuric nitrate; Millon's reagent

This reagent consists of 1 part of metallic mercury dissolved in 1 part of fuming nitric acid (w/w), the solution being diluted with 2 parts of water (v/v). A small quantity of the test material or a few drops of the test solution is warmed with a few drops of the reagent.

Red:

Phenolic compounds.

III

Hydrochloric Acid

Yellow:

Chlordiazepoxide. A few fragments are moistened with 2N hydrochloric acid and evaporated to dryness on a water-bath.

Triacetyloleandomycin. 10 mg of the substance is heated with 5 ml of the acid on a water-bath---- greenish-yellow.

III a

Hydrochloric Acid---- with wood shavings

Orange:

Sulphonamides. The substance is acidified with 2N hydrochloric acid and the mixture placed on wood shavings or newspaper. This test produces a positive response with many substances containing an aromatic amino-group.

III b

Hydrochloric Acid---- with furfuraldehyde

Violet:

Allantoin. A solution of the substance in ethanol is treated with a concentrated furfuraldehyde solution in ethanol to which a little of the concentrated acid is added.

Black:

Carbamates---- carbachol, ethinamate, mephenesin carbamate, meprobamate, methylpentynol carbamate, styramate, urea, urethane. A spot of an acetone or ethanol solution of the substance is placed on filter paper. After drying, the paper is sprayed lightly with a 10% v/v solution of furfuraldehyde in ethanol. The paper is then exposed to hydrochloric acid fumes.

III c

Hydrochloric Acid---- with potassium dichromate

Violet:

Paracetamol. A few mg of the test material is boiled gently with 2 to 3 ml of the concentrated acid for 3 minutes. The solution is cooled, diluted with water to about 3 times its volume, and a few drops of a cold dilute solution of potassium dichromate added to it. The violet colour persists; this is in contradistinction to phenacetin which also gives a violet colour that soon turns red.

III d

Hydrochloric Acid---- with sodium nitrite

Violet:

Amidopyrine. To a dried spot, or a few fragments on a white tile, is added a drop of water and a small droplet of the dilute acid. A few small crystals of sodium nitrite are added and the mixture stirred.

Green:

Phenazone (details as for Amidopyrinc).

Orange:

Sulphaphenazole and sulphasomizole. The substance is treated with the dilute acid, followed by a few crystals of sodium nitrite---- a bright orange precipitate.

III e

Hydrochloric Acid---- with potassium chlorate; amalic acid test

A few fragments of the substance under test are placed on a watch-glass and mixed with 2 to 3 drops of concentrated hydrochloric acid. A few crystals of potassium chlorate are added and, after stirring, the mixture on the watch-glass is evaporated to dryness on a boiling water-bath. The glass is cooled and the residue

moistened with a drop or two of 2N ammonium hydroxide. The colour of the residue becomes purple.

Purple:

Acepifylline, bamifyllinc, bufylline, caffeine, diprophyllinc, etamiphylline, proxyphylline, theobromine, theophylline.

IV

Sodium Hydroxide or Potassium Hydroxide

Violet:

Santonin. About 10 mg is warmed with 1 ml of a solution of potassium hydroxide in ethanol.

Blue:

m-Dinitrobenzene, 2,4-dinitrotoluenc. A drop of 2N alkali is added to an acetone solution of the substance. The blue colour changes to violet-red on addition of acetic acid.

Yellow:

Acetaldehyde. One or two drops are added to a large excess (1 to 2 ml) of 40% sodium hydroxide solution and the mixture is heated to boiling---- yellow turbid solution with a characteristic smell.

Nicotinamide. A little dry material is heated with 5% sodium hydroxide solution.

p-Nitrophenol. A drop of sodium hydroxide solution is added to a little dry material.

Orange:

m-Nitrophenol (the test is carried out as for p-Nitrophenol).

Orange-red:

Chloramphenicol. A little dry material is heated with 5% sodium hydroxide solution; ammonia vapour may be evolved.

Nitrofurantoin. 5 mg is dissolved in 5 ml of 0.1 N sodium hydroxide---- deep yellow changing to deep orange-red.

Nitrofurazone. 10 mg of the compound is dissolved in 10 ml of 20% sodium hydroxide solution.

Red:

o-Nitrophenol and phenolphthalein. A drop of sodium hydroxide solution is added to a little dry material.

2,4,6-Trinitrotoluene. A drop of sodium hydroxide solution is added to an acetone solution.

IV a

Sodium Hydroxide---- with iodine

Violet:

Aphanaphthol. The substance is added to a mixture of equal volumes of iodine solution in potassium iodide and 2N sodium hydroxide. The reagent mixture must contain a small excess of alkali. Betanaphthol does not react.

IV b

Sodium Hydroxide or Potassium Hydroxide---- with chloroform

Blue:

Alphanaphthol and betanaphthol. A little of the substance is heated with 2N sodium hydroxide and a few drops of chloroform.

Red:

Hydroquinone. A solution in chloroform is warmed with a small amount of solid potassium hydroxide. The mixture changes colour through yellow to red or red-brown.

IV c

Sodium Hydroxide---- with alphanaphthol or betanaphthol

Blue:

Chloroform. A drop of the liquid is heated with a small quantity of the naphthol in 2N sodium hydroxide.

IV d

Sodium Hydroxide---- with phenol

Blue:

p-Aminophenol. The substance is shaken with a solution of phenol in 2N sodium hydroxide.

IV e

Sodium Hydroxide---- with resorcinol

Red:

Chloroform. A drop of the liquid is heated with a small amount of resorcinol in 2N sodium hydroxide.

IV f

Sodium Hydroxide---- with potassium mercuric iodide; Nessler's reagent

The potassium mercuric iodide present in the reagent (see Appendix 1) reacts with ammonia to yield an orange flocculent mercuric complex. Substances which are hydrolysed in alkaline solution to yield ammonia give a positive response with this test. Equal volumes of the reagent and the test solution (aqueous) are mixed and heated gently.

Orange-brown:

Bromvaletone, chloramphenicol, and nicotinamide (all 3 yield the precipitate on heating above 40°), carbromal (on heating to 40°).

Dark brown:

Paracetamol (on heating to about 80°).

IV g

Sodium Hydroxide---- with sodium nitroprusside)

One or two drops of a test solution is added to a dilute aqueous solution of sodium nitroprusside and 1 or 2 drops of 2N sodium hydroxide is then added.

Orange-red:

Ketones.

Red:

Acetaldehyde.

IV h

Sodium Hydroxide---- with pyridine; Fujiwara's test

A little of the solid or a drop of a test solution is added to a mixture of pyridine (1 ml) and 20% sodium hydroxide solution (2 ml). The mixture is heated on a boiling water-bath for 3 to 5 minutes with vigorous periodic shaking. A control test must be carried out. The colour appears in the pyridine layer.

Red:

Chloral hydrate; chloramphenicol, chlorbutol, chloroform, trichloroethane, trichloroethanol, trichloroethylene.

Note. Chloralose does not respond to this test unless it is first heated to 100° with an equal volume of sulphuric acid to hydrolyse it.

IV i

Potassium Hydroxide---- with dimethylformamide

Blue:

Furazolidone. 1 mg is dissolved in 1 ml of dimethylformamide and 0.05 ml of 1 N ethanolic potassium hydroxide.

V

Sodium Hypochlorite

Violet:

Aniline. Aqueous sodium hypochlorite solution is added drop by drop to an aqueous solution of aniline. The initial colour is brown but this is very transient and rapidly becomes violet. The test is sensitive to 1 part of aniline in 66,000 parts of solution.

Orange:

Aminoguanidine, guanidine, sulphanilamide, sulphanilic acid (these compounds are treated as for Aniline, above).

VI

Ammonia---- with vanillin

Yellow:

Nitrazepam. A drop of an acetone solution is spotted on filter paper and exposed to ammonia vapour. The bright yellow colour fades when the vapour is withdrawn. Solutions of nitrazepam and its salts are turned yellow by the addition of ammonium hydroxide solution.

Aloin. 5 ml of dilute ammonia solution is added to 5 mg of the substance; the mixture turns yellow, green, and finally brown. The solution exhibits a yellow fluorescence in ultraviolet light.

VI a

Ammonia---- with copper sulphate

Purple:

Proguanil. To 10 ml of a saturated solution of proguanil hydrochloride is added 1 drop of 10% copper sulphate solution and 2-5 ml of dilute ammonia solution and (lie mixture is shaken well. 5 ml of benzene is then added and the mixture is again shaken. The upper benzene layer is coloured purplish-red.

VII

Ferric Chloride

Usually 1 or 2 drops of a 5% aqueous solution is added to a solution of the substance under test in ethanol or acetone. Water may be used as a solvent but only when the test material is readily soluble, since dissociation of the coloured iron complex may rapidly occur and the colour will not be seen.

Violet:

Salicylates---- aminosalicylic acid (in water), salicylaldehyde, salicylamide, salicylic acid, salicyluric acid; also amidopyrine, dipyrone (the ferric chloride solution is added to fragments on a tile), hexachlorophane (transient).

m- and p-Nitrophenol. The solution in water is boiled and cooled before the ferric chloride solution is added to it.

Saccharin. This substance is kept for ½ minute in a molten mixture with potassium hydroxide. The melt is then dissolved in water, acidified, and treated with the reagent.

Streptomycin. A little streptomycin sulphate is boiled with 1 N sodium hydroxide, then just acidified with hydrochloric acid and treated with ferric chloride solution.

Blue:

Alphanaphthol, gentisic acid (ink-blue), guaiacol, morphine, paracetamol, parachlorophenol, phenol.

Green:

Betanaphthol, hexylresorcinol, hydroquinone (transient), hydroxyquinoline, paraphenylenediamine (in water).

Adrenaline, ethylnoradrenaline, isoetharine, isoprenaline, noradrenaline, and norderrin (solutions on the gradual addition of 5% sodium bicarbonate solution turn from green to blue-violet and then red).

Yellow:

Gluconic acid (in water), glucuronic acid (in water), lactic acid (in water), pantothenic acid (after boiling with a few drops of sodium hydroxide and making just acid before adding the reagent).

Orange:

m-Aminophenol (in water).

Orange-red:

Acetates and propionates. A few drops of ferric chloride solution are added to neutral solutions of the substances. The colour due to propionate will pass into amyl alcohol on shaking but that due to acetate remains in the lower aqueous layer.

Red:

Pentachlorophenol, phenazone, thiourea (after keeping the solid molten for ½ minute).

Brown:

o- and p-Aminophenol (in water).

VIII

p-Dimethylaminobenzaldehyde

The test is usually carried out by spraying dried spots of the test solution on filter paper or chromatographic paper with a 1% solution of the reagent in a mixture of ethanol: hydrochloric acid (1:1). Heating the substance under test with a 1% solution of p-dimethylaminobenzaldehyde in sulphuric acid is sometimes used as a colour test.

Note. Most of these reactions can be carried out as Microtests---- see p. 133.

Purple:

Ergot alkaloids including lysergide and dimethyltryptamine and its derivatives (including psilocybin). The colour appears on warming the paper in hot air.

Chlordiazepoxide. Heating (he paper in a closed dish on a boiling water-bath for 30 to 40 minutes is necessary to bring up the colour.

Yellow:

Most compounds containing a primary aromatic amino-group, e.g. aminosalicylic acid, anileridine, aniline, procaine, and sulphanilamide, respond to this test.

Bromvaletone and carbromal. The colour develops overnight but is pale.

Carbamates.

Phenacetin. The colour develops on standing overnight.

Phenacemide. The colour develops on warming.

Sulthiame. The paper is dried in warm air and the background faded by dipping the paper in 2N ammonium hydroxide.

Orange:

Paracetamol. The colour forms on standing overnight.

Red:

Cannabis extracts. The material is warmed in a tube with 1 or 2 drops of ethanol and a few drops of a solution of p-dimethylaminobenzaldehyde in sulphuric acid.

IX

Copper Sulphate---- Copper sulphate test

If one drop of a 1% solution of copper sulphate is added to a 1% solution of the substance under test in 0.1N sodium hydroxide, the following colours are obtained.

Blue:

Phthalylsulphacetamide, sulphacetamide (pale blue), sulphaguanidine, sulphanilamide, sulphaphenazole (greyish-blue).

Green:

Phthalylsulphathiazole, succinylsulphathiazole (greyish-green), sulphadimethoxine and sulphaquinoxaline (yellowish-green), sulphadimidine (turning to reddish-brown), sulphaethidole, sulphafurazole, sulphamerazine (turning dark brown), sulphamethizole, sulphamethoxazole, sulphamoprinc (turning dull orange), sulphapyridine (turning brownish-green), sulphasomidine, sulphormethoxine (pale green).

Brown:

Sulphadiazine (turning purplish-brown), sulphamethoxydiazine (purplish-brown), sulphamethoxypyridazine (greenish-brown), sulphasalazine (orange-brown), sulphathiazole (purplish-brown), sulphasomizole.

IX a

Copper Sulphate---- with sodium hydroxide; Chen's test

To one drop of a 1% solution of the compound in 1% acetic acid add 1 drop of 1% copper sulphate solution followed by 1 drop of 2 N sodium hydroxide. This test can be carried out on the microscale but the technique is not very satisfactory.

Purple:

Bamethan, clorprenaline, ephedrine, metanephrine, metaraminol, methylephedrine, normetanephrine, norpseudoephedrine, orciprenaline (changing to green), oxedrine, phenylephrine, phenylpropanolamine, pseudoephedrine.

Green:

Adrenaline (changing to greenish-brown), adrenolone (changing to orange then yellow-brown), ethylnoradrenaline (grey-green), isoetharine (grey-green changing to red-brown and then to yellow-brown), isoprenaline (changing to brown), noradrenaline (changing to greenish-brown), nordefrin.

IX b

Copper Sulphate---- with sodium nitrite; McNally's test

About 1 mg of the substance is dissolved in a few drops of acetone and 1 to 2 ml of water is added. One or two drops of a 0.5% solution of copper sulphate in 10% acetic acid is added, followed by a pinch of solid sodium nitrite. The mixture is shaken, heated gradually to boiling, and maintained at boiling for a few minutes.

Red:

Aspirin and salicyclic acid.

Note. Other salicylates, such as salicyluric acid and salicylamide, give only a yellowish-orange colour, which is not diagnostic. Aminosalicylic acid gives a chocolate-brown precipitate.

X

Bromine and Ammonia---- Thalleioquin test

A drop of an aqueous solution of the substance is placed on a white tile and...

...has disappeared and the solution is just tinted yellow, the mixture is exposed to ammonia vapour. The test can be carried out on the microscale (see p. 133).

Green:

Hydroquinidine, hydroquinine, quinidine, quinine.

Cinchonidine and cinchonine do not give a positive reaction with this test. Note. A test involving the use of bromine and ammonia is also used for the identification of purines on paper chromatograms---- see the Clarke-Kalayc test. Appendix 2.

XI

Alkaline Cobalt Test---- Zwikker's test

This is a well-established presumptive test for barbiturates and structurally similar compounds. In essence it consists in mixing solutions of a cobalt salt (acetate or nitrate) with a solution of the substance under test in the absence of water and then making the mixture alkaline without introducing water. The colour produced by a positive response is a deep violet-blue. With barbiturates the colour is fairly persistent (i.e. it is retained for several minutes) but with certain other compounds it rapidly fades to a dirty slate-blue. The solvent medium used in this test is usually anhydrous ethanol or methanol and the alkalinising agent may be ammonia vapour, sodium hydroxide, lithium hydroxide, or even barium hydroxide.

Two methods have been found to be generally useful for rapid testing in a small tube. (1) Ethanolic solutions of cobalt acetate or nitrate and the substance under test are mixed and then shaken with a pellet of sodium hydroxide. The test produces better results when the cobalt solution is fairly weak and the barbiturate is in slight excess. (2) To a pinch of the powdered substance, 1 ml of 0.2% cobalt acetate solution in methanol is added and, after agitation to dissolve the solid, 1 ml of a 1% solution of lithium hydroxide in methanol is added.

Violet-blue:

Amylobarbitone and the other barbiturates (the colour persists for 5 to 10 minutes), bemegride (the colour persists for 1 to 2 minutes), glutethimide (the colour persists for a few minutes), phenytoin (the colour fades in 30 to 60 seconds), primidone (the colour is only transient).

The formation and persistence of the violet-blue colour with Zwikker's test is therefore a good indication of the presence of a barbiturate.

XII

Iodoform Test

To 2 ml of a dilute solution of the test material is added a few drops of 2 N sodium hydroxide; several drops of a 0.1 N solution of iodine in potassium iodide is then added until a persistent yellow colour appears. The appearance of yellow

crystals of iodoform on gentle heating indicates the presence of acetaldehyde, acetone, or ethanol. A positive response is also given by ethyl methyl ketone, isopropanol, lactic acid, and a number of quinones, quinols, and m-dihydric phenols.

XIII

Paraformaldehyde---- Phosphoric Acid Test

A drop of 1% paraformaldehyde in 90% phosphoric acid is added to a small crystal placed on a white tile. The test may also be carried out on the microscale and the reagent may be used to detect spots on paper chromatograms. The test is very specific for solanidine and solanine.

Purple:

Phenothiazines.

Steel-blue:

Solanidine, solanine.

Grey:

Deserpidine, yohimbine.

Yellow:

Colchicine, demecolcine.

Orange:

Ergotamine, pentaquin, propantheline, thebaine.

MICROTESTS

Many colour tests may be carried out on the microgram scale using the method developed by Clarke and Williams (1955) which involves a technique similar to that described for Microcrystal Tests (p. 135). Microrods are made by heating pieces of glass rod, each about 20 cm long and 0.5 cm in diameter, in (lie middle and pulling them out until the diameter at the thinnest point is about 0.1 cm. They

are then broken at this point, and the ends ground flat (Fig. la, p. 136). When the narrow end of such a rod is allowed to touch the surface of a liquid, and then removed, it brings away a small drop adhering to the glass. This pendant drop (fig. Ib, p. 136) is known as a microdrop, and is (lie standard quantity used for all the microcrystal and microcolour tests which are described in this book. It has a volume of about 0.1 md (Hawkins, 1959); a conventional drop (0.05 ml) will thus provide material for 500 different tests. It is imperative that the end of the rod should only touch the surface of the liquid and not penetrate it, as in the latter case there may be several droplets of indefinite size adhering to the sides of the rod and none to its end.

Tests are carried out by transferring a microdrop of the test solution to a small piece of opal glass and adding a microdrop of the appropriate reagent. The residue left after evaporation of the drop may be almost invisible: it is therefore advisable to indicate the location of the first drop on the glass with a pencil mark.

Sulphuric Acid---- formaldehyde Test (Marquis Test). A microdrop of the test solution is placed on the glass, allowed to evaporate to dryness, and a microdrop of Marquis reagent is added.

Ammonium Molybdate Test (Fröhde's Test). A microdrop of a 0.5% aqueous ammonium molybdate solution is added to a microdrop of the test solution and the residue after evaporation is moistened with sulphuric acid.

Ammonium Vanadate Test (Mandolin's Test). A microdrop of a 0.5% aqueous solution of ammonium vanadate is added to a microdrop of the test solution and the residue after evaporation is moistened with sulphuric acid.

Diazotisation Test. This test detects compounds containing a primary aromatic amino-group. To a microdrop of the test solution are added successively microdrops of 1 N hydrochloric acid, a 1% aqueous sodium nitrite solution, and a 4% solution of betanaphthol in 2 N sodium hydroxide.

Vitali's Test. A microdrop of the test solution is allowed to dry and a microdrop of fuming nitric acid is added. The colour (A) is noted; the drop is evaporated to dryness on a boiling water-bath and the colour (B) is noted; the residue is moistened with freshly prepared ethanolic potassium hydroxide solution and the colour (C) is noted. The colours are recorded as A/B/C. It should he noted that many compounds give various shades of yellow, orange, and brown which are of little diagnostic value.

OTHER TESTS

The following tests have been designated as micro tests in the monographs in Part 2 but they may also be performed on the macro scale. They are described in the General Tests section---- p-dimethylaminobenzaldehyde test (VIll, p. 130), ferric chloride test (VII, p. 129), paraformaldehyde-phosphoric acid test (XIII, p. 132), sulphuric acid test (I, p. 123), and thalleioquin test (X, p. 131).

Treatment of Bromides and Iodides

The colours recorded in Part 2 for the microtests with sulphuric acid are those obtained with the free base. In the case of a salt these may be modified by the nature of the acid radical present; bromides and iodides, for example, usually give a green or blue colour with the ammonium molybdate test. In the case of a compound extracted from biological material no difficulty will arise, as it will be extracted as the base, but care must be taken in applying colour tests directly to pharmaceutical preparations. These may, of course, be converted to the free base but it is often quicker to convert bromide or iodide to nitrate, which gives the same colour as the base. This may be done as follows. To 0.5 ml of a 1% solution of the salt in dilute acetic acid add 1 drop of an 8% solution of silver nitrate. Centrifuge off the precipitated silver halide, decant the supernatant liquid, and add to it 1 drop of a 2% solution of sodium chloride in order to remove excess silver. Centrifuge again, and use the supernatant liquid for the colour tests.

SENSITIVITY OF MICROTESTS

As the volume of a microdrop is 0.1 ml, it follows that a microdrop of a 1% solution will contain 1 mg, a microdrop of a 0.5% solution 0.5 mg, and so on. The sensitivity of a test refers to the smallest quantity of a substance that will give a positive result in that test. This may be stated as the concentration of the solution

used or the actual weight of the test material present in a microdrop of such a solution. Thus, if the most dilute solution to give a positive result is 1 in 1000, the sensitivity may be recorded as 1 in 1000 or as 0.1 mg. The former convention has been adopted for Microcrystal Tests (p. 135) which are carri