For some substances, the colour reaction with a particular chemical reagent may be quite specific, but it is much more common for the colour to be produced by a class of compounds. Moreover, compounds that do not fall into the class may also give colours. For some of the tests, the colour reactions can be correlated with certain aspects of the chemical structure of a compound or group of compounds. However, anomalous responses often occur that cannot be explained on that basis. Some of these are noted in the colour tests described below, but it should be borne in mind that many others may be found. It follows that colour tests are only an indication of the presence of a compound or class of compounds and that all tests must be confirmed by more specific methods. This is especially important in forensic cases!
The colour tests included here range from those that rely on reactions with certain functional groups (e.g. Folin–Ciocalteu for phenols), those that are almost specific for a given group (e.g. FPN reagent for phenothiazines) through to those that give diagnostic colours with a wide range of compounds (e.g. Mandelin’s test and the Marquis test).
Interpretation of colour tests
Colours exhibited by these tests cannot be described with any accuracy. They may vary in intensity or tincture with the concentration of compounds in the test samples and the presence of extraneous material. In addition, their assessment is always a subjective one, even in people with normal colour vision. Some of the complexes formed are unstable such that the colour changes or fades with time.
Effects of ionic form
Salts may give different colours to those of the corresponding acid or base. In general, free acids or bases that have been isolated from the test material by an extraction process give better colours than their salts. The colour of a salt may be modified by the nature of the other ions present. For example, all hydrochloride salts give a red colour in Mandelin’s test and a blue colour with Koppanyi–Zwikker reagent (prior to adding pyrrolidine). Basic salts of weak acids may produce different colours because of a change in pH. Where a compound has been extracted from biological material these factors should not create any difficulty, since it will be present in the form of the base. However, when applying the tests to pharmaceutical preparations, where the compounds are usually present as salts, this can cause problems. To overcome this, the material can be extracted in much the same way as for biological samples to derive the free base. Bromide and iodide salts can be converted into the nitrate before testing, which gives the same colour as the base, by the following method:
- To 0.5 mL of a 1% (w/v) solution of the salt in dilute acetic acid, add one drop of an 8% (w/v) solution of silver nitrate followed by one drop of a 2% (w/v) solution of sodium chloride to remove excess silver.
- Centrifuge to separate the precipitated silver halide and use the supernatant liquid, either as a solution or evaporated to dryness where necessary, for the colour tests.
The colours that are recorded in the tables and monographs are usually those obtained by testing either the free acid or base.
Use of the colour tests lists
The system adopted uses ten basic colours: the spectral colours (red, orange, yellow, green, blue and violet), together with pink, brown, grey and black. Where there is a variation in hue, this is indicated by combining two colours (e.g. red–brown). The second named colour is considered to be the dominant one and is the main colour used in the lists. For example, red–brown is listed under brown, whereas brown–red is listed under red. When interpreting results, it is often necessary to search the lists given under two main colours (e.g. for red–brown, the lists under both red and brown should be consulted). This takes account of the subjective nature of colour assessment. An arrow between two colours (e.g. red→brown) indicates that the colour changes during the course of the test. In the monographs, the notation brown/red is used where there are two parts to a test that produce two colours. Occasionally, the colour displayed by a test solution in reflected light may be different from that in transmitted light, in which case the solution is described as dichroic. A combined colour may be obtained when more than one drug is present or the drug itself is coloured, which limits the value of the tests for biological samples.
Performing the colour tests
The tests are carried out either in clear glass test–tubes or white glazed porcelain tiles (spotting tiles), which give a uniform background against which the colours can be assessed. For drugs, the tests are designed to work on about 1 mg, either as the solid form or a dried extract of this amount (see below), unless stated otherwise. Solutions should be made in water unless otherwise stated. Where an instruction, time, temperature, etc., appears in brackets after the drug name, such as (add water), (15 s) or (slowly at 100°), this indicates a change in the test procedure for that particular drug.
The following two recommendations are most important:
- A sample known not to contain the compound of interest should be tested at the same time as the test sample. This enables a comparison of the colours produced by the sample and by the reagent blank. Ideally, the blank sample should have the same matrix as the test sample (e.g. for urine tests use analyte–free urine), since this takes account of the effects of extraneous materials. Otherwise, water is usually adequate.
- Before making a final decision on the result of a test, the reaction of the unknown should be compared with that of a reference substance tested under exactly the same conditions.
Validation of a colour test
It is essential to validate all tests and test reagents for sensitivity and specificity; O’Neal et al. (2000) have outlined a suitable method for a chemical spot test.
Application of colour tests to sample extracts
Several solvent extraction schemes have been devised to fractionate compounds on the basis of their acidic, neutral or basic characteristics. The tests listed in Table 1 can be applied to the evaporated extracts.
Table.2 is formulated to give a quick lead to those tests that can be applied to detect some of the most important drug groups and other poisons.