Introduction

Chen-Kao test (also known as Chen) is used in the a testing procedure to distinguish ephedrine, pseudoephedrine, norephedrine, and methcathinone from amphetamine and methamphetamine. The latter two do not react with Chen’s test reagent. The Chen’s test results are shown in Table below.

Chen-Kao Reaction Theroy

The formation of a violet-coloured chelate complex with copper sulfate in alkaline medium is considered to be characteristic “selective” for phenylakylamines with vicinal amino- and hydroxyl-groups and has been in use in pharmaceutical analysis for the simple identification of ephedrine, pseudoephedrine, norephedrine and norpseudoephedrine. The result of the Chen-Kao reaction is a symmetrical (tetravalent) chelate complex (scheme below). The colour, solubility and the stability of this complex appear to be affected by the structural and steric differences of the alkylamine part of the molecule. As seen in the table, of all tested ephedrine-type compounds only ephedrine and pseudoephedrine give the typical violet solution. All other derivatives produce a blue precipitate.

A number of unrelated organic compounds, inter alia, pharmaceuticals, have been found to produce coloured, most often blue, complexes with Chen-Kao reagent, and minor modifications in the execution of the test have been introduced, e.g. variations in the nature and amount of the acid, the amount of copper sulfate and alkali used. To further improve selectivity, literature suggests the extraction of the complex formed in the first step of the reaction with ethyl ether or n-butanol. In this step, the violet complex is transferred to the organic layer, and the water phase turns usually blue.

Experiment

Chen-Kao Reagent Preparation

• Reagent 1: Glacial acetic acid 1ml is added to water 100ml (1%(volume/volume) an aqueous solution of acetic acid).
• Reagent 2: Copper(II) sulfate 1g is dissolved in water 100ml (1% (weight/volume) aqueous solution of CuSO4).
• Reagent 3: Sodium hydroxide 8g is dissolved in water 100ml (2N aqueous solution of sodium hydroxide).

Each reagent is stirred well until a complete dissolution.

The Chen’s Test

Chen’s reaction

A small amount (1-2mg of powder or 1-2drops of liquid) of the sample material is placed in the recess of the plate with holes.

• 2 drops of Chen’s 1st reagent (1% water solution of acetic acid) are added.
• 2 drops of Chen’s 2nd reagent (1% water solution of CuSO4*5H2O) are added.
• Then 2 drops of the 3rd reagent (2N water solution of NaOH) are added and mixed.

The coloring of the mixture is observed.

The ephedrine sample shows a characteristic purple color.

Samples 1, 2 and 3 are three different samples of ephedrine from different syntheses. They have different purity.

Their tests show a stable purple color.

Sample 4 is methamphetamine.

There is no reaction, the solution remained transparent, however, a bright blue precipitate is formed. It is copper hydroxide, which did not form a complex.

Sample 5 is crude methcathinone, has impurities. It shows a blue color, however, almost immediately changed color to orange-brown, which indicates the instability of the complex. The reaction is not too sensitive.

Among all ephedrine-related compounds, only ephedrine and pseudoephedrine make the typical stable violet color. Other ephedrine-related compounds make a blue to greenish-blue precipitate. The two ketoamines (cathinone and methcathinone) initially produce also blue-colored complexes with the Chen-Kao reagent. However, a slow transition of the initial color into yellow, followed by an orange-brown color can be observed with both compounds, thus indicating the instability of the complexes initially formed.

Conclusion

A brief analysis of the Chen-Kao test results allows the following conclusions:

• 1. The execution of the Chen-Kao reaction is simple, needs little practice and limited skills. Also, the violet colour obtained in the reaction is easy to define. For a correct execution, it is important to note that the typical colours develop relatively slowly, and that a good colour intensity requires a sample of a few milligrams of the substances tested.
• 2. Of all ephedrine-related compounds, only ephedrine and pseudoephedrine produced the typical, stable violet colour required by the testing procedure and the colour reference.
• 3. All other compounds tested produced a blue to greenish-blue precipitate. This precipitate could be seen as characteristic for the members of the ephedrine group other than pseudoephedrine and ephedrine itself. Hence, the Chen-Kao test appears to show a significant specificity within the ephedrine group. However, it is known from previously published cross-testing work that various pharmaceuticals not related to the ephedrine group may produce similar blue copper complexes.
• 4. The two keto-amines, cathinone and methcathinone, initially also produce blue-coloured complexes with the Chen-Kao reagent. However, a slow transition of the initial colour into yellow, followed by an orange-brown colour can be observed with both compounds, thus indicating the instability of the complexes initially formed, and an obvious decomposition of the two compounds under the alkaline conditions of this colour reaction.

Bibliography

• https://www.unodc.org
• https://en.wikipedia.org/wiki/Chen-Kao_reaction