Quantitative estimation of eugenol in volatile oils, extracts, formulations and body fluids may be undertaken by a number of spectrophotometric and HPLC methods. Being a simple phenol, it can be quantified in clove oil by direct titrimetric procedures.

A. Method I

One of the earliest official methods for the determination of eugenol content of cloves involves treatment of oil with a known volume of alkali. Eugenol forms water-soluble salts with alkalis and this water-miscible complex moves into the aqueous phase. The volume of immiscible oil remaining is measured. This subtracted from the volume of oil taken will indicate the volume of eugenol that has moved into the aqueous phase as an alkali salt. The percentage of eugenol may thus be estimated.

1. Take 5 ml of clove oil in a 25 ml burette. Add 5% sodium hydroxide solution up to the ‘0’ mark of the burette. Being denser than water, clove oil remains on the top. Set aside for 3 h.
2. Note the volume of the immiscible portion of the oil directly from the burette. Let it be ‘a’.
3. (5-a) × 20 gives the percentage of eugenol in the sample of clove oil taken for analysis.

B. Method II (Spectorphotometric assay)

1. Sample solution: Dissolve 1 ml of eugenol in methanol : chloroform (95:5) taken in a 10 ml standard flask. Add sufficient amount of the same solvent and make up the volume to the mark. Dilute 1 ml of the solution to 10 ml and further dilute 1 ml of this to 1 ml using the same solvent. The resulting 1 µg/ml solution is the test solution.
2. Standard solution: Take 10 ml of standard sample of eugenol in a 100 ml standard flask and make up the volume with the methanol: chloroform solvent. Mix well and from this stock solution prepare 1, 2, 4 6, 8, 20 and 40 µg/ml solutions.
3. Determine the absorbances of the test and standard samples at 281 nm. Prepare a calibration curve of the standard eugenol. Estimate the percentage purity of eugenol based on its absorbance values from the calibration curve.