Preparation of Standard Caffeine Solutions

Before preparation of a solution, all the glassware was soaked overnight with a chromic acid solution, washed thoroughly with water and detergent, then rinsed with distilled water and dried in the oven overnight, and transferred to desiccators before use.10

The standard solution was prepared according to the following procedure.

A 100 ppm stock standard solution of caffeine was prepared by dissolving 0.025 g of caffeine in 250 mL purified dichloromethane (CH₂Cl₂) or distilled water. After this, the aqueous phase working standard solutions of 1, 2, 3, 4, 5, and 6 ppm were prepared by serial dilution of the stock solution with distilled water in 100 mL volumetric flasks. The organic phase working standard solutions of 4, 8, 12, 16, and 20 ppm were prepared with dichloromethane in 50 mL volumetric flasks. After preparing both solutions, the content of caffeine solutions was thoroughly swirled for 15 min.

The λ_max was determined by scanning a single standard solution from the 200–600 nm range of the UV-Vis spectrometer and the absorbance versus wavelength of the standard solution was plotted against the corresponding reagent blank. Then, the calibration curve was analyzed using triplicate analysis to measure standard and sample solutions at a particular λ_max. The absorbance values were plotted against a working standard solution to generate a standard calibration curve at the specific λ_max of each solvent. Finally, the molar absorption coefficients of caffeine in dichloromethane and in distilled water were obtained. The quantitative amount of caffeine in samples (ppm) was then determined using the standard curve.18,21