Validated TLC-Densitometry Method for Determination of Chlorogenic Acid In Coffee Leaves Extract

Abstract

Thin Layer Chromatography method has been developed and validated for determination of chlorogenic acid in coffee leaves extract. The method was developed using a mobile phase prepared with analytical grade solvents: formic acid, ethyl acetate, aquabidest (1:8:1.5 v/v/v). Chlorogenic acid reference material and samples were chromatographed using pre-coated TLC silica gel 60 F254 plates and followed by densitometric measurements of their spots at 335 nm. Regression functions were established over the range of 5.02-194.58 ng/spot with r = 0.998. The limit of detection and limit of quantitation were 16.22 ng and 48.66 ng, respectively. The method was selective with resolution value more than 1.5 and specific with the spectra correlation value for purity and identity check more than 0.99. The percentage RSD was found 0.99% for repeatability precision and 1.75% for intermediet precision. The accuracy of the method was determined through standard addition method by adding known quantities of standard chlorogenic acid to the pre analyzed test solution and the mean recovery was 99.72% ± 1.58%. This TLC Densitometry method was linier, sensitive, selective, specific, precise, accurate and can be used for routine analysis of chlorogenic acid.