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An image analysis system for thin-layer chromatography quantification and its validation.

Quantitation of thin-layer chromatography (TLC) using image analysis is attractive for its low cost and convenience. The image analysis is investigated by designing a digital imaging system with simple equipment, developing an image analysis software based on our algorithm, and validated the system in the TLC quantitative assay of cichoric acid present in Echinacea purpurea (L.) Moench. TLC used a polyamide thin-layer plate with chloroform-methanol formic acid-water (3:6:1:1) as the mobile phase and 3% (m/v) aqueous aluminum chloride solution as the visualization reagent. Images are acquired with a standard digital camera under a UV viewing lamp (365 nm) in a dark room. The three-dimensional gray scale digital image dataset (x, y, gray) is reduced to two-dimensional dataset (distance, accumulative gray) and then plotted as a curve. The area under the peak corresponding to the cichoric acid spot is integrated and used for quantitation. The whole method was validated by the assay tests of detection limit, calibration curve, repeatability, reproducibility, and recovery. The results showed that our digital imaging method and image analysis algorithm were applicable for the quantification of TLC. The whole method is convenient, efficient, and moderately accurate for the quantitative assay of cichoric acid present in Echinacea purpurea (L.) Moench.