Enhanced pigment content estimation using the Gauss-peak spectra method with thin-layer chromatography for a novel source of natural colorants.

Alternative pigment sources that are harmless to human health and can be produced in an eco-responsible way are of great research interest. The experiments undertaken in this study were conducted using autumn leaves of Aesculus hippocastanum as potential novel colorant sources. This study focused on improving the Gauss-peak spectra method (a less expensive alternative to high-pressure liquid chromatography) in combination with thin-layer chromatography, leading to the development of a new methodology. The collected leaves were stored at two different temperatures: 20°C and -20°C. The data obtained by spectrophotometric scanning of the samples were analyzed using the Gauss-peak spectra method in the R program with three wavelength ranges: 350-750 nm, 390-710 nm, and 400-700 nm. The results were then assessed for statistically significant differences in the estimated concentrations for the different wavelength ranges regarding (1) total pigment, carotenoid, and chlorophyll concentration (two-sample t-test) and (2) concentration of each indicated pigment (two-way analysis of variance). The results were also tested for differences between the estimated concentrations of samples stored under the different conditions. The Gauss-peak spectra results with and without thin-layer chromatography were statistically compared using a paired t-test. The results showed that thin-layer chromatography greatly enhanced the efficiency of the Gauss-peak spectra method for estimating the major and minor pigment composition without generating high additional costs. A wavelength range of 400-700 nm was optimal for all Gauss-peak spectra methods. In conclusion, the proposed method is a more successful, inexpensive alternative to high-pressure liquid chromatography.