Orthogonal Separation of Propolis by Grafted Reversed‐Phase × Normal‐Phase Two‐Dimensional Thin‐Layer Chromatography With a Dedicated Image Processing Tool for Quantitative Performance Assessment

ABSTRACT

This study presents a grafted reversed‐phase × normal‐phase two‐dimensional thin‐layer chromatography (RP × NP 2D‐TLC) method for the analysis of propolis, a chemically complex natural resin. A dedicated MATLAB‐based application was developed to enable a systematic evaluation of 2D‑TLC separation performance in terms of positional repeatability, orthogonality, and resolution. The tool adapts a separation space method to assess orthogonality and implements three resolution algorithms specifically designed to accommodate the irregular spot shapes encountered in 2D planar chromatography. Applied to propolis analysis, the orthogonal system successfully separated 29 components, achieving an effective separation area of 44.8%, and 55.2% of the resolved components exhibited high resolution (>1.0). This platform provides a robust and accessible strategy for the rapid qualitative profiling and separation performance assessment of complex natural extracts.