Dye Retention on a Hybrid Fibroin-Silica Thin-Layer Chromatographic Stationary Phase: Mobile-Phase Dependent Selectivity and Dimensionless Transport Reconstruction

A microspherical hybrid fibroin-silica adsorbent (HFSA) was prepared and evaluated as a multifunctional stationary phase for the thin-layer chromatographic analysis of structurally diverse dyes. The material was synthesized by adding silk fibroin during the hydrolytic polycondensation of tetraethoxysilane (TEOS) and characterized using SEM-EDS, FTIR spectroscopy, XRD, and nitrogen adsorption-desorption analysis. HFSA exhibited a predominantly amorphous, microspherical, and porous structure, with a specific surface area of 318 m2/g and a total pore volume of 1.03 cm3/g. The chromatographic behavior of methyl orange, Sudan I, Sudan IV, and Rhodamine 6G was investigated by TLC using MPh1 [n-hexane:isopropanol (1:1, v/v)] and MPh2 [n-hexane:isopropanol:water:glacial acetic acid (1:1:0.5:0.1, v/v/v/v)]. Under MPh1, all four dyes exhibited greater apparent retention on HFSA than on microspherical silica gel (MSS), with the largest observed difference for Sudan IV. Introducing water and acetic acid markedly changed the migration order: Sudan IV and Sudan I became highly mobile, whereas methyl orange and Rhodamine 6G remained more strongly retained. A dimensionless retention transport reconstruction reproduced the experimental band-centroid positions and visualized condition-dependent migration without independently predicting experimental band broadening or resolution. Hydrogen bonding, hydrophobic association, van der Waals forces, π–π interactions, and possible electrostatic effects are proposed as contributions to retention; their individual roles were not experimentally distinguished. These findings demonstrate that HFSA provides a chemically diverse stationary phase that exhibits different retention behavior for the four investigated dyes under the two tested mobile-phase conditions.