Optimized Thin-Layer Chromatography Coupled with Digital Image Analysis for Semi-Quantitative Screening of Salidroside in Rhodiola crenulata

Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba is widely used in traditional Chinese and Tibetan medicine. Salidroside, its principal bioactive component, must not be less than 0.50% on a dry weight basis according to the 2025 Chinese Pharmacopoeia, which specifies high-performance liquid chromatography (HPLC) for quantitative determination. HPLC is a reliable but requires specialized equipment and trained personnel, restricting its use for rapid screening in resource-limited settings. This study presents an optimized thin-layer chromatography (TLC) method coupled with digital image analysis for the semi-quantitative screening of salidroside in R. crenulata. Mobile phase composition and development distance were adjusted to improve practical throughput while retaining usable spot appearance. Under the final conditions, a chloroform–methanol–acetone–water (5.5:3.5:1:1, v/v/v/v) mobile phase and 12 cm development distance were used with iodine visualization and ImageJ densitometry. The recommended working range was 0.20–0.70 mg/mL, supported by plate-specific calibration, residual analysis, and within-plate precision. Process recovery and matrix effect experiments indicated acceptable accuracy for a screening method. Application to three independent batches of root material gave results consistent with HPLC within a predefined relative window of ± 10%, and the complete workflow could be completed in less than two hours. This approach provides a practical alternative for preliminary screening and quality control of salidroside content in R. crenulata under limited-resource conditions.