In contrast to synthetic compounds, natural products exhibit a remarkable array of structures and a broad spectrum of bioactivities [[1], [2], [3]]. Over time, natural products (NPs) have consistently served as a significant wellspring for new drug discovery. Cannabidiol, isolated from the Cannabis sativa L. plant, is used to treat tuberous sclerosis-associated epilepsy [4]. Colchicine, an alkaloid isolated from the Gloriosa superba plant, is a crucial component of the colbenemid drug for gout [5]; and the anticholinergic agent scopolamine, isolated from the Solanaceae plant, is used as the main ingredient in Transderm Scop [6]. Remarkably, approximately 70 % of drugs approved by the conclusion of 2019 were either directly or indirectly linked to natural products [7,8]. As a result, the thorough exploration of natural products represents an immensely effective strategy for identifying potential new drug candidates, particularly those sourced from traditional Chinese medicine (TCM), characterized by intricate compositional diversity and structural variation [[9], [10], [11], [12]]. The conventional approach for identifying natural products involves untargeted open-column chromatography followed by step-by-step separation and chemical structure elucidation [[13], [14], [15]]. However, this method considerably constrains the efficiency of natural product discovery, underscoring the need for more innovative and streamlined approaches [16,17].

Currently, the methodologies employed to identify NPs include both one-dimensional liquid chromatography (1D-LC) and multidimensional liquid chromatography coupled with mass spectrometry (MDLC-MS) [[18], [19], [20]]. Traditional one-dimensional separation approaches have been hampered by notable limitations in chromatographic separation efficiency and peak capacity [21]. A primary drawback of 1D-LC/2D-LC is the potential contamination of the stationary phase, ascribed to the direct injection of liquid plant extracts into the system. To prepare samples for 1D-LC/2D-LC separation, additional sample pretreatment techniques must be applied to enrich target compounds and eliminate unnecessary substances. Medium pretreatment liquid chromatography (MPLC) employs materials such as styrene-divinylbenzene matrix (commercial name for sale: CHP20P), polyamide, sephadex (commercial name for sale: LH-20), or silica gel as column packing materials, which is one of the additional sample pretreatment techniques [[22], [23], [24]]. A suitable pretreatment method is pivotal for enriching target compounds, eliminating unnecessary substances, and streamlining subsequent purification steps. Styrene-divinylbenzene matrix MPLC, distinguished by its wide range of applications, compatibility of the mobile phase, and online detection capabilities, is widely employed for the bioactive enrichment of diverse natural products [25,26]. Therefore, it was selected as the ideal technique for pretreating samples.

Two-dimensional liquid chromatography combines distinct separation mechanisms of stationary phases to enhance resolution and peak capacity [[27], [28], [29], [30]]. Compared to online 2D chromatography, offline 2D chromatography systems offer notable advantages, including solvent compatibility and user-friendliness, rendering them a potent tool for investigating natural products [31]. Notably, supercritical fluid chromatography (SFC) boasts compelling benefits such as low viscosity, high efficiency at a high flow rate, and environmental sustainability owing to its utilization of supercritical CO2 as the mobile phase [[32], [33], [34]]. Significantly, SFC techniques have been applied in separating natural products and isomers, demonstrating unique advantages in isomeric separation due to their distinctive selectivity compared to traditional reversed-phase liquid chromatography (RPLC) [35]. This distinct selectivity may lead to a considerable degree of orthogonality in RPLC × SFC two-dimensional systems. The magnitude of orthogonality between the two dimensions remains a critical determinant in 2D separation. Greater orthogonality heightens the separation selectivity and peak capacity. Usually, RPLC × SFC two-dimensional systems use different columns with diverse separation mechanisms. RPLC × SFC two-dimensional systems have slightly greater peak capacities than RPLC × RPLC systems (with two identical stationary phases in the first and second dimensions) [36].

In previous studies, several authors detailed the preparative isolation of NPs through 2D-LC or 2D-LC × SFC employing diverse stationary phases, focusing on NP purification using two columns based on distinct chromatography separation mechanisms [37,38]. However, the current research introduces an innovative approach involving offline RPLC × SFC with only a 7-X10 stationary phase for the preparative isolation of NPs. Taking natural diarylheptanoids as an illustrative example, this work represents, to the best of our knowledge, an inaugural report on the use of 2D RPLC × SFC with one stationary phase for the preparative isolation of NPs.