关键词: 高效液相色谱, 混合模式, 固定相, 新型材料, 综述

Abstract:

High performance liquid chromatography （HPLC） remains a core separation and analytical technique in modern analytical chemistry. Boasting prominent advantages including high quantitative accuracy， rapid analysis speed， strong selectivity， and high sensitivity， it has deeply permeated key fields such as biochemistry， pharmaceutical R&D， food testing， environmental monitoring， and materials science， delivering crucial support for the accurate qualitative and quantitative analysis of target components in complex systems. However， with the increasing complexity of analytical samples in scientific research and industrial production， marked by enhanced matrix interference， an expanded polarity range of target compounds， and widespread coexistence of multiple components， the limitations of traditional single-mode chromatography （e.g.， reversed-phase liquid chromatography （RPLC）， hydrophilic interaction chromatography （HILIC）， ion-exchange chromatography （IEC）） have become increasingly pronounced. Specifically， RPLC exhibits insufficient retention and separation capabilities for highly polar compounds， HILIC struggles to handle hydrophobic substances effectively， and IEC is only applicable to the separation of ionic components. None of these single-mode techniques can meet the demand for efficient and comprehensive separation of complex samples. To address this technical bottleneck， mixed-mode chromatography （MMC） has emerged as a viable solution. Its core innovation lies in integrating two or more separation mechanisms into a single chromatographic column. Through the synergistic effects of functionalized stationary phases， MMC enables efficient separation and accurate analysis of complex systems， thereby significantly expanding the application scope of HPLC. This paper briefly elaborates on the separation mechanisms of four mainstream mixed modes， namely RPLC/IEC， RPLC/HILIC， HILIC/IEC， and RPLC/HILIC/IEC. It also summarizes in detail the key chemical reaction types for stationary phase preparation （e.g.， click chemistry reactions and free radical polymerization reactions）， typical packing structures， as well as the characteristics and preparation strategies of novel functional materials such as porous organic cages （POCs）， metal-organic frameworks （MOFs）， covalent organic frameworks （COFs）， carbon quantum dots （CQDs）， microporous organic networks （MONs）， and ionic liquids （ILs）. Based on the latest research findings from 2020 to 2024， this paper systematically reviews the application cases of the aforementioned four mixed-mode stationary phases in practical scenarios such as traditional Chinese medicine component analysis， environmental pollutant detection， food quality control， and pharmaceutical research and development. It also conducts an in-depth analysis of the technical advantages of these four mixed modes， as well as the limitations of some stationary phases， including insufficient stability under extreme pH conditions， complex preparation processes， and high costs for large-scale production. Finally， this paper outlines the core challenges currently confronting mixed-mode stationary phases， including cumbersome synthesis steps， easy degradation and inactivation of functional groups， and difficulties in mobile phase optimization. It further points out that future development trends should focus on simplifying preparation processes， developing environmentally friendly and smart responsive materials， and enhancing the feasibility of large-scale production. The aim is to provide theoretical reference and technical support for the design and development of novel high-efficiency stationary phases， and facilitate greater breakthroughs in mixed-mode chromatography technology in the field of complex sample separation.

Key words: high performance liquid chromatography （HPLC）, mixed-mode, stationary phase, new materials, review