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.

Current undergraduate environmental chemistry curricula often suffer from a disconnect between sample preparation and large-scale instrumental analysis， making it difficult to cultivate students’ systematic analytical capabilities. Furthermore， the experimental content predominantly focuses on conventional water quality parameters and traditional pollutants like heavy metals， lacking training in monitoring and remediation technologies for new pollutants， which creates a significant gap with national strategic needs. To address this issue， this study designed an integrated comprehensive experiment that combines material synthesis， sample pretreatment， and large-scale instrumental analysis， targeting typical new pollutants， such as organic ultraviolet filters （OUVFs） from pharmaceuticals and personal care products. A magnetic covalent organic framework （MCOF） was synthesized via a reflux heating method. Students were guided to apply it as the sorbent in magnetic solid-phase extraction （MSPE）. Owing to its facile magnetic separation， the rapid enrichment of OUVFs was achieved within 8 min， followed by quantitative determination of seawater samples using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. Through a three-stage teaching module encompassing “pre-class preview， in-class experiment， and post-class discussion”， the experiment emphasizes enhancing students’ independent exploration abilities in adsorbent synthesis and characterization， optimization of enrichment parameters， and data analysis. Teaching practice has shown that this design not only enabled students to deeply understand the complex sample pretreatment process and the mechanism of multi-factor synergistic effects， but also significantly enhanced their comprehensive innovation and practical abilities in solving new pollutant monitoring problems.

p-Phenylenediamine compounds （PPDs） are widely used as anti-aging agents and antioxidants in rubber industry. However， environmental concerns have arisen regarding the toxicity of their quinone derivatives （PPD-Qs）. Fine particulate matter （PM_2.5） serves as a crucial vector enabling PPDs and PPD-Qs to enter the human body via respiratory exposure pathway. Therefore， accurate monitoring of PPDs and PPD-Qs in PM_2.5 is essential. In this research， a high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） approach was established to quantify seven PPDs and seven PPD-Qs in PM_2.5. Samples were collected on quartz fiber filters with a diameter of 90 mm. The filter was cut into strips and subsequently spiked with 2 ng of 6PPD-d₅ and 6PPD-Q-d₅ internal standards. The target compounds were extracted with 10 mL of acetonitrile containing 1% （volume fraction） ammonia solution and 20 μmol/L glutathione （GSH）， and purified via a modified QuEChERS pretreatment method. Specifically， after salting out with 0.5 g of NaCl and 1.0 g of MgSO₄， the supernatant was subjected to dispersive solid-phase extraction for cleanup. In this process， 0.5 g of MgSO₄， 100 mg of octadecylsilane （C₁₈）， and 100 mg of N-propylethylenediamine （PSA） were employed as sorbents. Subsequently， 5 mL of the cleaned-up supernatant was evaporated to near dryness and then redissolved in 0.2 mL of a methanol-water mixture （1∶1， volume ratio） for instrumental determination. The compounds were separated on a Waters ACQUITY UPLC HSS T₃ column（100 mm×2.1 mm，1.8 μm） with mobile phases consisting of a 5 mmol/L ammonium formate aqueous solution containing 0.1% formic acid and methanol. Identification and quantification of the compounds were carried out under positive electrospray ionization in multiple reaction monitoring mode. The results indicated that all analytes exhibited good linear relationships within the range of 0.05-20.0 ng/mL， with correlation coefficients （r） exceeding 0.999. The method detection limits （MDLs） and method quantification limits （MQLs） of the compounds were within the ranges of 0.003-0.07 pg/m³ and 0.01-0.2 pg/m³， respectively. At three spiked levels of low， medium and high， the recoveries of the compounds were in the range of 66.3% to 119.0% ， and the relative standard deviations （RSDs） ranged from 1.5% to 13.2% （n=6）. Ultimately， the developed method was employed for the analysis of PPDs and PPD-Qs in PM_2.5 samples collected from Baotou during January and February 2025. The results indicated that seven PPDs and five PPD-Qs were detected， with mass concentrations ranging from 2.26 to 251.2 pg/m³ and 2.36 to 105.4 pg/m³， respectively. The proposed method is characterized by its simplicity， environmental friendliness and accuracy， making it suitable for the rapid quantitative analysis of PPDs and PPD-Qs in PM_2.5.

Carrimycin is a macrolide antibiotic widely used in the pharmaceutical and clinical fields， with its primary composition consisting of three distinct derivatives of spiramycin. For decades， macrolides like carrimycin have been valued for their efficacy against Gram-positive bacteria and certain atypical pathogens. However， as research has progressed， recent studies have revealed that carrimycin， along with various other structurally modified spiramycin derivatives， exhibits significant antitumor activity in multiple experimental models. This finding suggests that the spiramycin molecular scaffold possesses intrinsic potential beyond its conventional antibacterial role. By strategically modifying the substituents attached to this core structure， it may be feasible to develop novel compounds with enhanced and more selective antitumor properties. Despite this promising outlook， research into the specific targets and comprehensive mechanisms underlying the antitumor effects of spiramycin derivatives remains notably insufficient. A major gap exists in the precise identification of their molecular targets within cancer cells and the detailed signaling pathways they modulate. This lack of mechanistic understanding poses a substantial barrier to the rational design， optimization， and clinical translation of spiramycin-based antitumor therapeutics. To systematically address this knowledge gap and elucidate the antitumor mechanism of this compound class， the present study adopted an activity-based protein profiling （ABPP） strategy. ABPP is a chemoproteomic approach that enables the direct identification of functionally active proteins that interact with small-molecule probes in a native biological context. As a first step， we designed and synthesized a novel， high-activity spiramycin derivative termed n-hexanoyl spiramycin （h-SPM）. Building upon the structure of h-SPM， we subsequently engineered and synthesized a structurally analogous activity-based probe. This probe was specifically functionalized with chemical handles （such as an alkyne group） compatible with ABPP methodologies， allowing for downstream bioorthogonal conjugation and enrichment steps. The experimental workflow began by incubating this active probe with live cancer cells. During this co-incubation period， the probe engaged with and covalently bound to its potential protein targets within the complex cellular environment. Following the interaction， cells were lysed， and the probe-labeled proteins were efficiently isolated and purified using affinity-based enrichment techniques—specifically via copper-catalyzed azide-alkyne cycloaddition （click chemistry） to immobilize them onto a solid support. The enriched protein pool was then subjected to in-depth analysis using liquid chromatography-mass spectrometry （LC-MS）. This analytical phase yielded detailed， proteome-wide information on the identities of proteins that interact with the h-SPM-based probe. To extract biological insights from the list of identified proteins， we performed comprehensive bioinformatic analysis using Gene Ontology （GO） enrichment. This systematic classification provided crucial information regarding the biological processes， molecular functions， and cellular components associated with the captured proteins. The functional annotations derived from GO analysis allowed us to evaluate and prioritize several promising candidate target proteins for further experimental validation. Through this integrated proteomic and bioinformatic approach， we successfully identified multiple potential cellular targets of h-SPM. Notable among these were amyloid precursor protein （APP） and low-density lipoprotein receptor （LDLR）， both of which are implicated in diverse cellular processes such as cell adhesion， signal transduction， and metabolic regulation. To confirm the biological relevance of these interactions， we employed Western Blotting experiments. These studies verified that the expression or post-translational modification states of APP and LDLR were altered in response to h-SPM treatment， thereby confirming their status as responsive molecular targets. After identifying and preliminarily validating APP as a key interactor， we proceeded to investigate its functional role in the drug’s mechanism of action. Using short hairpin RNA （shRNA）-mediated protein knockdown， we generated cancer cell lines with significantly reduced APP expression. Comparative analysis of drug sensitivity between these knockdown cells and their wild-type counterparts revealed a marked attenuation of h-SPM’s antitumor effects in the absence of APP. Complementary to this， cell staining assays， including immunofluorescence， were conducted to visualize morphological changes， alterations in cell viability， and the subcellular localization of relevant biomarkers following h-SPM treatment. Collectively， these functional experiments provided compelling evidence that APP plays a critical and indispensable role in mediating the antitumor activity of h-SPM. Our findings thus outline a preliminary model of the drug’s mechanism， likely involving cellular pathways regulated or influenced by APP. In summary， this study achieves two significant objectives. First， it establishes and validates a robust， generalizable target-screening platform based on ABPP， specifically tailored for the investigation of spiramycin-class compounds. Second， and more importantly， it delivers novel biological insights by pinpointing specific protein targets such as APP. The identification of these targets provides invaluable mechanistic clues and a solid theoretical foundation for the future development of this family of compounds. This work effectively advances spiramycin derivatives from compounds with observed phenotypic activity toward agents with an emerging mechanistic understanding， thereby paving the way for more targeted drug design and informed combination therapy strategies in oncology. Future studies will focus on delineating the detailed downstream consequences of APP engagement by h-SPM and exploring the therapeutic potential of other identified targets in preclinical models.

This paper designs an experimental scheme to meet the undergraduate experimental teaching requirements of the Pharmaceutical Engineering major. The content focuses on the detection of bifenthrin pesticide residue in tea by QuEChERS-GC-MS. The experimental contents include sample pretreatment， single-factor screening and response surface methodology optimization of QuEChERS experimental conditions， establishment of a GC-MS analysis method， determination of bifenthrin pesticide residues in tea samples， and experimental data processing. It aims to cultivate students’ ability to comprehensively apply modern analytical techniques to solve practical problems. Through this experiment， students can master the basic principles and operational skills of GC-MS instruments and equipment， and understand the complete process of pesticide residue analysis in real samples， and recognize the importance of sample pretreatment for the analysis of trace components in complex systems. Under the QuEChERS experimental conditions screened in the single-factor experiment， the recovery rate of bifenthrin in the tea samples was 85.9%. Under the QuEChERS experimental conditions optimized by the response surface methodology， the recovery rate of bifenthrin in tea samples can reach 90.33%. Among the 38 tea samples， bifenthrin was detected in 12 samples， with the maximum detected value of 0.911 mg/kg. None of them exceeded the maximum residue limit of bifenthrin in tea stipulated in the National Food Safety Standard GB 2763-2021. This teaching design involves learning relevant content before the experiment， training operational skills during the experiment， and conducting data analysis after the experiment. It can not only stimulate students’ interest in learning， but also cultivate their rigorous scientific thinking. It enables students to proactively face problems， actively analyze them and try to solve them throughout the entire learning process.

A supercritical fluid chromatography （SFC） method coupled with UV detection was developed for the separation of linagliptin and its S-enantiomer. The method was validated and successfully applied to detect the S-enantiomer in real pharmaceutical samples. The separation of the enantiomer was investigated using six different chromatographic columns， and different cosolvents were studied. Chromatographic conditions， including column temperature， backpressure， and flow rate， were optimized. The DAICEL CHIRALPAK AD-H column （250 mm×4.6 mm， 5 μm） was used for separation. Supercritical CO₂ served as mobile phase A， and ethanol-isopropanol （1∶1， volume ratio） containing 0.25% diethanolamine and 0.25% trifluoroacetic acid was used as mobile phase B. Isocratic elution was carried out at a ratio of A∶B=73∶27 （volume ratio） with a flow rate of 1.5 mL/min. The column temperature was set at 40 ℃， back pressure at 15 MPa， injection volume at 6 μL， and detection wavelength at 220 nm. Under these conditions， linagliptin and its S-enantiomer were separated with a resolution of 3.1 and good peak shapes. Both linagliptin and its S-enantiomer exhibited good linearity in the concentration range of 2–90 μg/mL， with correlation coefficients of 0.999　7 and 0.999　9 （n=8）， respectively. The limit of detection （LOD） for both was 0.8 μg/mL （S/N=3）， and the limit of quantification （LOQ） for both was 2 μg/mL （S/N=10）. The average recoveries of the S-enantiomer spiked at low， medium， and high concentrations in active pharmaceutical ingredients and tablets were 97.4% （RSD=1.1%， n=9） and 101.6% （RSD=1.2%， n=9）， respectively. S-Enantiomer was not detected in three batches of active pharmaceutical ingredients or in three batches of tablets from two different manufacturers. This study represents the first application of SFC for the separation of linagliptin and its S-enantiomer. The method is environmentally friendly， sensitive， and highly efficient， offering good repeatability of peak area. It provides a solid foundation for the quality control of linagliptin and the inclusion of the SFC method in pharmaceutical quality standards， while also offering a useful approach for the rapid separation and impurity control of other chiral drugs.

In analytical chemistry， separation and enrichment steps serve not only as critical stages in the analytical workflow， but also as core technologies that determine the accuracy， reliability， and applicability of analytical methods. However， in conventional experimental instruction， this step is frequently overlooked owing to time constraints and an overemphasis on instrumental techniques， leading to fragmented knowledge structures and hindering the development of integrated analytical thinking that connects theory with practice. To address this issue， this study， based on the “knowledge-ability-practice” three-dimensional integrated teaching objective， innovatively establishes a “positive-negative case-based teaching” model， using the solid-phase microextraction （SPME） of six nitrogen-containing pesticides from environmental water samples as the instructional case. By comparing fibers coated with non-polar polydimethylsiloxane （PDMS） and strongly polar polyacrylate （PA）， and employing high performance liquid chromatography with ultraviolet detection （HPLC-UV）， this study systematically investigates the matching relationship between analyte polarity and coating properties. In the positive case， the PDMS coating demonstrates superior extraction performance for weakly polar pesticides based on the “like dissolves like” principle， with a linear coefficient of determination R²≥0.993 7 and detection limits ranging from 0.019 to 0.17 μg/L. In contrast， in the negative case， the PA coating shows weaker extraction efficiency due to differences in the extraction mechanism， yielding detection limits between 0.066 to 1.069 μg/L. Based on spiked recovery tests conducted on actual river water samples from Lanzhou （recoveries： 81.5% to 117%）， a multi-dimensional evaluation system of “knowledge internalization， competency enhancement， and practical literacy” is established accordingly. Teaching feedback demonstrates that the students show a marked improvement in their understanding of the core principles， and their innovative thinking and problem-solving skills are effectively cultivated. The “contrasting-case-based” pedagogical approach effectively bridges theory and experiment while stimulating students’ investigative thinking. The study provides a replicable and scalable practical model for the reform of analytical chemistry experimental teaching， which holds significant value in cultivating high-caliber talents with rational method selection and problem-solving abilities.

Alkaloids represent a class of naturally-occurring nitrogen-containing compounds widely distributed across diverse plant species. Owing to their well-documented potential to induce adverse effects on human health， certain alkaloids are explicitly prohibited from being used in cosmetic formulations. The escalating global popularity of essential oil-based cosmetics， which commonly incorporate complex botanical extracts， presents a potential avenue for the inadvertent introduction of these prohibited substances. Consequently， the development of robust， sensitive， and efficient analytical methods for their monitoring is of utmost significance for consumer safety and regulatory compliance. This study devises a reliable， high-throughput approach for the simultaneous determination of 13 prohibited alkaloids in essential oil-based cosmetics. It integrates optimized QuEChERS sample preparation with ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. The sample preparation procedure was meticulously designed to maximize efficiency and minimize analyte loss. Chromatographic separation was accomplished on a Waters ACQUITY UPLC HSS T3 column （100 mm × 2.1 mm， 1.8 μm） maintained at a constant temperature of 30 ℃. The mobile phase was composed of （A） acetonitrile and （B） 0.1% （volume fraction） formic acid aqueous solution. A gradient elution program was implemented at a stable flow rate of 0.3 mL/min according to the following profile： an initial 5， a linear increase to 15 （0-2 min）， a rapid rise to 70 （2-4.5 min）， followed by an immediate reversion to the initial 5 （4.5-5.5 min）， which was maintained for re-equilibration until 7.0 min. The injection volume was 5 µL. Detection and quantification were conducted using a triple quadrupole mass spectrometer equipped with an electrospray ionization （ESI） source operating in positive ion mode （ESI⁺）. Data acquisition was carried out in the multiple reaction monitoring （MRM） mode to ensure superior specificity and sensitivity. For each of the 13 alkaloids， two specific ion transitions were monitored： one for quantitative analysis and the other for confirmatory identification. The method was rigorously validated in accordance with accepted analytical guidelines. All 13 target alkaloids displayed excellent linearity within a mass concentration range of 0.2 to 50 ng/mL， with correlation coefficients （R²） consistently exceeding 0.99. The limits of detection （LODs） and limits of quantification （LOQs）， determined with acceptable accuracy and precision， ranged from 1 µg/kg to 4 µg/kg and 2 µg/kg to 10 µg/kg， respectively. Method accuracy and precision were assessed through recovery tests at three spiking levels， with six replicates at each level. The mean recoveries for all analytes ranged from 83.9% to 119.1%， with associated relative standard deviations （RSDs） all being ≤7.3%， validating the method’s high reliability and repeatability. Systematic evaluation indicated that the matrix effects for the 13 analytes were negligible； hence， the solvent calibration curve was adopted for quantitative analysis. The practical applicability of the validated method was demonstrated through the analysis of 50 batches of commercially available essential oil-based cosmetics. This market survey encompassed 15 products specifically marketed for infants or children and 35 products intended for adult use. As a result， none of the 13 target prohibited alkaloids were detected in any of the tested samples above their respective LOQs. A particularly notable accomplishment of this work is the successful development of a sensitive and reliable quantification strategy for oleandrin， a potent cardiotoxic alkaloid for which standardized detection methods in complex cosmetic matrices such as essential oils have been conspicuously absent. In conclusion， this study successfully establishes a simple， rapid， sensitive， and robust QuEChERS-UPLC-MS/MS method. It is comprehensively validated and clearly well-suited for the routine screening， risk monitoring， and quality control of 13 prohibited alkaloids in a wide array of essential oil-based cosmetics. The method offers reliable technical support for regulatory bodies to enforce safety standards and for manufacturers to ensure the safety and compliance of their products， thereby effectively contributing to the protection of consumer health.

Organic ultraviolet absorbers （OUVAs） are a class of emerging contaminants that have garnered significant attention in recent years. In response to the characteristics of high detection frequency and low concentrations of OUVAs in water bodies， this study established an analytical method based on solid phase extraction （SPE） coupled with ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） for the simultaneous determination of 16 OUVAs （nine UV stabilizers and seven UV filters） in drinking water. The 500 mL water sample was pretreated with 25 mg ascorbic acid， acidified to pH 2-3， and spiked with 1 mL of 5 μg/L mixed internal standard working solution. Target compounds were enriched and concentrated using an HLB solid phase extraction cartridge （200 mg/6 mL）. Separation was achieved on an Acquity Premier BEH C₁₈ column （100 mm×2.1 mm， 1.7 μm） using a gradient elution with methanol and 2 mmol/L ammonium acetate aqueous solution as the mobile phases. Detection was performed using electrospray ionization in positive mode and multiple reaction monitoring （MRM）， with quantification carried out by the internal standard method. The precision and accuracy of the established method were evaluated using drinking water as the matrix. The results showed that all 16 OUVAs exhibited good linearity within their respective mass concentration ranges， with correlation coefficients （r） greater than 0.992. The method detection limits （MDL， S/N=3） were 0.03-5 ng/L， and the method quantification limits （MQL， S/N=10） were 0.1-15 ng/L. At spiked levels of 5， 20 and 50 ng/L， the recoveries of the target analytes ranged from 75.0% to 130.6%， with relative standard deviations （RSDs） ranging from 0.9% to 12.9% （n=6）. A total of seven source water samples and seven drinking water samples were analyzed using this method. The results showed that two OUVAs， 2-hydroxy-4-methoxybenzophenone （UV-9） and octocrylene （OC） were detected. In source water， the mass concentrations of UV-9 ranged from <MQL to 13.4 ng/L， while OC mass concentrations ranged from 5.0 to 32.23 ng/L. In drinking water， UV-9 was detected at 0.4 ng/L， and OC mass concentrations ranged from <MQL to 13.2 ng/L. This method is suitable for the trace analysis of 16 OUVAs in drinking water， demonstrating good accuracy and precision. It effectively improves the detection efficiency of OUVAs in water and can be used for the environmental monitoring and analysis of OUVAs in drinking water in China.

Quality control of traditional Chinese medicine （TCM） has always been a key and challenging issue in the field of its modernization research. This has posed a high demand for advanced separation materials due to the complex compositions. Covalent organic framework materials （COFs） are a new class of porous crystalline materials composed of multidentate organic units connected by covalent bonds. They have demonstrated significant application value in areas such as catalysis and chromatographic analysis. This study focused on developing a novel core-shell-type chromatographic stationary phase using 2，4，6-tris（4-aminophenyl）-1，3，5-triazine （TAPT） and 1，4-benzenedicarboxaldehyde （TA） as building units. The TAPT-TA-COF@SiO₂ core-shell composite materials were successfully prepared on the surface of silica microspheres using a multi-step polymerization strategy， in which the SiO₂ cores were fabricated using the polymerization-induced colloidal aggregation （PICA） method. The imine-linked TAPT-TA-COF@SiO₂ core-shell stationary phase was subjected to comprehensive physicochemical characterization and chromatographic evaluation experiments. The analytical techniques employed included scanning electron microscopy （SEM）， transmission electron microscopy （TEM）， energy dispersive spectroscopy analysis （EDS）， nitrogen adsorption-desorption isotherms， Fourier transform infrared spectroscopy （FT-IR）， and powder X-ray diffraction （PXRD）. The systematic characterization results clearly indicate that the prepared stationary phase exhibits excellent monodispersity， and the COF layer is uniformly coated on the surface of the SiO₂ core. TEM characterization demonstrated that the thickness of the COF materials on the surface of SiO₂ is approximately 110 nm. Furthermore， FT-IR spectra were collected and the results demonstrated that the characteristic stretching vibrations at 3 209， 2 927， and 1 515 cm^-1， attributed to N-H， C-H， and C=N stretching， confirm the condensation reaction between TAPT and TA. In the XRD pattern， the peaks observed at 16.6°， 18.9°， 25.2° and 27.5° were attributed to the COF material and were consistent with previous reports， thereby confirming the successful synthesis of this derivative. The N₂ adsorption-desorption isotherm analysis confirmed that the material possesses a typical mesoporous structure. Its specific surface area and pore size distribution are similar to those of the original porous SiO₂ microspheres， subsequently providing a structural basis for efficient chromatographic mass transfer kinetics. In addition， the chromatographic performance was investigated. And it was confirmed that the stationary phase was effectively used for the separation of representative neutral polar or non-polar compounds such as benzenes， alkylbenzenes， phthalate esters， formamides， and aniline mixtures. These compounds are separated due to hydrophobic interactions， π-π interactions， and the unique mesoporous structure in the reversed-phase chromatography mode. ACN-water （30∶80 or 40∶60， volume ratio） was selected as the mobile phase at a flow rate of 1 mL/min. The results of the methodological validation indicate that the intra-batch relative standard deviations （RSDs） of one TAPT-TA-COF@SiO₂ packed chromatographic column were less than 1.6%， demonstrating excellent preparation reproducibility. Furthermore， this stationary phase was applied to the quality control of traditional Chinese medicine. Specifically， it was used to determine the content of astragaloside Ⅳ in Astragalus according to pharmacopoeial records. The measured result met the pharmacopoeia standard of ≥0.08%. This research work not only provides new opportunities for advancing fundamental and applied research on novel COF stationary phases， but also helps to further promote in-depth research on the application of COF materials at the intersection of separation science and pharmaceutical sciences.