Solvent mismatch between the two dimensions is one of the main limitations in two-dimensional liquid chromatography (2D-LC) and presents a significant challenge for method development. Although 2D-LC provides a powerful means to increase peak capacity for oligonucleotides analysis compared to conventional one-dimensional LC, solvent incompatibility remains a major obstacle that discourages broader development of such methods. In this work, we investigate a technical solution that can be easily implemented and that eliminates solvent mismatch effects during 2D-LC analysis of oligonucleotides. This approach is based on the total breakthrough behavior of oligonucleotides, which is a phenomenon that allows the injection of large volumes into the second dimension (2D) without peak distortion. In this work, we showed that under appropriate conditions, oligonucleotides exhibit total breakthrough behavior in HILIC. This behavior in HILIC is particularly advantageous, as the IP-RPLC × HILIC configuration offers improved mass spectrometry (MS) compatibility compared to IP-RPLC or HILIC × IP-RPLC. Assuming an IP-RPLC × HILIC configuration, we systematically investigated the composition of first-dimension (1D) fractions and the 2D-HILIC parameters influencing total breakthrough to identify the key factors promoting this behavior. Our results offer clear guidance for implementing successful IP-RPLC × HILIC conditions that avoid mismatch effects for oligonucleotides analysis while maintaining a high injection volume in the second dimension. This work demonstrates the potential of the total breakthrough strategy for implementing 2D-LC methods with HILIC as the second dimension.

The quantification of trace amounts of steryl glucoside (SG) in biodiesel presents a significant analytical challenge due to its low solubility and tendency to crystallize. This study introduces a dispersive liquid-liquid extraction method utilizing a deep eutectic solvent (DES) for the efficient pretreatment and extraction of SG from a palm oil (PO) biodiesel model. The optimized extraction conditions employed a DES composed of choline chloride and ethylene glycol (1:2 molar ratio), combined with isopropanol as a disperser solvent in a 1:1 weight ratio, and vortex-assisted mixing. High-performance size exclusion chromatography coupled with an evaporative light scattering detector was employed for the analysis of SG, using a mobile phase consisting of toluene, tetrahydrofuran, and acetic acid (100:6:0.25, v/v/v). The method demonstrated a limit of detection of 1.92µg/mL, a limit of quantification of 5.82µg/mL, and excellent linearity (R2=0.9928). When applied to commercial PO biodiesel (both crude and refined), the method yielded recovery rates ranging from 86.99% to 96.97%. These results demonstrate that dispersive liquid-liquid extraction with DES is an effective and environmentally friendly approach for SG extraction and analysis in biodiesel.

Wax deposition in petroleum systems is intrinsically connected with paraffin wax composition, yet their quantitative characterization by one-dimensional high-temperature gas chromatography with flame ionization detection (HTGC-FID), as prescribed by ASTM D5442, remains challenging. In this work, we systematically quantify the main sources of error affecting HTGC-FID analysis of paraffinic systems and benchmark the chromatographic results against differential scanning calorimetry (DSC). The results demonstrate that solvent-based sample preparation leads to nonhomogeneous solutions at the colloidal scale due to paraffin aggregation, introducing significant sampling errors that intensify with increasing molecular weight, whereas an additional error source arises from incomplete volatilization of heavy paraffins in HTGC. A trade-off between loss of detectability at high dilution and aggregation effects at high concentration was observed, impacting quantitative analysis. The absence of reliable retention time patterns for nonlinear paraffins highlights the intrinsic limitations of one-dimensional HTGC-FID for their structural assignment, emphasizing the need for more advanced chromatographic approaches for comprehensive wax characterization.

A chiral stationary phase-high performance liquid chromatography-tandem mass spectrometry (CSP-HPLC-MS/MS) approach was developed and validated for the first time to quantify quantification of eight components: the enantiomers of three chiral components notopterol, oxypeucedanin hydrate, oxypeucedanin (in total six configurations), and achiral components nodakenin, imperatorin, isoimperatorin, bergapten, and ferulic acid, in Notopterygii Rhizoma et Radix. By adjusting the types of chiral stationary phase and the composition ratio of the mobile phase, methanol-acetonitrile (75:25, v/v) was selected as the mobile phase (flow rate 0.5mL/min), and three chiral components notopterol, oxypeucedanin hydrate, and oxypeucedanin were successfully separated into their enantiomers with Chiralpak IG. The method was applied to analyze both plant extracts and rat plasma samples. A considerable variation in content was observed among the eight components in the plant extracts, with their individual concentrations covering a wide range from 1.04 to 20400 µg/mL (see Section3 for details). Similarly, their plasma concentrations also spanned from 0.2 to 262.76ng/mL. The results demonstrated significant differences in the contents of the components. Notably, the chiral components exhibited marked differences between their enantiomers, suggesting that chiral components should be considered in the quality assessment and control of natural products.

American ginseng fibrous roots, often discarded as bioprocessing waste, constitute a valuable source of ginsenosides with high recovery potential. This study established a systematic strategy to enrich ginsenosides from the fibrous roots using macroporous adsorption resins and to investigate the adsorption mechanisms. Among the nine resins tested, the non-polar HPD100 resin, characterized by its styrene-divinylbenzene copolymer matrix and high specific surface area, exhibited superior performance, with an adsorption capacity of 194.1 ± 5.8mg/g and desorption efficiency above 98.0 ± 1.9%. Dynamic column experiments optimized the operating conditions, achieving 74.9 ± 3.0% purity and 84.9 ± 3.7% recovery at a flow rate of 3 bed volumes per hour, 2.3 ± 0.2mg/mL sample concentration, and 80% ethanol eluent. The resin maintained stable performance over five adsorption-desorption cycles. Kinetic and thermodynamic analyses revealed that adsorption followed a pseudo-second-order model and Langmuir isotherm, with spontaneous and endothermic characteristics. Structural analyses, including scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy, revealed hydrogen bonding, van der Waals forces, and π-π stacking as key interactions. Molecular dynamics simulations revealed a thermally enhanced binding effect, demonstrating that elevated temperatures strengthen ginsenoside-resin interactions by increasing binding energy even as hydrogen bonding diminishes. This work elucidates the fundamental adsorption mechanism and establishes a theoretical basis for the high-value valorization of American ginseng by-products through a rationally designed, temperature-controlled strategy.

Magnolia officinals, owing to its significant anti-inflammatory effect, has been widely utilized in the field of traditional Chinese medicine. This study employed ultrafiltration-LC in conjunction with a deep eutectic solvent-enhanced counter-current chromatography method to achieve the separation and purification of three cyclooxygenase-2 inhibitors from M. officinals plant material. First, cyclooxygenase-2 inhibitors were discovered from M. officinals via ultrafiltration-LC. Response surface methodology was further employed to optimize ultrasonic-assisted extraction parameters. It was found the discovered cyclooxygenase-2 inhibitors could be significantly enriched under the optimized conditions. Using the deep eutectic solvent-enhanced counter-current chromatography, three target cyclooxygenase-2 inhibitors were successfully separated with a solvent system composed of n-hexane/ethyl acetate/DES/water (3:4:5:2, v/v/v/v). Ultimately, three cyclooxygenase-2 inhibitors, including 91mg of honokiol, 41 mg of 8-obovatol, 95 mg of magnolol were obtained from 500 mg sample. The prioritized magnolol was found to exhibit anti-inflammatory activity via NF-κB pathway phosphorylation cascade activation inhibition and NF-κB/p65 nuclear translocation prevention. It demonstrated the integration of ultrafiltration-LC with deep eutectic solvent-enhanced counter-current chromatography enables the efficient separation of bioactive molecules. It not only advanced the academic understanding of the anti-inflammatory active molecules of M. officinalis, but also created preconditions for the subsequent research, development, and clinical application of M. officinalis.

Automated liquid handling can achieve higher precision for low-volume dispensing than hand-held pipetting while increasing sample processing throughput and avoiding human error. Current commercial liquid-handling platforms range in cost from tens to hundreds of thousands of dollars, which can be prohibitive for smaller research groups. Home-built systems offer customized functionality but require significant technical knowledge and specialized parts. We reduced the burden of developing homebuilt systems by adapting the hardware and software of a low-cost open-source platform, the Opentrons OT-2. The modifications extend accurate pipetting for the OT-2 to the low nanoliter range, enabling sensitive sample preparation. We then augmented the capabilities of this platform by incorporating control of two-position valves and selector valves for autosampling applications. Our modified system is flexible and can be readily configured to meet the needs of novel LC applications. We demonstrated the system for both sub-microliter sample preparation and autosampling for nano-LC-MS applications. The software developed to control the modified system can be adapted to other custom liquid handling and separations platforms.

Metal-organic frameworks (MOFs) have emerged as highly promising stationary phases in chromatographic separation technologies due to their exceptional structural tunability, high surface areas, and tailored pore environments. However, the transformation of these intrinsic advantages into practical chromatographic performance is often hindered by kinetic mass-transfer limitations, hydrolytic instability, and restricted accessibility for bulky analytes. This review systematically summarizes recent advances in the design and application of MOFs for chromatography, focusing on three critical material development directions: nano-sized MOFs, highly stable MOFs, and hierarchically porous MOFs. We first summarize the synthesis strategies, scientific challenges, and representative breakthroughs for each category, highlighting the transition between material properties and performance. The subsequent discussion critically evaluates their applications in gas chromatography (GC), liquid chromatography (HPLC), and capillary electrochromatography (CEC), with a particular focus on structure-performance relationships and the realization of material stability and nanoscale effects into enhanced separation efficiency, selectivity, and operational lifetime. By bridging the gap between advanced material synthesis and practical separation needs, this review aims to provide a roadmap for the development of next-generation and high-efficiency MOF-based stationary phases.