Journal of separation science最新文献

The selection of a biphasic solvent system is the first and most important step for a successful countercurrent chromatography (CCC) separation. The partitioning coefficient of targets and their related impurities on one side, and the stationary phase retention on the other, are among the parameters used for solvent system selection. At constant temperature, the stationary phase retention of a selected solvent system in any CCC instrument is dominated by the rule of Du's plot, which describes the correlation between stationary phase retention and flow rate. In a biphasic liquid system, physico-chemical properties such as density, viscosity, and interfacial tension between two phases are temperature dependent. In this article, the effect of temperature on the retention of several typical solvent systems was investigated using a high-speed CCC (HSCCC) instrument. The results showed that temperature changes could induce density, viscosity, and interfacial tension variation of the biphasic system with consequent changes in the system's hydrodynamic behavior. As the temperature increased, the density and viscosity of the selected solvent system decreased. This led to a variation in the interfacial tension between two phases depending on the solvent system composition. The stationary phase retention increased when the temperature increased for the tested solvent systems. Based on the obtained results, an improved Du's plot was suggested, accounting for the contribution of operating temperature. The prospect of this article provides hands-on strategies for the development of high-performance separation method for HSCCC.

An integrated strategy combining comprehensive two-dimensional gas chromatography with chemometrics was established to characterize Aucklandiae radix (AR, Mu-xiang) and its complex formulations. By employing contour visualization, intelligent deconvolution, and in-silico retention index prediction, the method effectively resolved severe co-elution, separating at least five components from single peak vertices. A total of 55 characteristic compounds were identified in the AR essential oil. Comparative analysis revealed that AR and its substitute, Vladimiriae radix (Chuan-Muxiang), shared 21 components, while the former possessed 12 unique constituents compared to 47 in the latter. Chemical divergence was even more pronounced in other substitutes. Furthermore, although costunolide is a major component in the herb powder, it constitutes only 0.4% of the essential oil; thus, dehydrocostus lactone (or dihydrodehydrocostus lactone) and 1,8,11,14-heptadecatetraene were selected as quality markers. The application of these markers to Xiangsha-Yangwei pills from eight manufacturers yielded similarity values of 0.22-0.76. This quantitative profiling revealed significant quality inconsistencies, including the absence of critical components, demonstrating the method's utility for the comprehensive quality assessment of traditional formulas.

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 ± 5.8 mg/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 ± 0.2 mg/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.

Amlodipine besylate, a third-generation dihydropyridine calcium channel modulator, is routinely prescribed as a racemic mixture in the clinical management of arterial hypertension and ischemic chest pain. Among its enantiomeric pair, (S)-amlodipine is the pharmacologically active form and is primarily responsible for the antihypertensive effect. Given that both (R)- and (S)-amlodipine circulate at extremely low concentrations following oral administration, a highly sensitive analytical method is required for enantioselective monitoring. In this study, we developed a simple, sensitive, and cost-effective capillary electrophoresis (CE) method for clinical application, which integrates hydroxypropyl-β-cyclodextrin-mediated chiral recognition with acetonitrile-driven field-amplified sample stacking. Chiral resolution of trace amlodipine (AML) present in human plasma samples was performed in an uncoated bare fused silica capillary (effective length 31.2 cm, internal diameter 75 µm), thermostated at 25°C and operated with an imposed potential of 15 kV. The running buffer consisted of borate (6.25 mM) and phosphate (25 mM), adjusted to pH 2.5, containing 30 mg/mL hydroxypropyl-β-cyclodextrin, and ultraviolet monitoring was conducted at 200 nm. This assay was fully validated for the quantitative measurement of each AML enantiomer in plasma. For (R)- and (S)-amlodipine, linear calibration was achieved across the range 0.2475-19.80 ng/mL and met the commonly accepted bioanalytical criteria. Comparative pharmacokinetic evaluation demonstrated no statistically significant differences between the concentration-time profiles obtained with the proposed CE method and those generated by a reference liquid chromatography-tandem mass spectrometry assay, confirming the accuracy and reliability of the developed procedure for routine therapeutic drug monitoring.

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.

Lysionotus pauciflorus Maxim. is a traditional Chinese medicinal herb with antitussive, antiasthmatic, and antioxidant activities. However, systematic studies on its quality control methods remain limited, which restricts its further development and application. In this study, an integrated strategy combining separation and analytical techniques with chemometric methods was established to screen quality markers related to the antiasthmatic activity of L. pauciflorus. The chemical constituents of the extracts were systematically characterized using ultra-high performance liquid chromatography coupled with Orbitrap high-resolution mass spectrometry, and plasma pharmacochemistry was employed to investigate potential in vivo bioactive constituents. In addition, high-performance liquid chromatography fingerprint profiles of samples from different batches were established. Principal component analysis and orthogonal partial least squares discriminant analysis were applied to identify key differential components. To evaluate antioxidant activity and link it with chemical constituents, radical scavenging assays combined with high-performance liquid chromatography with diode array detection were conducted to identify potential antioxidant-related components. Furthermore, network pharmacology and molecular docking were used to construct a compound-target interaction network, from which candidate components related to antiasthmatic effects were screened. By integrating all the above results, a multidimensional "spider-web model" was constructed, and the regression area of each component was calculated to evaluate its overall contribution. Ultimately, eight quality markers were identified: chlorogenic acid, neochlorogenic acid, nevadensin 5-gentiobioside, nevadensin 5-O-β-D-glucopyranoside, forsythoside B, paraboside B, acteoside, and lysionotin.

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 0.5 mL/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 20 400 µg/mL (see Section 3 for details). Similarly, their plasma concentrations also spanned from 0.2 to 262.76 ng/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.

Minor deviations in biopharmaceutical manufacturing processes, such as those used for monoclonal antibodies (mAbs), can introduce structural modifications that alter protein efficacy and safety. Monitoring these changes is critical to ensure product consistency and efficacy. Ion-exchange chromatography (IEC) is widely employed to assess product quality by separating charge variants. However, the use of non-volatile salts in IEC makes direct identification and characterization of these variants difficult. In this study, we developed a multi-heart-cut IEC-reversed-phase liquid chromatography (RPLC) platform that enables the separation, online concentration, desalting, and fractionation of mAb charge variants. The system integrates a 10-port valve and a six-column selector valve, allowing automated collection of up to five charge variants within a single analytical workflow. Moreover, the platform can be directly coupled to mass spectrometry for the characterization of charge heterogeneity and glycosylation. Systematic optimization of the capture and buffer-exchange conditions, including evaluation of stationary phase types and mobile phase compositions, was performed to maximize overall recovery. This platform can also be used for other chromatographic modes, such as hydrophobic interaction chromatography or size-exclusion chromatography. This integrated multiple heart-cut IEC-RPLC platform provides a high-resolution and efficient platform for detailed characterization of mAb charge variants, facilitating the characterization of critical product quality attributes.

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.

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 91 mg 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.