[Development progress of stationary phase for supercritical fluid chromatography and related application in natural products].

Supercritical fluid chromatography (SFC) is an environment-friendly and efficient column chromatography technology that was developed to expand the application range of high performance liquid chromatography (HPLC) using a supercritical fluid as the mobile phase. A supercritical fluid has a temperature and pressure that are above the critical values as well as relatively dynamic characteristics that are between those of a gas and liquid. Supercritical fluids combine the advantages of high solubility and diffusion, as their diffusion and viscosity coefficients are equivalent to those of a gas, while maintaining a density that is comparable with that of a liquid. Owing to the remarkable compressibility of supercritical fluids, analyte retention in SFC is significantly influenced by the density of the mobile phase. Thus, the column temperature and back pressure are crucial variables that regulate analyte retention in SFC. Increasing the back pressure can increase the density and solubility of the mobile phase, leading to reductions in retention time. The column temperature can affect selectivity and retention, and the degree to which different analytes are affected by this property varies. On the one hand, increasing the temperature reduces the density of the mobile phase, thereby extending the retention time of the analytes; on the other hand, it can also increase the energy of molecules, leading to a shorter retention time of the analyte on the stationary phase. CO2, the most widely employed supercritical fluid to date, presents moderate critical conditions and, more importantly, is miscible with a variety of polar organic solvents, including small quantities of water. In comparison with the mobile phases used in normal-phase liquid chromatography (NPLC) and reversed-phase liquid chromatography (RPLC), the mobile phase for SFC has a polarity that can be extended over a wide range on account of its extensive miscibility. The compatibility of the mobile phase determines the diversity of the stationary phase. Nearly all stationary phases for HPLC, including the nonpolar stationary phases commonly used for RPLC and the polar stationary phases commonly used for NPLC, can be applied to SFC. Because all stationary phases can use the same mobile-phase composition, chromatographic columns with completely different polarities can be employed in SFC. The selectivity of SFC has been effectively expanded, and the technique can be used for the separation of diverse analytes ranging from lipid compounds to polar compounds such as flavonoids, saponins, and peptides. The choice of stationary phase has a great impact on the separation effect of analytes in SFC. As new stationary phases for HPLC are constantly investigated, specialized stationary phases for SFC have also been continuously developed. Researchers have discovered that polar stationary phases containing nitrogen heterocycles such as 2-EP and PIC are highly suitable for SFC because they can effectively manage the peak shape of alkaline compounds and provide good selectivity in separating acidic and neutral compounds.The development of various stationary phases has promoted the applications of SFC in numerous fields such as pharmaceuticals, food production, environmental protection, and natural products. In particular, natural products have specific active skeletons, multiple active groups, and excellent biological activity; hence, these materials can provide many new opportunities for the discovery of novel drugs. According to reports, compounds related to natural products account for 80% of all commercial drugs. However, natural products are among the most challenging compounds to separate because of their complex composition and low concentration of active ingredients. Thus, superior chromatographic methods are required to enable the qualitative and quantitative analysis of natural products. Thanks to technological improvements and a good theoretical framework, the benefits of SFC are gradually becoming more apparent, and its use in separating natural products is expanding. Indeed, in the past 50 years, SFC has developed into a widely used and efficient separation technology. This article provides a brief overview of the characteristics, advantages, and development process of SFC; reviews the available SFC stationary phases and their applications in natural products over the last decade; and discusses prospects on the future development of SFC.

Synthesis and chromatographic evaluation of pyrazinedicarboxylic anhydride bonded stationary phase.

A silica based hydrophilic stationary phase bonding with 2,3-pyrazinedicarboxylic anhydride and amino groups was synthesized via amino-acid anhydride ring opening reaction. The bonded groups could not only provide hydrophilic interaction, but also electrostatic, π-π and hydrogen bonding interactions, etc. The results of characterization with elemental analysis and solid-state 13C cross-polarization magic-angle-spinning NMR indicated the successful preparation of amino and carboxyl bonded stationary phase named ZAC. The ζ-potential of ZAC stationary phase showed the negatively charge was dominate at pH larger than 3.5. Chromatographic evaluation revealed that ZAC stationary phase behaved well under HILIC mode. It showed different selectivity and retention compared to some typical commercial columns, and it was validated by the separation of chitooligosaccharides, flavonoid glycosides, organic acids and alkaloid samples. Based on the different selectivity between ZAC stationary phase and C18 columns, ZAC stationary phase also showed different selectivity with C18. And it was verified by the separation of Lonicerae Japonicae Flos and Menispermi Rhizoma extracts.

Biomimetic Polymer-Based Method for Selective Capture of C-Reactive Protein in Biological Fluids.

Selective capturing and purification of C-reactive protein (CRP) from complex biological fluids plays a pivotal role in studying biological activities of CRP in various diseases. However, obvious nonspecific adsorption of proteins was observed on current affinity sorbents, and thus additional purification steps are often required, which could compromise the recovery of the target protein and/or introduce new impurities. In this study, inspired by the highly specific interaction between CRP and the cell membrane, an excellent anti-biofouling compound 2-(methacryloyloxy)ethyl phosphorylcholine and a highly hydrophilic crosslinker N, N'-methylenebisacrylamide were employed to fabricate a novel cell membrane biomimetic polymer for selective capture of CRP in the presence of calcium ions. Based on the polymer described above, a facile enrichment approach was established after systematic optimization of the washing and elution conditions. With its favorable properties, such as good porosity, weak electrostatic interaction, high hydrophilicity, and biocompatibility, the novel biomimetic polymer exhibits good specificity, selectivity, recovery (near 100%), purity (95%), and a lower nonspecific protein adsorption for CRP in comparison with commercial immobilized p-aminophenyl phosphoryl choline gel and other purification materials. Furthermore, the structural integrity and functionality of CRP in the elution fraction were well preserved and confirmed by circular dichroism spectroscopy, fluorescence spectroscopy, and immunoturbidimetric assay. Finally, the biomimetic polymer was successfully applied to the selective enrichment of CRP from sera of patients with inflammation and rats. The proposed novel enrichment approach based on the versatile biomimetic polymer can be used for effective CRP purification, which will benefit the in-depth study of its biological roles.

Biomimetic small peptide functionalized affinity monoliths for monoclonal antibody purification.

The rapid development of monoclonal antibodies (mAbs) in therapeutic and diagnostic applications has necessitated the advancement of mAbs purification technologies. In this study, a biomimetic small peptide ligand 3,5-di-tert-butyl-4-hydroxybenzoic acid-Arg-Arg-Gly (DAAG) functionalized monolith was fabricated through a metal ion chelation-based multi-step approach. The resulting monolith showed good chromatographic performance. Compared with the Ni2+ based IMAC monolith, the DAAG functionalized monolith exhibited not only excellent specificity but also higher dynamic binding capacity (DBC). The 10% DBC and 50% DBC for hIgG reached as high values as 26.0 and 34.6 mg/mL, respectively, at a ligand density of 8.8 µmol/mL, due to the high porosity and accessibility of the monolithic matrix. Moreover, the stability of the DAAG functionalized monolith in successive breakthrough experiments indicates that it has a promising potential for long-term use in mAbs purification. Finally, the DAAG functionalized monolith was successfully applied to the purification of trastuzumab or human immunoglobulin G (hIgG) from biological samples.