Chromatographic Stationary Phases Research Articles

Homochiral organic molecular cage RCC3-R-modified silica as a new multimodal and multifunctional stationary phase for high-performance liquid chromatography.

In this work, homochiral reduced imine cage was covalently bonded to the surface of the silica to prepare a novel high-performance liquid chromatography stationary phase, which was applied for the multiple separation modes such as normal phase, reversed-phase, ion exchange, and hydrophilic interaction chromatography. The successful preparation of the homochiral reduced imine cage bonded silica stationary phase was confirmed by performing a series of methods including X-ray photoelectron spectroscopy, thermogravimetric analysis, and infrared spectroscopy. From the extracted results of the chiral resolution in normal phase and reversed-phase modes, it was demonstrated that seven chiral compounds were successfully separated, among which the resolution of 1-phenylethanol reached the value of 3.97. Moreover, the multifunctional chromatographic performance of the new molecular cage stationary phase was systematically investigated in the modes of reversed-phase, ion exchange, and hydrophilic interaction chromatography for the separation and analysis of a total of 59 compounds in eight classes. This work demonstrated that the homochiral reduced imine cage not only achieved multiseparation modes and multiseparation functions performance with high stability, but also expanded the application of the organic molecular cage in the field of liquid chromatography.

GC-MS analysis of eight aminoindanes using three derivatization reagents.

Aminoindanes are a class of novel psychoactive substances (NPSs) that have become more prevalent over the past decade. GC-MS is often utilized for identifying seized drugs and is well regarded for its ability to separate mixtures. However, certain aminoindanes have similar mass spectral data and require specific gas chromatographic stationary phases for separation. Derivatization is an alternative method that can be applied to GC-MS to enhance chromatographic results, providing more selective analysis in seized-drug identification. This study investigates derivatization techniques to provide options for forensic science laboratories in accurately identifying aminoindanes. Three derivatization reagents, N-methyl-bis(trifluoroacetamide) (MBTFA), heptafluorobutyric anhydride (HFBA), and ethyl chloroformate (ECF) were evaluated for the analysis of eight aminoindanes by GC-MS using two common gas chromatographic stationary phases, Rxi®-5Sil MS and Rxi®-1Sil MS. All three derivatization methods successfully isolated eight aminoindanes, including the isomers 4,5-methylenedioxy-2-aminoindane (4,5-MDAI), and 5,6-methylenedioxy-2-aminoindane (5,6-MDAI) that could not be differentiated prior to derivatization. Reduced peak tailing and increased abundance were observed after derivatization for all the compounds, and mass spectra of the derivatives contained individualizing fragment ions that allowed for further characterization of the aminoindanes. This excluded 4,5-MDAI and 5,6-MDAI as they shared the same characteristic ions and were only distinguishable by their retention times. All three derivatization techniques used in this study allow for successful characterization of the aminoindanes and give forensic science laboratories flexibility in their analysis approach when they encounter these compounds.

Preparation, characterization, and separation mechanism of a dehydroabietic-acid-based shape-selective chromatographic stationary phase1

Chromatographic stationary phases with molecular-shape selectivity are advantageous for the separation and analysis of geometric isomers. Herein, dehydroabietic acid is bonded on the surface of silica microspheres via 3-glycidoxypropyltrimethoxysilane to form a monolayer dehydroabietic-acid stationary phase (Si-DOMM) with a racket-shaped structure. Various characterization techniques indicate that Si-DOMM is successfully prepared, and the separation performance of a Si-DOMM column is evaluated. The stationary phase has a low silanol activity and metal contamination and a high hydrophobicity and shape selectivity. The resolutions of lycopene, lutein, and capsaicin on the Si-DOMM column confirm that the stationary phase exhibits high shape selectivity. The elution order of n-alkyl benzene on the Si-DOMM column indicates its high hydrophobic selectivity and suggests that the separation is an enthalpy-driven process. Repeatability experiments reveal highly stable preparation processes of the stationary phase and column and indicate that the relative standard deviations of retention time, peak height, and peak area are less than 0.26%, 3.54%, and 3.48%, respectively. Density functional theory calculations using n-alkylbenzenes, polycyclic aromatic hydrocarbons, amines, and phenols as model solutes provide an intuitive and quantitative description of the multiple retention mechanisms. The Si-DOMM stationary phase exhibits superior retention and high selectivity for these compounds via multiple interactions. The bonding phase of the monolayer dehydroabietic acid stationary phase with a racket-shaped structure has a unique affinity for benzene, strong shape selectivity, and good separation performance for geometrical isomers with different molecular shapes.

Particle Size Regulation of Single-Crystalline Covalent Organic Frameworks for High Performance of Gas Chromatography.

Although polycrystalline covalent organic frameworks (PCOFs) have already shown great potential as stationary phases for chromatography, irregular shape and size distribution of PCOFs make regulation of particle size of PCOFs for high separation performance impossible, which is accessible by the application of single-crystalline COFs (SCOFs). Herein, we showed preparation of three-dimensional SCOF (SCOF-303) bonded capillaries (SCOF-303-capillary) with different particle sizes (about 0.4-1.6 μm) and further investigated gas chromatographic separation ability of these SCOF-303-capillaries for isomers of xylene, dichlorobenzene, and pinene. It was found resolution and column efficiency of SCOF-303-capillaries for isomers decreased with the increase in particle size, mainly resulting from the weaker size-exclusion effect and higher mass transfer resistance of the larger particle size of flexible SCOF-303. The obtained SCOF-303-capillary (particle size of ∼0.4 μm) offered baseline separation of xylene isomers with the high resolution of 2.26-3.52, great efficiency of 7879 plates m-1 for p-xylene, better than PCOF-303-capillary, and commercial DB-5 and HP-FFAP capillary columns as well as many reported capillaries. This work not only shows the great potential of SCOFs for gas chromatography but also provides the theoretical direction for the design of the efficient COF based stationary phase by adjusting the particle sizes.

Extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance using supercritical fluid extraction followed by supercritical fluid chromatography.

In this paper, an off-line combination method of supercritical fluid extraction and supercritical fluid chromatography was developed for the selective extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance. The enrichment of target components was successfully achieved using supercritical fluid extraction with the following conditions (8% ethanol as co-solvent at 45°C and 30MPa for 30min). Taking full advantage of the complementarity of supercritical fluid chromatography stationary phases, a two-step preparative supercritical fluid chromatography strategy was constructed. The extract was firstly divided into seven fractions on a Diol column (250 × 20mm internal diameter, 10μm) within 8min by gradient elution increasing from 5% to 20% modifier (methanol) at 55ml/min and 15MPa. Then the seven fractions were separated by using a 1-AA or a DEA column (250 × 19mm internal diameter, 5μm) at 50ml/min and 13.5MPa. This two-step strategy showed superior separation ability for structural analogs. As a result, seven compounds, including four diphenylheptanes and three flavonoids with high purity, were successfully obtained. The developed method is also helpful for the extraction and isolation of other structural analogs of traditional Chinese medicines.

A Metal Organic Framework + Ionic Liquid Pseudophase System as a Gas Chromatography Stationary Phase

A Metal Organic Framework + Ionic Liquid Pseudophase System as a Gas Chromatography Stationary Phase

Performance evaluation of 2-undecylimidazole/propyl methacrylate bifunctional silica gel for mixed-mode reversed-phase/anion-exchange chromatography

In this work, 2-undecylimidazole (UI) and propyl methacrylate (PMA) dual-functionalized silica gel was prepared and applied as a mixed-mode chromatographic stationary phase. Successful preparation of this stationary phase (SiO2-UI-PMA) was proved through a range of characterization techniques. The chromatographic performance of the obtained SiO2-UI-PMA was investigated for the separation of environmental endocrine disruptors (phthalates, steroid hormones, bisphenols and alkylphenols), sulfonamides and food-borne stimulants in reversed-phase chromatography, as well as for the separation of inorganic anions in anion-exchange chromatography. The effect of column temperature, acetonitrile content, salt concentration and mobile phase pH on retention was studied to explore the separation mechanism, and the results indicated that this stationary phase provided multiple interactions with analytes. Compared with the commercial C18 column, the SiO2-UI-PMA demonstrated more preferable separation ability under the mobile phase conditions with a low content of acetonitrile, thereby acting as a good alternative candidate for the conventional reversed-phase stationary phase in chromatographic analysis. Furthermore, the SiO2-UI-PMA stationary phase exhibited high column efficiency (45 860 plates m−1 for progesterone) even though it was prepared from the non-serviceable silica based chromatographic packing materials.

Per-aqueous liquid chromatography stationary phase with poly(ethylene oxide-co-dimethylsiloxane) immobilized on mesoporous aluminized silica support

This work proposes an alternative to searching for HPLC stationary phases to separate polar compounds. The method involves preparing an aluminized silica support and thermally immobilizing a thin layer of pre-synthesized poly(ethylene oxide-co-dimethylsiloxane) to create a chemically stable and polar stationary phase. The polymer immobilized aluminized silica-based stationary phase has a mesoporous structure (10.5 nm of mean size pore), high surface area (374 m2 g−1), and highly regular spheres. The presence of alumina (1.6% Al) ensures high chemical stability of the material under phosphate mobile phases at high pH and temperature. Using highly aqueous mobile phases and retention mechanisms of per-aqueous liquid chromatography mode, separations of polar and non-polar compounds using the proposed stationary phase are fast and efficient (N/m > 70,000 plates m−1). The aluminized silica-based stationary phase provides a viable and effective alternative to commercial reversed phases designed for polar compounds.

Silica microparticles modified with ionic liquid bonded chitosan as hydrophilic moieties for preparation of high-performance liquid chromatographic stationary phases.

Two novel stationary phases, 1-(4-bromobutyl)-3-methylimidazolium bromide bonded chitosan modified silica and 1-(4-bromobutyl)-3-methylimidazolium bromide bonded chitosan derivatized calix[4]arene modified silica stationary phase, were synthesized using 1-(4-bromobutyl)-3-methylimidazolium bromide bonding chitosan as a polarity regulator solvingthe limitation of the strong hydrophobicity of calixarene in the application of hydrophilic field. The resulting materials were characterized by solid-state nuclear magnetic resonance, Fourier-transform infrared spectra, scanning electron microscopy, elemental analysis, and thermogravimetric analysis. Based on the hydrophilicity endowed by 1-(4-bromobutyl)-3-methylimidazolium bromide bonded chitosan, the retention mode of ILC-Sil and ILCC4-Sil could be effectively switched from the hydrophilic mode to a hydrophilic/hydrophobic mixed mode and could simultaneously provide various interactions with solutes, including hydrophilic, π-π, ion-exchange, inclusion, hydrophobic, and electrostatic interactions. On the basis of these interactions, successful separation and higher shape selectivity were achieved among compounds that vary in polarity under both reverse-phase and hydrophilic interactive liquid chromatography conditions. Moreover, the ILCC4-Sil was successfully applied tothe determination of morphine in actual samples using solid-phase extraction and mass spectrometry. The LOD and LOQ were 15 pg/mL and 54 pg/mL, respectively. This work presents an exceptionally flexible adjustment strategy for the retention and selectivity of a silica stationary phase by tuning the modification group.

End-modification of poly(ether-carbonate) copolymer by adamantane cages: An effective approach for improving the selectivity of gas chromatographic stationary phases

This work reports the investigation of a new poly(propylene-carbonate) copolymer terminated by the adamantane cages (APPC) as the stationary phase for gas chromatographic (GC) analyses. In GC, the selectivity of a stationary phase is the key factor that governs the column separation performance for analytes, particularly those of high similarity in structures and properties. As such, we employed more than a dozen of isomer mixtures of separation difficulty for investigating the separation performance of the APPC column, involving the isomers of alkanes, alkylbenzenes, halobenzenes phenols and anilines. Meanwhile, the column coated with poly(propylene carbonate) diol (PPCD) differing from APPC only in their terminal groups and two commercial columns coated with polyethylene glycol (PEG) and polysiloxane, respectively, were used as the reference columns. The separation results evidenced the distinctly advantageous performance of the APPC column over the reference columns. Moreover, the APPC column showed excellent repeatability and reproducibility with the relative standard deviation (RSD) values over the range of 0.01%–0.04% for run-to-run, 0.15%–0.28% for day-to-day and 3.4%–3.9% for column-to-column (n = 4). Its application to GC–MS analysis of the verbena essential oil demonstrated its separation advantages for a wide range of components in practical samples. Up to date, the adamantyl-terminated poly(ether-carbonate) copolymers have not been reported in any fields. Its high-resolution performance demonstrates the feasibility of adamantyl-terminated block copolymers as highly selective stationary phases for GC analyses, which offers a vast room for fundamental researches and applications.

Screening of a short chain antimicrobial peptide-LKLHI and its application in hydrogels for wound healing

Antibacterial peptides have been widely used in the field of antibacterial due to their biocompatibility. In this work, owing to quickly screen out peptides with antibacterial effects, the bacterial membranes of E. coli and S. aureus were extracted and fixed on self-made silica gel microspheres to prepare bacterial membrane chromatography stationary phase. We successfully screened antimicrobial peptides from a peptide library composed of one-bead-one-compound by bacterial membrane chromatography. The antibacterial peptide has an effective defense effect on gram-positive bacteria, gram-negative bacteria, and fungi. In addition, the antibacterial peptide has almost no hemolysis and cytotoxicity and other excellent biocompatibility and has excellent properties such as stability, broad-spectrum antibacterial, and promotion of wound healing，and HA hydrogel carrier loaded with antimicrobial peptides was prepared, which provided the application direction of antimicrobial dressings for antimicrobial peptides. In summary, this method can screen out polypeptides with antibacterial effects, and the screened-out antibacterial peptides are expected to be applied in clinical applications.

Synthesis and evaluation of ginsenosides imprinted polymer-based chromatographic stationary phase.

Molecular imprinting technique has aroused great interest in preparing novel stationary phases, and the resulting materials named molecularly imprinted polymers coated silica packing materials exhibit good performance in separating diverse analytes based on their good characteristics (including high selectivity, simple synthesis, and good chemical stability). To date, mono-template is commonly used in synthesizing molecularly imprinted polymers-based stationary phases. The resulting materials always own the disadvantages of low column efficiency and restricted analytes, and the price of ginsenosides with high purity was very high. In this study, to overcome the weaknesses of molecularly imprinted polymers-based stationary phases mentioned above, the multi-templates (total saponins of folium ginseng) strategy was used to prepare ginsenosides imprinted polymer-based stationary phase. The resulting ginsenosides imprinted polymer-coated silica stationary phase has a good spherical shape and suitable pore structures. Additionally, the total saponins of folium ginseng were cheaper than other kinds of ginsenosides. Moreover, the ginsenosides imprinted polymer-coated silica stationary phase packed column performed well in separation of ginsenosides, nucleosides, and sulfonamides. The ginsenosides imprinted polymer-coated silica stationary phase possesses good reproducibility, repeatability, and stability for seven days. Therefore, a multi-templates strategy for synthesizing the ginsenosides imprinted polymer-coated silica stationary phase is considered in the future. This article is protected by copyright. All rights reserved.

Preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate) monolithic column for hydrophilic interaction chromatography.

A method for the preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate copolymerization)-based monolithic capillary column was reported for the separation of polar small molecular weight compounds with nano-liquid chromatography in hydrophilic interaction chromatography mode. The monolithic columns were prepared by in situ copolymerization of N-vinylpyrrolidone and a cross-linker pentaerythritol triacrylate in a binary porogenic agent consisting of methanol and water. The composition of the polymerization solution was systematically optimized in terms of column permeability, theoretical plate number, asymmetric factor, and retention factor. A typical hydrophilic chromatography retention mechanism was observed with a mobile phase composed of a high content of organic solvent. The preparation method is simple and robust, the precursor N-vinylpyrrolidone is chemically stable, cheap, and easily available. The N-vinylpyrrolidone-based hydrophilic interaction chromatography stationary phase displays satisfactory separation selectivity for a range of polar test analytes, including benzoic acid derivatives, nucleosides, and phenols.

Separation of chiral compounds using high performance liquid chromatography stationary phase based on covalent organic framework material TpPa-NH2-Glu

Covalent organic frameworks （COFs） are an emerging class of porous crystalline materials composed of multidentate organic units connected by covalent bonds. COFs have been demonstrated to exhibit great potential and research value in many fields， including gas storage and separation， photoelectric devices， fluorescence sensors， catalysis， drug delivery， dye and pollutant adsorption， and electronic devices， and so on. The COFs obtained by post-synthesis modification tend to exhibit high crystallinities and porosities， thereby rendering them suitable materials for use in the fields of chiral resolution， asymmetric catalysis， and chromatography. In this work， TpPa-NO2 was synthesized from 1，3，5-tricarbaldehyde phloroglucinol and 2-nitro-1，4-phenylenediamine， which was then reduced to TpPa-NH2. Subsequently， this material was modified with D-glucose via a post-synthesis modification strategy to obtain the TpPa-NH2-Glu. TpPa-NH2-Glu were characterized by nuclear magnetic resonance （NMR） spectroscopy， Fourier transform-infrared （FT-IR） spectroscopy， X-ray powder diffraction （XRD） analysis， etc. In the XRD pattern， the peaks observed at 4.7°， 8.1°， 11.1°， and 27° were attributed to the TpPa-NH2-Glu， and these peaks are consistent with previous reports， thereby confirming the successful synthesis of this derivative. In addition， circular dichroism experiments indicated that the TpPa-NH2-Glu exhibited a Cotton effect， further confirming the chiral COF was prepared. Subsequently， this material was immobilized on the surface of spherical silica gel particles via the net-wrapping method to prepare a stationary phase for high performance liquid chromatographic column. Using n-hexane-isopropanol （9∶1， v/v） or methanol-water （9∶1， v/v） as mobile phases at a flow rate of 0.5 mL/min， 16 racemates and two benzene-based positional isomers （o，m，p-nitroaniline and o，m，p-Iodoaniline） were successfully resolved by this chiral column. In addition， under methanol-water （9∶1， v/v） mobile phase conditions， five racemates were separated， among which propranolol hydrochloride， warfarin， and metoprolol reached baseline separation. Furthermore， under n-hexane-isopropanol （9∶1， v/v） mobile phase conditions， 11 racemates were resolved， among which ethyl 2-bromopropionate and 3-butyn-2-ol reached baseline separation. Meanwhile， the effect of temperature on the TpPa-NH2-Glu liquid chromatography column and the repeatability of the TpPa-NH2-Glu liquid chromatography column were also explored. The HPLC column prepared by TpPa-NH2-Glu had good repeatability， and its relative standard deviation （RSD） was 1.55% and 1.46%， respectively. It is demonstrated that the TpPa-NH2-Glu material has good resolution ability for chiral compounds.

Ionic liquid-functionalized dendrimer grafted silica for mixed-mode chromatographic separation and online solid-phase extraction

The efficient sample pretreatment and high-performance chromatographic separation are essential for an accurate analysis of the complex sample. Finding a material that can apply as both sorbent for sample preparation and stationary phase for chromatography is greatly important for analytical chemistry. With a large number of active sites and designable properties, the dendrimers displayed potential performance for sample preparation and chromatographic separation. In order to improve its selectivity restricted by the limited type of functional groups (amide, ethylidene, amino groups), ionic liquid (1,1′-(1,4-butyricdiyl)bis(1-aminopropyl imidazolium)dibromide) were introduced to functionalize dendrimers. With porous silica as the base, ionic liquid-functionalized dendrimer grafted silica (SiO2@G1.5-IL) was obtained and characterized by X-ray photoelectron spectroscopy, elemental analyzer, as well as specific surface and porosity analyzer. The material was used as stationary phase for mixed-mode chromatography and sorbent for SPE, respectively. Under reversed-phase liquid chromatography (RPLC), it successfully separated some hydrophobic analytes (five polycyclic aromatic hydrocarbons (PAHs), two bisphenols, diphenylpropane, and diphenylmethane), by utilizing hydrophobic, π-stacking and hydrogen bonding interactions. Based on the hydrophilic effect, it could separate hydrophilic analytes (nucleosides and alkaloids), which coincident with hydrophilic interaction liquid chromatography (HILIC). And it exhibited better separation capacity than NH2@SiO2 and SiO2@G1.5 under the above two modes. Furthermore, the thermodynamic parameters (ΔH, ΔS, and ΔG) of the above types of analytes on the SiO2@G1.5-IL column were also investigated in detail, revealing the controlling factors during the chromatographic separation. And the relative standard deviations (RSDs, 0.03–2.95%, n = 10) of the repeatability of chromatographic separation were satisfactory. After that, SiO2@G1.5-IL-based SPE column was online combined with high-performance liquid chromatography to investigate the extraction selectivity for three types of hydrophobic organic pollutants (PAHs, estrogens, and bisphenols), and compared to SiO2 and C18 columns. According to their extraction performance, PAHs were selected as the target analytes, and the online analytical method was established. The limits of detection were as low as 0.001 μg L-1 due to the high enrichment factor reach to 3869, limits of quantity were 0.003–0.17 μg L-1, linear ranges were from 0.003 to 15.0 µg L-1, the adsorption capacity was 16.10 μg g−1, as well as RSDs were 0.5–3.7%, 0.6–6.7%, and 4.2–9.0% for intraday, interday tests and reproducibility. Finally, it was applied to analyze the environmental water samples (tap water, snow water, and rainwater), and satisfactory results were obtained (spiked recoveries were 86.8%-120.9%). Therefore, SiO2@G1.5-IL was a powerful material that can be used for the chromatographic separation of both hydrophobic and hydrophilic samples by RPLC or HILIC mode, as well as the efficient online extraction of hydrophobic targets prior to C18 column-based high-performance liquid chromatography.

Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization

Hydrophilic interaction chromatography (HILIC) has been proposed as a valuable alternative to ion-pairing reversed-phase chromatography (IP-RPLC) for oligonucleotide (ON) analysis. In this context, the potential of seven zwitterionic HILIC columns has been evaluated against amide- and poly-hydroxy fructan-functionalized HILIC columns and a C18 column operated under IP-RPLC mode.Based on the retention characteristics of key small molecule pairs, each zwitterionic HILIC column showed a unique radar-shaped profile, suggesting different selectivities for distinct structural differences. Unmodified DNA and RNA samples were then evaluated, and the columns classified based on their retentivity. Two zwitterionic columns were particularly promising in terms of overall resolution, especially for the largest ONs (> 40-mer). Finally, separations between a chemically modified drug-like ON and its closely related impurities were performed. Although the ZIC-cHILIC column showed similar selectivity values as compared to the reference IP-RPLC technique, all columns demonstrated a general decrease in selectivity due to the minor structural differences present in the highly complex samples.This work highlights the utility of zwitterionic HILIC mode for ON analysis and it reveals the importance of understanding columns characteristics – in terms of retention and selectivity – when selecting a stationary phase for specific ON applications.

Preparation and chromatographic evaluation of a mixed polymer brush-silica stationary phase with temperature-sensitive property.

In this study, a developed chromatographic stationary phase combines the high selectivity of mixed-mode retention with a temperature-responsive property to boost separation efficiency. Copolymer brushes were grafted onto silica gels through surface initiated-atom transfer radical polymerization by polymerizing two types of monomer, temperature-responsive vinylcaprolactam (VCl) and quinine (Qun) containing benzopyridine, a tertiary ammonium positive center, and hydroxyl groups. The obtained silica@poly(Qun-co-VCl) stationary phases were packed as a chromatographic column, and the retention behavior of hydrophobic polycyclic aromatics, highly polar nucleosides, charged organic acids and β-agonists was studied for this column under different separation modes. The ability to separate different types of analyte shows that the silica@poly(Qun-co-VCl) column provides multiple hydrophobic, hydrophilic and electrostatic interactions toward analytes, achieving the separation of various compounds in one column. In addition, temperature-dependent resolution of polycyclic aromatics, nucleosides, organic acids and β-agonists was investigated using modulation of the column temperature, and the column exhibited adjustable separation selectivity by simply changing the column temperature. These results demonstrate that the grafting of copolymer brushes on a silica surface, consisting of temperature-responsive poly-VCl and multifunctional groups of poly-Qun, is useful as a mixed-mode chromatographic stationary phase for thermally-modulated multiple interactions. Additionally, this column was also used for the quantitative detection of uridine and inosine from cordyceps.

Increase in cysteine-mediated multimerization under attractive protein–protein interactions

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein–protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein–protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.

Effect of silver(I) ion reduction on the selectivity of olefins, paraffins, and aromatic compounds by gas chromatographic stationary phases consisting of silver(I) salts in ionic liquids

The olefin/paraffin selectivity offered by ionic liquid (IL) stationary phases can be enhanced through the addition of silver(I) ion, which is well-known to undergo selective complexation with unsaturated compounds. However, such stationary phases often suffer from the loss of chromatographic selectivity as silver(I) ion can be reduced to elemental silver. To maintain the separation performance of silver(I) ion/IL stationary phases, an understanding of factors and conditions that promote the reduction of silver(I) ion is needed. In this study, capillary gas chromatography columns featuring a stationary phase consisting of the 1-decyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([C10MIM+][NTf2−]) IL impregnated with [Ag+][NTf2−] were examined to investigate the effects of temperature, hydrogen content in exposure gas stream, and time of heating/exposure events on olefin selectivity. Retention factors of representative analytes, such as C6 olefins and paraffins as well as aromatic compounds, were measured after subjecting the columns to the aforementioned conditions, followed by an evaluation of selectivity factors over time. Selectivity factors of olefins and aromatic compounds were observed to decrease significantly when the stationary phases were heated to temperatures higher than 110°C as well as being subjected to mixed gas streams containing greater than 50 mol% of hydrogen. As constant column heating temperatures were applied under exposure gas mixtures containing hydrogen and nitrogen, a gradual decrease in analyte selectivity factors was observed under prolonged periods of time. However, application of a ternary gas mixture comprised of 25/50/25 mol% hydrogen/nitrogen/methane resulted in an increase in the 3-hexyne/cis-2-hexene selectivity when measured at 120°C for 60 h, due to a smaller decrease in the retention factor of 3-hexyne compared to cis-2-hexene.

Fabrication and evaluation of dodecyl imide maleopimaric acid glycidyl methacrylate ester modified silica with multiple retention mechanisms for reversed phase liquid chromatography

As green, less toxic, and abundant ligands with rich functional groups, natural products are widely used in synthesis of chromatographic stationary phases. In this work, dodecyl imide maleopimaric acid glycidyl methacrylate ester (C12-MPAGN) was prepared from maleopimaric acid through the imidization and ring-opening based esterification reaction. By using “thiol-ene” click chemistry, it was chemically bonded to the silica and (3-mercaptopropyl) trimethoxysilane (γ-MPS) was used as the coupling agent to obtain dodecyl imide maleopimaric acid glycidyl methacrylate ester bonded silica stationary phase (Sil-C12-MPAGN). Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopies (SEM), and elemental analysis (EA) were utilized to verify that the Sil-C12-MPAGN stationary phase was successfully prepared with C12-MPAGN immobilized on the silica surface. In order to evaluate the chromatographic performance and retention mechanisms of the Sil-C12-MPAGN column and compared with C18 column, a variety of compounds were used, including stander mixture of Tanaka, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), phenols and flavonoids. Based on these multiple interactions, including hydrophobic, hydrogen-bonding, and π-π interactions, high selectivity and superior separation performance were demonstrated by the Sil-C12-MPAGN column for probe molecules what had previously been mentioned. In addition, the quantitative determination of paclitaxel content in Yew bark extract was conducted with this column, which was found that the concentration was 83.67 mg/L, respectively. In short, the present study proposes a new strategy for introducing rosin to liquid chromatography with high selectivity and separation performance.