An improved subtraction model applied in supercritical fluid chromatography to characterise polar stationary phases.

Herein, an improved subtraction model was proposed to characterise the polar stationary phases in supercritical fluid chromatography (SFC). Fifteen stationary phases were selected, including two types of aromatic columns, Waters Torus and Viridis series columns, as well as silica and amino columns. Ethylbenzene and Torus 1-AA were defined as the reference solute and column, respectively. Identifying the interaction with the maximum contribution to retention in SFC separation and using it as the initial term is a key step in modelling. The dipole, or induced dipole interaction (θ'P), replaced the hydrophobic interaction (η'H) as the starting term. The improved model was expressed as logα=η'H+ß'A+α'B+κ'C+θ'P+ε'E+σ'S, where the term ε'E indicated that anion exchange interaction was intentionally supplemented. A 7-step modelling process, including bidirectional fitting and residual analysis, was proposed. The obtained column parameters had reasonable physical significance, with the adjusted determination coefficient (R2adj) greater than 0.999 and the standard error (SE) less than 0.029. Methodological validation was further performed using the other four columns and 12 solutes that were not involved in the modelling. The result revealed good predictions of solutes' retention, as demonstrated by R2adj from 0.9923 to 0.9979 and SE from 0.0636 to 0.1088. This study indicated the feasibility of using the improved subtraction model to characterise polar stationary phases in SFC, with the most crucial being the determination of an initial term, followed by the addition of a new descriptor and the selection of an appropriate reference column. The study expanded the application scope of the subtraction model in SFC, which will help gain an in-depth understanding of the SFC separation mechanism.

Separation of carbohydrates using dynamically adsorbed borate stationary phase for hydrophilic interaction liquid chromatography.

In this work, a chromatographic method for the separation of carbohydrates was proposed. Tris-(hydroxymethyl)-amine (TRIS) functionalized silica-based hydrophilic interaction liquid chromatography (HILIC) stationary was synthesized. The dynamically absorbed borate layer is generated by using borate buffer as a polar modifier due to the complexation of borate with TRIS ligand in the stationary phase. The chromatographic systems were analyzed by the linear solvation energy relationship model. The calculated system constants revealed the enhancement of anionic exchange by the addition of borate in the mobile phase system. In addition, ligand exchange is critical for the retention and elution order of sugars and sugar alcohols. Carbohydrates displayed prolonged retention with different selectivity profiles relating to their complexation coefficients with borate. Experiment results showed that the effect of borate in this chromatographic system was stable within the range of pH 3-7 and borate concentration of 5-15 mM. This work provides a complementary solution for the separation of carbohydrates. It can also be extended to the separation of glycosides.

Synthesis and evaluation of aromatic stationary phases based on linear solvation energy relationship model for expanded application in supercritical fluid chromatography.

In the last decade, the separation application based on aromatic stationary phases has been demonstrated in supercritical fluid chromatography (SFC). In this paper, four aromatic stationary phases involving aniline (S-aniline), 1-aminonaphthalene (S-1-ami-naph), 1-aminoanthracene (S-1-ami-anth) and 1-aminopyrene (S-1-ami-py) were synthesized based on full porous particles (FPP) silica, which were not end-capped for providing extra electrostatic interaction. Retention mechanism of these phases in SFC was investigated using a linear solvation energy relationship (LSER) model. The aromatic stationary phases with five positive parameters (a, b, s, e and d+) can provide hydrogen bonding, π-π, dipole-dipole and cation exchange interactions, which belong to the moderate polar phases. The LSER results obtained using routine test solutes demonstrated that the aforementioned interactions of four aromatic stationary phases were influenced by the type and bonding density of the ligand, but to a certain extent. Furthermore, the LSER data verified that the S-1-ami-anth column based on full porous particles silica had higher cation exchange capacity (d+ value), compared to the commercialized 1-AA column (based on the ethylene-bridged hybrid particles). The relationship between the d+ value and SFC additive was quantitatively proved so as to regulate electrostatic interaction reasonably. This value was greatly increased by phosphoric acid, slightly increased by trifluoroacetic acid and formic acid, but significantly reduced by ammonium formate and diethylamine. Taking the S-1-ami-naph column as an example, better peek shape of the flavonoids was obtained after the addition of 0.1 % phosphoric acid in MeOH while isoquinoline alkaloids were eluted successfully within 11 min after adding 0.1 % diethylamine in MeOH. Combined with the unique π-π interaction and controllable electrostatic interaction, the aromatic stationary phases in this study have been proven to have expandable application potential in SFC separation.

Supercritical fluid-based method for selective extraction and analysis of indole alkaloids from Uncaria rhynchophylla.

The development of an approach based on simultaneous supercritical fluid extraction-sample cleanup, followed by supercritical fluid chromatography/tandem mass spectrometry (SFE-SFC-MS/MS) was as a tool for the extraction, separation and characterization of indole alkaloids of Uncaria rhynchophylla. A two-step SFE method was designed. A mixture of the U. rhynchophylla sample and an adsorbent named C18SCX with the ratio of 1:1 (w/w) was placed into an extraction cell. The extraction temperature was 40 °C and the pressure was 25 Mpa. In the first step, 10 % EtOH as the co-solvent was used to extract for 60 min, which was considered as a cleanup process to remove non-alkaloid components. In the second step, 0.1 % DEA was added to 10 % EtOH and it extracted for 60 min to obtain the desired extract. By introducing an additional adsorbent, the specificity of SFE towards alkaloids was greatly improved. An SFC-MS/MS method was then utilized for analysis of the SFE extract. Using 2-EP as stationary phase with the gradient elution of 0-10 min, 5-25 % EtOH (+0.05 % DEA) in CO2, column temperature 40 °C, and back pressure 13.8 Mpa, 10 peaks were separated within 8 min. Further MS/MS analysis confirmed that nine of the 10 peaks in the SFE extract were indole alkaloids. This study developed a supercritical fluid-based method specifically towards extraction and analysis of alkaloids, which is helpful to the study of alkaline compounds in complex samples.

Supercritical fluid chromatography based on reversed-phase/ ion chromatography mixed-mode stationary phase for separation of spirooxindole alkaloids.

The present paper illustrates the versatility of the supercritical fluid chromatography (SFC) since, for the first time, four spirooxindole alkaloids (SOAs) including two pairs of isomers were separated by using two types of reversed-phase/ ion chromatography (RP/IC) mixed-mode stationary phases. Two mixed-mode stationary phases (C8SAX and C8SCX) was simultaneously provided dispersive and electrostatic interactions, which were suitable for the separation of such alkaloids. This study tried to provide an in-depth understanding of the SFC separation mechanism of the mixed-mode stationary phase through investigation of the impact of changes in mobile phase composition on alkaloids' retention behavior. On C8SAX, due to the strong electrostatic repulsion, there was a very narrow elution window of the alkaloids, of which behaviors were hardly affected by adding diethylamine in mobile phase. When adding formic acid or acidic ammonium formate, the prolonged retention time of alkaloids was presented because of the shielded effect of formate anions on the electrostatic repulsion. In particular, better peak shape and improved resolution were obtained by using acidic ammonium formate due to the deactivation of silanol groups by ammonium cations. On the other hand, both formic acid and acidic ammonium formate can strengthen the electrostatic attraction of C8SCX, causing difficult elution of the alkaloids. Ammonium cations from either the protonated diethylamine or the ionized ammonium formate, were considered as counter ions to effectively mask the electrostatic attraction of C8SCX, to significantly reduce the retention of alkaloids, but improve the resolution. Finally, utilizing two developed SFC methods, i.e., C8SAX with EtOH+ 10 mM acidic ammonium formate in CO2, or C8SCX with EtOH+0.1% diethylamine in CO2, the baseline separation of corynoxeine and isocorynoxeine, rhynchophylline and isorhynchophylline was achieved within 5 min.

An attempt to apply a subtraction model for characterization of non-polar stationary phase in supercritical fluid chromatography.

This study verified the feasibility of using a subtraction model to characterize the non-polar stationary phases (including C4, C8, and phenyl-type) in supercritical fluid chromatography (SFC). The model with 6 terms was expressed as log α = Î·'H + Î¸'P + ß'A + α'B + κ'C + σ'S, where a term θ'P indicating dipole or induced dipole interaction was intentionally supplemented. Ethylbenzene and SunFire C8 were respectively defined as the reference solute and column. A 7-step modeling procedure was proposed: in the first 6 steps, except σ'S, by the use of a bidirectional fitting method, other parameters were calculated based on the equation: log α = log (ki/kref) ≈ η'H + Î¸'P + ß'A + α'B + κ'C; and in the 7th step, residual analysis was employed to describe the σ'S term according to the equation: σ'S = log αexp. - log αpre. Furthermore, six columns that were not involved in modeling process and 12 compounds with unknown retention were used for methodology validation. It showed good predictions of log k, as demonstrated by adjusted determination coefficient (R2adj) from 0.9927 to 0.9998 (column) and from 0.9940 to 0.9999 (compound), respectively. The subtraction model emphasized the contribution of dipole or induced dipole interaction to the retention in SFC, and it obtained the σ'S term through residual analysis. Moreover, it made reasonable physical-chemical sense as the linear solvation energy relationship (LSER) model did, with the distinct advantages of better fitting and more accurate prediction. This study provided some new insights into the characterization of non-polar stationary phases in SFC.

An in-depth investigation of supercritical fluid chromatography retention mechanisms by evaluation of a series of specially designed alkylsiloxane-bonded stationary phases based on linear solvation energy relationship.

Fundamental research on supercritical fluid chromatography (SFC) has gained considerable interest, with many studies focusing on its retention mechanism based on the linear solvation energy relationship (LSER) model. In this paper, a series of alkylsiloxane-bonded stationary phases were specifically designed and synthesized, then evaluated using the mobile phase composed of CO2 with 10% (v/v) methanol. The study demonstrated the close relationship between the interactions (manner and magnitude) of stationary phases and the C-chain length, bonding density and the endcapping treatment. All C8 phases provide positive e, v and negative s, whose magnitude was regularly affected by bonding density. It was worth mentioning the non-endcapped C8 phases could provide H-bonding (positive a and b) by reducing the bonding density of the alkyl chain. Once it was endcapped, the interaction manner did not vary with bonding density adjustment. The non-endcapped C4 phases with higher bonding density could establish additional dispersion interaction (positive v). It can be seen that two synthesis strategies, 1) non-endcapped, long C-chain (C8) combined with low bonding density, and 2) non-endcapped, short C-chain (C4) combined with high bonding density, can obtain the alkylsiloxane-bonded stationary phases (C8-1 and C4-3) to provide both polar and dispersion interactions, showing different separation selectivity. Furthermore, the LSER model with ionic terms was applied to evaluate partial C8 columns, and its rationality was verified. The non-endcapped C8 showed great d+ values, which originated from the silanol groups. C8SCX also possessed a great d+ value due to the benzenesulfonic acid groups. A remarkable result showed that C8SAX exhibited prominent d- and d+ values simultaneously due to the combined effect of silanol and quaternary ammonium groups, which indicates the unique selectivity when separating ionic compounds. This study provides in-depth insights into the retention mechanism of alkylsiloxane-bonded stationary phases in SFC, as well as a reference for the design of SFC stationary phases.

Pseudomorphic synthesis of bimodal porous silica microspheres for size-exclusion chromatography of small molecules.

In this work, mesoporous silica microspheres with bimodal porous structures for size exclusion chromatography (SEC) supports were synthesized via a pseudomorphic transformation method by using 3.5 and 5 µm commercial silica particles as sources and cetyltrimethylammonium bromide (CTAB) as a template. The effects of the synthetic conditions on the pore size distribution were examined, including the temperature, reaction time and the molar ratio of SiO2:NaOH. Bimodal porous silicas (BPSs) with pore sizes of 3.01 and 12.80 nm were obtained with SiO2:NaOH:CTAB:H2O=1:0.1:0.1:20 at 80 °C for 24 h. The BPSs were bonded with diol groups to produce a stationary phase for SEC. The column performance was evaluated with three types of samples, namely, dextran (70 KDa-62 Da), polyethene glycol (PEG) (20 KDa-32 Da) and three biomolecules (36 KDa-1.36 KDa). The column that was packed with a 3.5 µm stationary phase showed excellent resolution for molecular weights of less than 1 KDa with high column efficiency. Carbohydrate samples (dextran (MW=1296), dextran (MW=972), sucrose (MW=342), glucose (MW=180) and glycerol (MW=92)) were separated. Heptaethylene glycol, hexaethylene glycol, pentaethylene glycol, tetraethylene glycol, triethylene glycol, and diethylene glycol were resolved in a PEG200 sample. In summary, this work shows the advantages of bimodal mesopores in SEC for small molecules less than 1 kDa. In the pseudomorphic synthesis, the pore size can be regulated by template micelles. Thus, the development SEC supports with high accuracy for a specified molecular weight range is expected since the pore size can be regulated by the surfactant template.

[Covalent organic framework functional materials and their applications in glycopeptide enrichment].

Protein glycosylation is among the most important post-translational modifications in living organisms and the research in the field of protein glycosylation continues to garner attention. Currently, the efficient separation and enrichment of glycoproteins and glycopeptides is the primary challenge of glycoproteomics research. The number of glycoproteins is small in complex biological samples. Moreover, the presence of highly-abundant, non-glycosylated, and modified peptides makes the detection of low-abundance glycopeptides more difficult. Therefore, efficient glycopeptide enrichment methods are required to improve the detection of these compounds. The development of highly selective glycopeptide enrichment tools is important to efficiently capture glycoproteins or glycopeptides at the molecular level. Compared with traditional glycopeptide-enriched materials, covalent organic framework materials have the advantages of large specific surface area and rich modification sites, thereby exhibiting great application potential in the field of glycopeptide enrichment. In this study, a novel covalent organic framework material (O-T-D-COFs) was prepared and applied for selective glycopeptide enrichment. We applied the solvothermal method, using 2,5-dimethoxy benzene-1,4-2 formaldehyde and 1,3,5-Tris(4-aminophenyl) benzene, to synthesize imino-based COFs. The Schiff base generated via copolymerization condensation reaction constitutes the framework of the material. Next, the synthesized intermediate material was oxidized to improve the enrichment performance of the material. The functional, specific glycopeptide-binding groups were modified on the COF channels and the structure of the material was characterized using scanning and transmission electron microscope, as well as infrared spectrum and solid-state nuclear magnetic resonance. The enrichment conditions comprised the loading and elution steps, including the optimization of the elution conditions. We could observe the clear profile of 32 glycopeptides derived from human serum immunoglobulin G (IgG) tryptic digests with a significantly improved signal-to-noise (S/N) ratio. We applied a complex sample system to verify the enrichment selectivity of the material when the molar ratios of the IgG and bovine serum albumin (BSA) tryptic digest mixtures reached 1∶50. In addition, we investigated the enrichment performance of the detection limit, enrichment capacity, recovery rate of the material, and the application potential in glycopeptides enrichment using real samples. The material showed a good detection limit (2.5 fmol/µL), an ideal enrichment capacity (120 mg/g), and enrichment recovery (103.5%±6.6% and 101.5%±10.4%). We identified a total of 86 glycopeptides derived from 53 glycoproteins with 94 N-glycosylation sites from only 1 µL human serum. The O-T-D-COFs exhibited a good glycopeptide separation and enrichment potential, indicating that the COF material has promising application potential in glycoproteomics.

Enantioseparation of cloprostenol on the polysaccharide chiral stationary phase: Influence of the mobile phase on enantioselective adsorption.

Cloprostenol (CLO) is an important chiral drug widely used in veterinary practice. In high-performance liquid chromatography (HPLC), the acetonitrile/water system is more effective in the chiral resolution of CLO. The change in the ratio of acetonitrile and water has a great influence on the chiral selectivity of CLO. The enantioselective adsorption and nonselective adsorption of CLO on the polysaccharide stationary phase with three groups of mobile phases (Acetonitrile / 0.1 TFA; 95 Acetonitrile/5 H2O/0.1 TFA; 45 Acetonitrile/55 H2O/0.1 TFA) were determined and fitted with the subtraction fitting method (SFM). Adding a small amount of water to the mobile phase mainly reduced the enantioselective equilibrium adsorption constants Ks of (-)-CLO and thus changed the selectivity. Among them, the true separation factor (αtrue) of the 95 Acetonitrile/5 H2O mobile phase was as high as 92.86. Chiral preparation was performed on the basis of this mobile phase. The preparation performance in kkd (kilograms per kilogram of stationary phase purified compound per day) was 0.25 kg racemate/kg CSP/day.

A ternary eluent strategy to tune the peak shape of steviol glycosides in reversed-phase liquid chromatography.

In this paper, an effective strategy of using acetonitrile-methanol-water as mobile phase was developed to achieve acceptable peak shape of steviol glycosides in reversed-phase liquid chromatography (RPLC). The change of elution profiles of rebaudioside A (RA) was systematically investigated. Two classical distributions, namely, tailing and fronting peaks resulting from injections of RA solution in range of 0.5-5 mg were both observed in a ternary eluent of acetonitrile-methanol-water (21:43:36, v/v). Next, a three-phase diagram of tailing factor (Tf) was illustrated, showing high dependence of elution profile of RA on the ternary composition. The peak shape of RA can be adjusted mainly based on the additive effect, that is, acetonitrile is more strongly adsorbed to the stationary phase than RA in the pure weak solvent (H2O). Therefore, with the increase of acetonitrile concentration in the ternary eluent, the RA band profiles went from being tailing to fronting shapes. At the same time, due to the large RA-RA interactions, there was anti-Langmuir adsorption isotherm in acetonitrile-water mobile phase, which is the reason for the fronting peaks of RA. It could be concluded that the way of using the ternary eluent of acetonitrile-methanol-water does control and tune the peak shape of steviol glycosides in RPLC separation.

Synthesis of Al3+-doping-TiO2 monodisperse microspheres and their application for phosphopeptides and glycopeptides enrichment.

Glycosylation and phosphorylation are two of the most common and important post-translational modifications (PTMs) of proteins, which play critical roles in regulating a variety of complex biological processes and involvement in many diseases. Due to the low abundance of phosphopeptides and glycopeptides, highly selective enrichment methods are crucial to the identification of protein phosphorylation and glycosylation by mass spectrometry (MS). Here, monodisperse uniform Al3+-doping-TiO2 mixed oxide microspheres were easily synthesized. The morphology was controlled by a sol-gel method, during the hydrothermal treatment. The obtained microspheres with uniform particle size distribution (about 1-2 µm)，high surface area and improved pore structures, were characterized by SEM, TEM, XRD and N2 adsorption-desorption isotherms. Al3+-doping-TiO2 was applied in enriching glycopeptides and phosphopeptides respectively or simultaneously by using different enrichment conditions, achieving selective enrichment of glycopeptides and phosphopeptides. 20 glycopeptides and 25 phosphopeptides enriched from the tryptic digest mixtures of human serum immunoglobulin G (IgG) and α-casein (molar ratio of 1:1) were obviously observed with greatly improved signal-to-noise (S/N) ratio. Meanwhile, the enrichment results of non-fat milk and human serum also show the enrichment selectivity from complex biological samples. This study will provide a novel insight for selective enrichment of glycopeptides and phosphopeptides in post-translational modification proteomics research.

Highly Efficient Separation of Methylated Peptides Utilizing Selective Complexation between Lysine and 18-Crown-6.

Protein methylation is one of the most common and important post-translational modifications, and it plays vital roles in epigenetic regulation, signal transduction, and chromatin metabolism. However, due to the diversity of methylation forms, slight difference between methylated sites and nonmodified ones, and ultralow abundance, it is extraordinarily challenging to capture and separate methylated peptides from biological samples. Here, we introduce a simple and highly efficient method to separate methylated and nonmethylated peptides using 18-crown-6 as a mobile phase additive in high-performance liquid chromatography. Selective complexation between lysine and 18-crown-6 remarkably increases the retention of the peptides on a C18 stationary phase, leading to an excellent baseline separation between the lysine methylated and nonmethylated peptides. A possible binding mechanism is verified by nuclear magnetic resonance titration, biolayer interferometry technology, and quantum chemistry calculation. Through establishment of a simple enrichment methodology, a good selectivity is achieved and four methylated peptides with greatly improved signal-to-noise (S/N) ratios are successfully separated from a complex peptide sample containing 10-fold bovine serum albumin tryptic digests. By selecting rLys N as an enzyme to digest histone, methylation information in the histone could be well identified based on our enrichment method. This study will open an avenue and provide a novel insight for selective enrichment of lysine methylated peptides in post-translational modification proteomics.

A subtraction fitting method for independent determination of enantioselective and nonselective adsorption isotherms based on the single-component isotherms in the framework of the two-site model.

Based on theory of two-site model, a subtraction fitting method (SFM) is proposed to independently fit enantioselective adsorption sites and the nonselective adsorption sites on the surface of chiral stationary phases (CSP) based on polysaccharide derivatives. Compared with the direct fitting method, the method allows independent fitting the isotherm models of two types of adsorption sites. By the SFM, the adsorption data of methyl mandelate on the CSP based on cellulose tri-(3, 5-dimethylphenylcarbamate) was measured. The adsorption on enantioselective sites was well fitted with Langmuir model, and the adsorption on nonselective sites was well fitted with Tóth model or Langmuir-Freundlich model. The adsorption isotherms of the enantiomers of Corey PG-lactone diol on chiral stationary phase coated with different amounts of amylose tri-(3,5-dimethylphenylcarbamate) (Wchiral selector: Wsilica = 20 : 100, 30: 100 and 40: 100, respectively) were also studied. It was found that the saturated adsorption capacities of both sites increase with the increase of the chiral selector loading. The chiral stationary phase with low chiral selector loading (Wchiral selector: Wsilica = 20 : 100) has a high true separation factor (αtrue = 16.8) and the lowest apparent separation (αapp = 1.70) due to the large nonselective adsorption, which indicates that a chromatographic support with an inert surface is important for this type of CSP.

Monodisperse core-shell silica particles as a high-performance liquid chromatography packing material: Facile in situ silica sol-gel synthesis.

In recent years, core-shell silica particles (CSSPs) have been increasingly used for highly efficient separation at fast flow rates and relatively low back pressures in high-performance liquid chromatography (HPLC). However, material synthesis techniques for producing CSSPs economically in batch processes remain elusive. In this report, a practical and straightforward method for the preparation of CSSPs is presented. By refluxing freshly prepared nonporous silica particles in ammonia-water solution in the presence of poly(diallyldimethylammonium chloride) at 70-100 °C, CSSPs with shell thicknesses of up to 300 nm and pore sizes from 8 to 25 nm were easily prepared. The effects of the synthetic conditions on the shell thickness, surface area, and pore size were investigated in detail, and the method reproducibility was evaluated in scale-up experiments. A mechanism of CSSP formation is also proposed. The CSSPs were characterized via scanning electron microscopy, transmission electron microscopy, laser particle size (dynamic light scattering) analysis, and nitrogen adsorption and desorption experiments. The synthesized 3.4-µm CSSPs were functionalized with dimethyloctadecylchlorosilane and used as an HPLC packing material, exhibiting excellent separation performance for both small molecules and large biomolecules. In summary, we report the simplest method developed thus far for the preparation of monodisperse core-shell silica particles suitable for HPLC column packing.

Novel chiral stationary phases based on 3,5-dimethyl phenylcarbamoylated ß-cyclodextrin combining cinchona alkaloid moiety.

Novel chiral selectors based on 3,5-dimethyl phenylcarbamoylated ß-cyclodextrin connecting quinine (QN) or quinidine (QD) moiety were synthesized and immobilized on silica gel. Their chromatographic performances were investigated by comparing to the 3,5-dimethyl phenylcarbamoylated ß-cyclodextrin (ß-CD) chiral stationary phase (CSP) and 9-O-(tert-butylcarbamoyl)-QN-based CSP (QN-AX). Fmoc-protected amino acids, chiral drug cloprostenol (which has been successfully employed in veterinary medicine), and neutral chiral analytes were evaluated on CSPs, and the results showed that the novel CSPs characterized as both enantioseparation capabilities of CD-based CSP and QN/QD-based CSPs have broader application range than ß-CD-based CSP or QN/QD-based CSPs. It was found that QN/QD moieties play a dominant role in the overall enantioseparation process of Fmoc-amino acids accompanied by the synergistic effect of ß-CD moiety, which lead to the different enantioseparation of ß-CD-QN-based CSP and ß-CD-QD-based CSP. Furthermore, new CSPs retain extraordinary enantioseparation of cyclodextrin-based CSP for some neutral analytes on normal phase and even exhibit better enantioseparation than the corresponding ß-CD-based CSP for certain samples.

Enantiomeric analysis of simendan on polysaccharide-based stationary phases by polar organic solvent chromatography.

Herein, the enantiomeric separation of simendan by high-performance liquid chromatography with ultraviolet detection using polysaccharide-based chiral stationary phases in polar organic mode is described. Three chiral columns (Chiralpak AD-H, Chiralcel OD-H, and Chiralpak AS) were screened using pure methanol and acetonitrile without additives under isocratic conditions. A reversed elution order was observed on the Chiralpak AD-H column when the methanol content in the mobile phase (methanol-acetonitrile mixtures) was above 10%, whereby levosimendan eluted prior to dextrosimendan. Further, it was found that increasing temperature effectively improved the enantioresolution on the Chiralpak AD-H column. Van't Hoff analysis was performed to evaluate the contribution of enthalpy and entropy to the chiral discrimination process. The best enantioseparation (α = 3.00, Rs = 12.85) was obtained on the Chiralpak AD-H column with methanol as the mobile phase at 40°C. Thus, a quantitative method for the resolution of dextrosimendan was established and validated, which could be used as a reference for the determination of dextrosimendan in levosimendan products.

A novel C2 symmetric chiral stationary phase with N-[(4-Methylphenyl)sulfonyl]-l-leucine as chiral side chains.

In this study, a series of chiral stationary phases based on N-[(4-methylphenyl)sulfonyl]-l-leucine amide, whose enantiorecognition property has never been studied, were synthesized. Their enantioseparation abilities were chromatographically evaluated by 67 enantiomers. The chiral stationary phase derived from N-[(4-methylphenyl)sulfonyl]-l-leucine showed much better enantioselectivities than that based on N-(4-methylbenzoyl)-l-leucine amide. The construction of C2 symmetric chiral structure greatly improved the enantiorecognition performance of the stationary phase. The C2 symmetric chiral stationary phase exhibited superior enantioresolutions to other chiral stationary phases for most of the chiral analytes, especially for the chiral analytes with C2 symmetric structures. By comparing the enantioseparations of the enantiomers with similar structures, the importance of hydrogen bond interaction, π-π interaction, and steric hindrance on enantiorecognition was elucidated. The enantiorecognition mechanism of trans-N,N'-(1,2-diphenyl-1,2-ethanediyl)bis-acetamide, which had an excellent separation factor on the C2 symmetric chiral stationary phase, was investigated by 1 H-NMR spectroscopy and 2D 1 H-1 H nuclear overhauser enhancement spectroscopy.

Adsorption mechanism of triterpenoid saponins in reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography: Mogroside V as test substance.

In this paper, adsorption mechanism of triterpenoid saponins in reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) was proposed based on the study of the retention behavior of mogroside V as test substance. The change of peak shape of mogroside V and its influencing factors was first investigated. As the increase of sample loading, a tailing peak of mogroside V was observed in MeOHH2O of both two modes. It was the fronting peak in ACNH2O of HILIC while there was a transition from fronting peak to tailing peak in ACNH2O of RPLC that was largely affected by column temperature and ACN concentration. The adsorption isotherm of mogroside V in ACNH2O of RPLC was fitted by Moreau model, where a monolayer adsorption with large inter-molecular interaction was formed on the C18 surface. While in ACNH2O of HILIC, the adsorption of mogroside V was in accordance with BET model, showing multilayer adsorption behavior. In MeOHH2O of both HILIC and RPLC, there was always monolayer adsorption, which was fitted by Langmuir model. At last, by choosing the suitable chromatographic mode, controlling the key factors such as the solvent concentration and column temperature, and predicting the broadening trend of peak, three methods were screened out, namely, C18 column with 22% ACN (30 °C), Click XIon column with 90% MeOH or 70% ACN, to get mogroside V of purity greater than 98% from Siraitia grosvenorii extract. Among them, the RPLC method of 22% ACN that showed the highest loading sample per hour (1.92%) and the lowest solvent consumption emerged as the best approach.

Evaluation of a series of phenyl-type stationary phases in supercritical fluid chromatography with the linear solvation energy relationship model and its application to the separation of phenolic compounds.

In recent years, supercritical fluid chromatography (SFC) has become a powerful tool in modern analytical chemistry, and the diversity of stationary phases in SFC promotes phenyl-type phases to confront with a significant resurgence of interest. In this paper, a series of phenyl-type stationary phases with different substituted benzenes involving N-propylbenzamide (PB), 4-fluoro-N-propylbenzamide (PB-F), and 4-ethyl-N-propylbenzamide (PB-ET) were synthesized. Retention mechanism of these phases in SFC was investigated using a linear solvation energy relationship (LSER) model. The phenyl-type stationary phases with all positive parameters can provide all types of interaction, typically involving hydrogen bonding, dipole-dipole and dispersive interactions. The different benzene's substituents of the stationary phases mainly affected their hydrogen bonding and dipole-dipole interactions, which could be reflected by the angle between the solvation vectors to some extent. The k-k plot showed that the selectivity difference of phenyl-type stationary phases was closely related to the type of solute. Thus, based on twenty-five natural phenolic compounds, two systems with high orthogonality (63.49%) were constructed using three columns, namely phenyl column (PHE) × PB-F and PB × PB-F. Finally, after investigating the influence of chromatographic conditions, ten flavonoids could be separated by using PB, PB-F and PHE columns in SFC.