High-resolution performance of pillar[6]arene functionalized with imidazolium ionic liquids for gas chromatography.

Pillar[n]arenes (P[n]A, n = 5-10) have attracted much attention because of their highly symmetric pillar-shaped architecture with π-electron rich cavity. Nevertheless, the use of ionic liquid functionalized P[n]A in chromatography has not been reported up to data. This work reports the investigation of the imidazolium ionic liquids functionalized pillar[6]arene (P6A-C10-IM-C8[NTf2]) as the stationary phase for gas chromatography (GC). The statically coated P6A-C10-IM-C8[NTf2] column (0.25 mm i.d.) showed moderate polarity and high column efficiency of 4733 plates/m determined by n-dodecane at 120 °C (k = 2.29). Owing to its unique amphiphilic conformation, the P6A-C10-IM-C8[NTf2] showed good column inertness and resolving capability for a wide range of analytes and isomers. Particularly, the P6A-C10-IM-C8[NTf2] column exhibited distinctly advantageous performance for the challenging isomers of halogenated benzenes, benzaldehydes, phenols and anilines over the common commercial columns, namely 5% phenyl methyl polysiloxane (HP-5) and 35% phenyl methyl polysiloxane (HP-35). In addition, it exhibited good column repeatability and reproducibility with RSD values on the retention times less than 0.05% for run-to-run, 0.38% for day-to-day and 2.94% for column-to-column, respectively. This work demonstrates the promising future of ionic liquid P[n]A stationary phases for chromatographic separations.

Multipurpose new gas chromatography column based on pillararenes functionalized with imidazolium ionic liquids.

BACKGROUND: Gas chromatography is worldwide recognized as one of the most important analytical techniques, due to its high versatility and reliability. The heart of a gas chromatograph is the column, that allows analyte peak separations and, consequently, accurate qualitative and qualitative analyses. New and more efficient columns are always requested to satisfy new and challenging analytical needs. RESULTS: In this work, imidazolium ionic liquids functionalized pillar [5] arenes have been used for the first time as gas chromatographic stationary phases, considering their highly symmetric pillar-shaped architecture with cavities rich in π-electrons. Four imidazolium ionic liquids functionalized pillar [5] arenes have been tested as stationary phases with numerous analytes and isomers. In particular, one of these showed superior performances if compared to commercial columns, enabling challenging isomeric separations of halogenated benzenes, aromatic aldehydes, and aromatic anilines. SIGNIFICANCE AND NOVELTY: To our knowledge, this is the first report on the use of the ionic liquid P[n]A as a stationary phase in chromatography, either in GC or liquid chromatography (LC) separations. This work demonstrates the promising potential of ionic liquid P[n]A stationary phases for chromatographic separations.

Hydroxyl-functionalized pillar[5]arene with high separation performance for gas chromatography.

Here we report the first example of employing hydroxyl-functionalized pillar[5]arene (P5A-C10-OH) as stationary phase for capillary gas chromatographic (GC) separations. The statically coated P5A-C10-OH capillary column possessed moderate polarity and column efficiency of 3233 plates per m determined by n-dodecane. As a result, the P5A-C10-OH column exhibited high-resolution capability for the mixture of 17 analytes from apolar to polar nature. Importantly, it exhibited advantageous performance for high resolution of the challenging isomers of bromonitrobenzene, chloroaniline, bromoaniline, iodoaniline and dimethylaniline with good peak shapes over the P5A-C10 and commercial HP-35 columns. In addition, eight cis-/trans-isomers with diverse types were baseline separated on the P5A-C10-OH column. And the application of detecting isomeric impurities in real samples gave strong evidence of its potential and feasibility for the viable GC analysis.

The red/far-red light photoreceptor FvePhyB regulates tissue elongation and anthocyanin accumulation in woodland strawberry.

Light is an important environmental signal that influences plant growth and development. Among the photoreceptors, phytochromes can sense red/far-red light to coordinate various biological processes. However, their functions in strawberry are not yet known. In this study, we identified an EMS mutant, named P8, in woodland strawberry (Fragaria vesca) that showed greatly increased plant height and reduced anthocyanin content. Mapping-by-sequencing revealed that the causal mutation in FvePhyB leads to premature termination of translation. The light treatment assay revealed that FvePhyB is a bona fide red/far-red light photoreceptor, as it specifically inhibits hypocotyl length under red light. Transcriptome analysis showed that the FvePhyB mutation affects the expression levels of genes involved in hormone synthesis and signaling and anthocyanin biosynthesis in petioles and fruits. The srl mutant with a longer internode is caused by a mutation in the DELLA gene FveRGA1 (Repressor of GA1) in the gibberellin pathway. We found that the P8 srl double mutant has much longer internodes than srl, suggesting a synergistic role of FvePhyB and FveRGA1 in this process. Taken together, these results demonstrate the important role of FvePhyB in regulating plant architecture and anthocyanin content in woodland strawberry.

Copper (II) Ions Induced Self-Disproportionation of Enantiomers in Capillary Electrophoresis for the Quantification of Atenolol Enantiomers.

Despite the fact that the self-disproportionation of enantiomers (SDE) has been found for several decades and has been widely used in crystallization, sublimation and chromatography for the purification or separation of nonracemic compounds, the phenomenon of SDE in capillary electrophoresis (CE) has never been reported up to now. Here, a new approach to separate enantiomers in CE based on SDE was demonstrated by introducing copper (II) ions into the separation media. The enantiomers of atenolol interact with copper ions to produce positively charged complexes with different electrophoretic mobilities from the single molecules. The dynamic equilibrium between homo- or heterochiral complexes (associates) and single molecules of atenolol enantiomers supports the manifestation of SDE. Different mobilities of the single molecules and associates, and different distribution of two enantiomers between the single molecules and associates caused by their different concentrations, produce a net difference in electrodriven migration velocities of the two enantiomers. The relative movement of two enantiomers causes a zone depleted in one enantiomer at the rear end of sample segment, giving a trapezoidal CE curve with a step at the end. Quantification of enantiomers is achieved according to the step height. The analysis does not rely on the use of enantiomerically pure chiral selector and the result agrees with that obtained by conventional chiral CE using a chiral selector.

Roles and evolution of four LEAFY homologs in floral patterning and leaf development in woodland strawberry.

The plant-specific transcription factor LEAFY (LFY), generally maintained as a single-copy gene in most angiosperm species, plays critical roles in flower development. The woodland strawberry (Fragaria vesca) possesses four LFY homologs in the genome; however, their respective functions and evolution remain unknown. Here, we identified and validated that mutations in one of the four LFY homologs, FveLFYa, cause homeotic conversion of floral organs and reiterative outgrowth of ectopic flowers. In contrast to FveLFYa, FveLFYb/c/d appear dispensable under normal growth conditions, as fvelfyc mutants are indistinguishable from wild type and FveLFYb and FveLFYd are barely expressed. Transgenic analysis and yeast one-hybrid assay showed that FveLFYa and FveLFYb, but not FveLFYc and FveLFYd, are functionally conserved with AtLFY in Arabidopsis (Arabidopsis thaliana). Unexpectedly, LFY-binding site prediction and yeast one-hybrid assay revealed that the transcriptional links between LFY and the APETALA1 (AP1) promoter/the large AGAMOUS (AG) intron are missing in F. vesca, which is due to the loss of LFY-binding sites. The data indicate that mutations in cis-regulatory elements could contribute to LFY evolution. Moreover, we showed that FveLFYa is involved in leaf development, as approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Phylogenetic analysis indicated that LFY homologs in Fragaria species may arise from recent duplication events in their common ancestor and are undergoing convergent gene loss. Together, these results provide insight into the role of LFY in flower and leaf development in strawberry and have important implications for the evolution of LFY.

A GT-1 and PKc domain-containing transcription regulator SIMPLE LEAF1 controls compound leaf development in woodland strawberry.

Leaves are strikingly diverse in terms of shapes and complexity. The wild and cultivated strawberry plants mostly develop trifoliate compound leaves, yet the underlying genetic basis remains unclear in this important fruit crop in Rosaceae. Here, we identified two EMS mutants designated simple leaf1 (sl1-1 and sl1-2) and one natural simple-leafed mutant monophylla in Fragaria vesca. Their causative mutations all reside in SL1 (FvH4_7g28640) causing premature stop codon at different positions in sl1-1 and sl1-2 and an eight-nucleotide insertion (GTTCATCA) in monophylla. SL1 encodes a transcription regulator with the conserved DNA-binding domain GT-1 and the catalytic domain of protein kinases PKc. Expression of SL1pro::SL1 in sl1-1 completely restored compound leaf formation. The 35S::SL1 lines developed palmate-like leaves with four or five leaflets at a low penetrance. However, overexpressing the truncated SL1ΔPK caused no phenotypes, probably due to the disruption of homodimerization. SL1 is preferentially expressed at the tips of leaflets and serrations. Moreover, SL1 is closely associated with the auxin pathway and works synergistically with FveLFYa in leaf morphogenesis. Overall, our work uncovered a new type of transcription regulator that promotes compound leaf formation in the woodland strawberry and shed new lights on the diversity of leaf complexity control.

Factor of DNA methylation 1 affects woodland strawberry plant stature and organ size via DNA methylation.

RNA-directed DNA methylation (RdDM) is an epigenetic process that directs silencing to specific genomic regions and loci. The biological functions of RdDM are not well studied in horticultural plants. Here, we isolated the ethyl methane-sulfonate-induced mutant reduced organ size (ros) producing small leaves, flowers, and fruits in woodland strawberry (Fragaria vesca) due to reduced cell numbers compared with that in the wild-type (WT). The candidate mutation causes a premature stop codon in FvH4_6g28780, which shares high similarity to Arabidopsis (Arabidopsis thaliana) Factor of DNA Methylation1 (FDM1) encoding an RdDM pathway component and was named FveFDM1. Consistently, the fvefdm1CR mutants generated by CRISPR/Cas9 also produced smaller organs. Overexpressing FveFDM1 in an Arabidopsis fdm1-1 fdm2-1 double mutant restored DNA methylation at the RdDM target loci. FveFDM1 acts in a protein complex with its homolog Involved in De Novo 2 (FveIDN2). Furthermore, whole-genome bisulfite sequencing revealed that DNA methylation, especially in the CHH context, was remarkably reduced throughout the genome in fvefdm1. Common and specific differentially expressed genes were identified in different tissues of fvefdm1 compared to in WT tissues. DNA methylation and expression levels of several gibberellic acid (GA) biosynthesis and cell cycle genes were validated. Moreover, the contents of GA and auxin were substantially reduced in the young leaves of fvefdm1 compared to in the WT. However, exogenous application of GA and auxin could not recover the organ size of fvefdm1. In addition, expression levels of FveFDM1, FveIDN2, Nuclear RNA Polymerase D1 (FveNRPD1), Domains Rearranged Methylase 2 (FveDRM2), and cell cycle genes were greatly induced by GA treatment. Overall, our work demonstrated the critical roles of FveFDM1 in plant growth and development via RdDM-mediated DNA methylation in horticultural crops.

Chromatographic Separation of Aromatic Amine Isomers: A Solved Issue by a New Amphiphilic Pillar[6]arene Stationary Phase.

In this work, the fabrication, synthesis, and characterization of a new stationary phase based on an amphiphilic pillar[6]arene (P6A-C10-2NH2) for gas chromatographic analyses are reported. The gas chromatography (GC) column prepared with P6A-C10-2NH2 stationary phase exhibited a medium polarity, an efficiency of 3219 plates/m, and unmatched resolving capabilities toward chloroaniline, bromoaniline, iodoaniline, and toluidine isomers. Furthermore, the P6A-C10-2NH2 column showed excellent repeatability with maximum relative standard deviations equal to 0.02, 0.07, and 2.56% for run-to-run, day-to-day, and column-to-column, respectively, demonstrating a great potential as a new stationary phase in separation science.

A New Capillary Gas Chromatography Column Based on Poly(ethylene glycol) Methyl Ether-Functionalized Calix[4]arene.

In this work, a novel capillary column (C4A-mPEG) with a calixarene-based polymer stationary phase (poly(ethylene glycol) methyl ether-functionalized 4-tert-butylcalix[4]arene) was designed and used for gas chromatographic (GC) separations. The C4A-mPEG capillary column, prepared by the static coating method, showed moderate polarity and a column efficiency of 2332 plates/m, determined by 1-octanol at 120 °C. The separation features of C4A-mPEG stationary phase, resulting from its unique structure and multiple molecular recognition processes with analytes, including π-π, H-bonding, dipole-dipole, and van der Waals interactions, allowed to obtain high-resolution performances for a wide range of compounds and their isomers, especially benzaldehydes, phenols, and anilines. Moreover, compared with 4-tertbutyl calix[4]arene (C4A) and polyethylene glycol (PEG) stationary phases, a higher resolving capability was also observed for the separation of toluidine and xylidine isomers.

Comparison of the Performance of Different Bile Salts in Enantioselective Separation of Palonosetron Stereoisomers by Micellar Electrokinetic Chromatography.

Bile salts are a category of natural chiral surfactants which have ever been used as the surfactant and chiral selector for the separation of many chiral compounds by micellar electrokinetic chromatography (MEKC). In our previous works, the application of sodium cholate (SC) in the separation of four stereoisomers of palonosetron (PALO) by MEKC has been studied systematically. In this work, the parameters of other bile salts, including sodium taurocholate (STC), sodium deoxycholate (SDC), and sodium taurodeoxycholate (STDC) in the separation of PALO stereoisomers by MEKC were measured and compared with SC. It was found that all of four bile salts provide chiral recognition for both pairs of enantiomers, as well as achiral selectivity for diastereomers of different degrees. The structure of steroidal ring of bile salts has a greater impact on the separation than the structure of the side chain. The varying separation results by different bile salts were elucidated based on the measured parameters. A model to describe the contributions of the mobility difference of solutes in the aqueous phase and the selectivity of micelles to the chiral and achiral separation of stereoisomers was introduced. Additionally, a new approach to measure the mobility of micelles without enough solubility for hydrophobic markers was proposed, which is necessary for the calculation of separation parameters in MEKC. Under the guidance of derived equations, the separation by SDC and STDC was significantly improved by using lower surfactant concentrations. The complete separation of four stereoisomers was achieved in less than 3.5 min by using 4.0 mM of SDC. In addition, 30.0 mM of STC also provided the complete resolution of four stereoisomers due to the balance of different separation mechanisms. Its applicability for the analysis of a small amount of enantiomeric impurities in the presence of a high concentration of the effective ingredient was validated by a real sample.

Performance and selectivity of amphiphilic pillar[5]arene as stationary phase for capillary gas chromatography.

Pillar[n]arenes possess highly symmetrical and rigid pillar-shaped architecture with π-electron rich cavity that afford their reliable host-guest recognition interactions towards matched guests. In this work, a novel amphiphilic pillar[5]arene (P5A-C10-2NH2) was designed, synthesized and employed as the stationary phase for capillary gas chromatography. To date, they have not been reported in the field of chromatography. The P5A-C10-2NH2 capillary column (10 m × 0.25 mm i.d.) was prepared by static coating method. Its capillary column exhibited moderate polarity and column efficiency of 2265 plates/m determined by naphthalene at 120 °C. As evidenced, the P5A-C10-2NH2 column achieved advantageous separation performance for a mixture of 24 analytes of diverse types and exhibited different chromatographic selectivity from two pillar[5]arene derivatives columns and commercial HP-35 column with 35%-phenyl-methylpolysiloxane. Moreover, the P5A-C10-2NH2 column baseline resolved more than a dozen positional and cis-trans isomers. Furthermore, the separation mechanism of P5A-C10-2NH2 column was discussed by quantum chemical calculations. In addition, the P5A-C10-2NH2 column had high thermal stability and excellent separation repeatability 0.01-0.04% for run-to-run, 0.03-0.17% for day-to-day and 3.2-3.9% for column-to-column. The special amphiphilic structure and high resolution for various analytes reveal the good potential of pillararenes as a new class of stationary phases for chromatographic analyses. Moreover, the TPG column achieved improved thermal stability over the GIL column and excellent repeatability.

The MADS-box gene FveSEP3 plays essential roles in flower organogenesis and fruit development in woodland strawberry.

Flower and fruit development are two key steps for plant reproduction. The ABCE model for flower development has been well established in model plant species; however, the functions of ABCE genes in fruit crops are less understood. In this work, we identified an EMS mutant named R27 in woodland strawberry (Fragaria vesca), showing the conversion of petals, stamens, and carpels to sepaloid organs in a semidominant inheritance fashion. Mapping by sequencing revealed that the class E gene homolog FveSEP3 (FvH4_4g23530) possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain. Additional fvesep3CR mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27. Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect. Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry. Moreover, both R27 and fvesep3CR mutants exhibited parthenocarpic fruit growth and delayed fruit ripening. Transcriptome analysis revealed that both common and specific differentially expressed genes were identified in young fruit at 6-7 days post anthesis (DPA) of fvesep3 and pollinated wild type when compared to unpollinated wild type, especially those in the auxin pathway, a key hormone regulating fruit set in strawberry. Together, we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.

Amphiphilic Block Copolymer PCL-PEG-PCL as Stationary Phase for Capillary Gas Chromatographic Separations.

This work presents the first example of utilization of amphiphilic block copolymer PCL-PEG-PCL as a stationary phase for capillary gas chromatographic (GC) separations. The PCL-PEG-PCL capillary column fabricated by static coating provides a high column efficiency of 3951 plates/m for n-dodecane at 120 °C. McReynolds constants and Abraham system constants were also determined in order to evaluate the polarity and possible molecular interactions of the PCL-PEG-PCL stationary phase. Its selectivity and resolving capability were investigated by using a complex mixture covering analytes of diverse types and positional, structural, and cis-/trans-isomers. Impressively, it exhibits high resolution performance for aliphatic and aromatic isomers with diverse polarity, including those critical isomers such as butanol, dichlorobenzene, dimethylnaphthalene, xylenol, dichlorobenzaldehyde, and toluidine. Moreover, it was applied for the determination of isomer impurities in real samples, suggesting its potential for practical use. The superior separation performance demonstrates the potential of PCL-PEG-PCL and related block copolymers as stationary phases in GC and other separation technologies.

ß-Cyclodextrin-copper (II) complex as chiral selector in capillary electrophoresis for the enantioseparation of ß-blockers.

The Copper (II) complex of ß-cyclodextrin (ß-CD), ß-CDCu2, was prepared and used as a chiral selector for the enantioseparation of 11 ß-blockers by capillary electrophoresis. Resolution for 10 pairs of enantiomers was greater than 1.9 and for the four stereoisomers of labetalol (with two chiral centers), the closest two peaks had a resolution of 1.2. The resolution was superior to that obtained by ß-CD itself as well as using anionic cyclodextrin, sulfated-ß-cyclodextrin (S-ß-CD). In order to elucidate the separation mechanism, the conditional binding constants between the enantiomers and the complex chiral selector and the elctrophoretic mobilities of the diastereomeric complexes were calculated by nonlinear regression. It was found that binding difference and mobility difference are two effects that are responsible for enantiomeric separation. When the concentration of selector is low, the differences in binding were dominant in the separation, while at high concentrations, differences in mobility were dominant in the separation. It was possible to achieve changes in migration orders for two of the drugs tested, with the remainder showing robust resolution due to compound effects of mobility and conditional formation constant. The complex chiral selector provides good separation for the enantiomers or stereoisomers of all the drugs tested which cannot be resolved by ß-CD itself.

Performance and selectivity of lower-rim substituted calix[4]arene as a stationary phase for capillary gas chromatography.

This work presents the investigation of p-tert-butyl(tetradecyloxy)calix[4]arene (C4A-C10) as stationary phase for capillary gas chromatographic (GC) separations. The statically-coated C4A-C10 capillary column showed weak polarity and column efficiency of 2566 plates per m determined by n-dodecane at 120 °C. Impressively, the C4A-C10 column exhibited extremely high resolving capability for a wide range of analytes from nonpolar to polar, including n-alkanes, esters, ketones, aldehydes, alcohols and bromoalkanes. Most importantly, the C4A-C10 column exhibited an excellent separation performance for positional, structural and cis-/trans-isomers. Among them, the column displayed advantageous resolving capability over the commercial polysiloxane stationary phase for aromatic amine isomers. Moreover, the C4A-C10 column showed good column repeatability with RSD values below 0.06% for run-to-run, 0.12-0.27% for day-to-day and 2.8-5.3% for column-to-column.

Star-poly(ε-caprolactone) as the stationary phase for capillary gas chromatographic separation.

This work presents the separation performance of star-poly(ε-caprolactone) (star-PCL) as the stationary phase for capillary gas chromatography (GC). The statically coated star-PCL column showed a column efficiency of 3345 plates per m and moderate polarity. Importantly, the star-PCL column exhibited high selectivity and resolving capability for more than a dozen mixtures covering a wide-ranging variety of analytes and isomers. Among them, the star-PCL column displayed advantageous resolving capability over the commercial DB-1701 column for aromatic amine isomers such as toluidine, chloroaniline and bromoaniline. Moreover, it was applied for the determination of isomer impurities in real samples, showing good potential in GC applications.

Separation performance of p-tert-butyl(tetradecyloxy)calix[6]arene as a stationary phase for capillary gas chromatography.

This work presents the first example of the utilization of p-tert-butyl(tetradecyloxy)calix[6]arene (C6A-C10) as a stationary phase for capillary gas chromatographic (GC) separation. The statically coated C6A-C10 column showed a column efficiency of 3293 plates per m and had a nonpolar nature. Its selectivity and retention behaviour were investigated using a number of mixtures of diverse analytes and their isomers. As a result, the C6A-C10 column exhibited high resolving capability for aliphatic and aromatic analytes from apolar to polar nature, especially for positional, structural and cis-/trans-isomers. Moreover, the C6A-C10 column showed thermal stability up to 240 °C. In addition, it was applied for the determination of isomer impurities in real samples, proving its good potential for practical GC analysis.

Performance of palm fibers as stationary phase for capillary gas chromatographic separations.

Herein we report the first example of exploring bio-based materials, palm fibers (PFs), as a stationary phase for capillary gas chromatographic separations. The PFs capillary column was fabricated by the sol-gel coating method and showed a weak polar nature and high column efficiency over 4699 plates per m for n-dodecane, naphthalene and n-octanol. Importantly, the column exhibited high selectivity and resolving capability for more than a dozen mixtures covering a wide-ranging variety of analytes and isomers. In addition, it was applied for the determination of isomer impurities in real samples, proving its good potential for practical gas chromatographic analysis.

Thermodynamic models to elucidate the enantioseparation of drugs with two stereogenic centers by micellar electrokinetic chromatography.

An equilibrium model depicting the simultaneous protonation of chiral drugs and partitioning of protonated ions and neutral molecules into chiral micelles in micellar electrokinetic chromatography (MEKC) has been introduced. It was used for the prediction and elucidation of complex changes in migration order patterns with experimental conditions in the enantioseparation of drugs with two stereogenic centers. Palonosetron hydrochloride (PALO), a weakly basic drug with two stereogenic centers, was selected as a model drug. Its four stereoisomers were separated by MEKC using sodium cholate (SC) as chiral selector and surfactant. Based on the equilibrium model, equations were derived for a calculation of the effective mobility and migration time of each stereoisomer at a certain pH. The migration times of four stereoisomers at different pHs were calculated and then the migration order patterns were constructed with derived equations. The results were in accord with the experiment. And the contribution of each mechanism to the separation and its influence on the migration order pattern was analyzed separately by introducing virtual isomers, i.e., hypothetical stereoisomers with only one parameter changed relative to a real PALO stereoisomer. A thermodynamic model for a judgment of the correlation of interactions between two stereogenic centers of stereoisomers and chiral selector was also proposed. According to this model, the interactions of two stereogenic centers of PALO stereoisomers in both neutral molecules and protonated ions with chiral selector are not independent, so the chiral recognition in each pair of enantiomers as well as the recognition for diastereomers is not simply the algebraic sum of the contributions of two stereogenic centers due to their correlation.