Preparation of a Sulfoalkylbetaine-Based Zwitterionic Monolith with Enhanced Hydrophilicity for Capillary Electrochromatography Separation Applications

A novel monolithic stationary phase based on in situ copolymerization of zwitterionic monomer N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (DMMSA), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2-methylpropene-1-sulfonate (MPS) was designed as a multifunctional separation column for hydrophilic interaction capillary electrochromatography (HI-CEC). A significantly enhanced hydrophilicity was obtained on the poly(DMMSA-co-PETA-co-META or MPS) monolith, which was contributed by the high percentage of DMMSA in the polymerization mixture. A column efficiency of 200,000 plates/m was obtained and the monolithic column also displayed a satisfactory repeatability in terms of migration time with RSD values less than 1.1% (intra-day, n = 5) and 2.0% (inter-day, n = 3). Most importantly, the column was successfully applied to separation of a pool of neurotransmitters which are not well separated on commercial HILIC packing materials. A baseline separation of the 12 model components was obtained with good selectivity, symmetrical peak shape and high column efficiency with BGE consisting of 20 mM ammonium formate (pH 3.0) in ACN/H₂O (80/20, v/v).     

Possibilities and Limitations of Computer-Assisted Method Development in HILIC: A Case Study

In the present study, we investigated the possibilities and limitations of computer-assisted method development (CAMD) for the HILIC separation optimization of a mixture of 13 isomeric hydroxy- and aminobenzoic acids on a ZIC-HILIC column. The isocratically obtained Neue and Kuss retention parameters enabled the accurate gradient retention modeling for peaks eluting well within the gradient (mean error of 2.7 %). The prediction errors for peaks eluting at the end of the gradient could be reduced from 8.8 to 6.1 % by implementing the isocratic regime after the gradient into the expression for the gradient retention factor. The prediction of the corresponding peak widths improved significantly for certain compounds and gradient profiles using individual gradient N values for each compound compared to employing a single N value for all compounds and gradient profiles. Two gradient optimization strategies (constructing the R _s map based on individual retention modeling and predictive elution stretching and shifting, PEWS²) resulted in a reasonable separation of the challenging mixture of 13 isomeric hydroxy- and aminobenzoic acids on the ZIC-HILIC column. Overall, the optimization was limited by the steep decrease in N (dropping to the isocratic N value) and corresponding increase in peak width when increasing the gradient time. The discrimination factors d ₀ were used to assess the resolution between peaks varying widely in height. The best separation was found to be obtained via the PEWS² approach. Both the individual retention modeling and PEWS² strategies corresponded to a total instrument time less than 12 h (including equilibration). Finally, it was found that the salt concentration had a significant effect on both the retention and the peak shape of the compounds, resulting in a small “solution domain” at 10 mM. Coupled columns with higher efficiencies are suggested to improve the resolution and robustness of the separation.     

Phase Ratio and Equilibrium Constant in RP-HPLC Obtained from Octanol/Water Partition Constant Through Solvophobic Theory

The experimentally known dependence in RP-HPLC of the retention factor k′ on octanol/water partition coefficient (K _ow) has been examined based on solvophobic theory. The result showed that the dependence provides a means for the evaluation of phase ratio (Φ) of RP-HPLC columns, and of the equilibrium constant for a given compound and mobile phase. Using this theory, the phase ratio was evaluated for a set of seven different C18 columns (five having fully porous particles and two core–shell particles), and the equilibrium constants were calculated for four homologous series of compounds in two mobile phase systems. One mobile phase was methanol/aqueous solution of 0.1% H₃PO₄, and the other was acetonitrile/aqueous solution of 0.1% H₃PO₄. Besides providing the values for Φ for the evaluated columns, the results of the study indicated that for a specific composition of the mobile phase and for a given compound displaying only hydrophobic interactions, the equilibrium constant K(X) for different C-18 columns is basically the same. The data were further used to provide guidance in the selection of a chromatographic column for a specific separation based on K _ow values and chemical structure of the analytes. The study indicated that the separation of compounds with identical polar groups (or no polar groups) and with very close values for the K _ow cannot be achieved based only on hydrophobic interactions that dominate the separation on RP-type columns. Only column that displays polar interactions may provide a solution to such separations. For hydrocarbons with close K _ow values, the separation cannot be achieved even on columns with some polarity. On the other hand, even compounds with equal K _ow values, but with different functionalities can be separated on RP-HPLC columns without involving polar interactions. The compounds with different K _ow values are expected to be easily separated on RP-HPLC columns.     

Spectrometric analysis,phenolics isolation and cytotoxic activity of <Emphasis Type="Italic">Stipagrostis plumosa</Emphasis> (Family Poaceae)

Grasses (family Poaceae) are economically important plants; they are used as crops and animal foods. Stipagrostis plumosa (L.) Munro ex T. Anderson is a member of this family and subjected to chemical and biological studies. The chromatographic techniques,  LC–ESI–MS and GC/MS were used for identification of polar and non-polar compounds in its extract. Ten compounds, including one new flavone glycoside; tricin 7-O-galactoside, three known flavones, three C-glycosyl flavones and three phenolic acids, were isolated from S. plumosa for the first time except tricin. Their structures were elucidated on the basis of extensive spectroscopic interpretation. In addition to the isolated compounds, eleven compounds were tentatively identified using LC–ESI–MS, five of them were detected for the first time from this species. 29 non polar compounds were identified using GC–MS analysis, representing 83.13% of S. plumosa diethyl ether extract. In addition to the DPPH activity evaluation, the crude extract and the isolated compounds were investigated against five human carcinoma cell lines; A549, HCT-116, HepG2, MCF-7 and PC3 at a concentration of 100 μg/ml. From the isolated compounds tricin and luteolin 6,8-di-C-glucoside could be considered as natural-free radical scavenging agents.     

Alkylation of 1-Alkyl-3-methyl-1,4-dihydropyrazolo[4,3-<Emphasis Type="Italic">c</Emphasis>]pyrazoles with Halocarboxylic Acids Esters

A preparative method for the synthesis of 1-alkyl-3-methyl-1,4-dihydropyrazolo[4,3-c]pyrazoles was developed. The alkylation of the obtained compounds with halocarboxylic acid esters was also investigated. A principal possibility of creating libraries of compounds based on pyrazolo[4,3-c]pyrazole derivatives was shown.     

Investigation of Euphrasia rostkoviana Hayne Using GC–MS and LC–MS

Gas and liquid chromatography coupled with mass spectrometry was applied to solve difficulties and reinvestigate the serious matrix problems affecting analysis of the active compounds in Euphrasia rostkoviana Hayne. The main groups of compounds were obtained by extracting the herb stepwise with n-hexane, chloroform, ethyl acetate and methanol. Polyamide column chromatography facilitated further separation. Phenolic/flavonoid- and terpenoid-type molecules were studied by GC–MS, HPLC and LC–MS–MS. The β-sitosterol content of the herb was determined by gas chromatography with flame ionisation detection (GC-FID). Caffeic acid, chlorogenic acid, coumaric acid and flavonoid glycosides of apigenin, luteolin, rhamnetine (hexoside), kaempferol (both hexoside and rutinoside) and quercetin (rutinoside) were identified in the fractions of the methanolic extract.     

Nonaqueous CE for Rapid and Sensitive Determination of Matrine and Oxymatrine in <Emphasis Type="Italic">Sophora flavescens</Emphasis> and Its Medicinal Preparations

A simple, rapid, and sensitive non-aqueous capillary electrophoresis procedure for the quantitative determination of matrine and oxymatrine is established. Optimum separation conditions were obtained when the sample was injected under pressure for 3 s at 50 mbar and separated with the buffer containing 70 mM ammonium acetate, 7.0% (v/v) acetic acid, and 10% (v/v) acetonitrile in methanol medium at 25 kV applied voltage. The analytes were detected at 205 nm. The two alkaloids can be separated within 12 min and quantified with high sensitivity. The method was validated in terms of reproducibility, linearity, and accuracy when applied to the analysis of matrine and oxymatrine in Sophora flavescens and its medicinal preparations.     

Validated LC Method for Determination of Enantiomeric Purity of Apremilast Using Polysaccharide-Type Stationary Phases in Polar Organic Mode

Enantioseparation of an anti-psoriatic agent, apremilast (APR), was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode for the first time. The separation capability of six different chiral columns (Chiralpak AD, Chiralpak IA, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) was investigated using neat MeOH and ACN. During the preliminary experiments the best results were obtained on Chiralpak IA column with ACN (R_s?=?5.4). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN. U-shaped retention pattern was obtained when plotting the retention factors of the APR enantiomers versus the MeOH content of the binary mobile phases on Chiralpak IA column. For further method optimizations an L25 orthogonal array table was employed altering the concentration of MeOH in ACN, column temperature, and flow rate. The best result was achieved on Chiralpak IA column with 80/20 (v/v%) MeOH/ACN with 0.7 mL min^?1 flow rate at 25 °C (R_s?=?5.4, t₂?=?7.45 min). Thermodynamic analysis revealed an enthalpy-driven enantioseparation. The developed HPLC method was validated according to the ICH guideline Q2(R1) and proved to be reliable, linear, precise and accurate for the determination of 0.1% R-enantiomer as chiral impurity in S-APR as well as quantification of the S-enantiomer.

Graphical Abstract

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How medicinal chemists learned about log <Emphasis Type="Italic">P</Emphasis>

Although log P is now recognized to be a key factor that determines the bioactivity of a molecule, the focus of medicinal chemists on hydrophobicity and log P started with the quantitative structure–activity relationships (QSAR) publications of Hansch and Fujita. Their original publication represents a dramatic change of focus to incorporate consideration of log P after a decade of work unsuccessfully attempting to use the Hammett equation to explain the structure-activity relationships of plant growth regulators. QSAR allows one to explore the quantitative relationship between log P and biological activity even when other factors also influence potency. In particular, Hansch’s publications of thousands of QSAR equations demonstrate that a relationship of biological activity with log P is indeed a general phenomenon. Hansch’s group also provided data and tools that enable others to explore the relationship between log P and the biological activity of compounds of interest.     

Study of the growth of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>, <Emphasis Type="Italic">Escherichia coli</Emphasis> and their mixtures by microcalorimetry

In a majority of environments, microbes live as interacting communities. Microbial communities are composed of a mix of microbes with often unknown functions. Polymicrobial diseases represent the clinical and pathological manifestations induced by the presence of multiple infectious agents. These diseases are difficult to diagnose and treat and usually are more severe than monomicrobial infections. The interaction relationship between Enterococcus faecalis and Escherichia coli was researched using a Calvet calorimeter. Three mixtures of both bacteria were prepared in the following proportions: 20 + 80 % (0.2 mL E. faecalis + 0.8 mL E. coli), 50 + 50 % (0.5 mL E. faecalis + 0.5 mL E. coli) and 80 + 20 % (0.8 mL E. faecalis + 0.2 mL E. coli). Experiments were carried out at concentration of 10⁶ CFU mL^?1 and a constant temperature of 309.65 K. The differences in shape of graph of E. faecalis, E. coli and their mixtures were compared. Also, the thermokinetic parameters such as detection time (t _d), growth constant (k), generation time (G) and the amount of heat released (Q) were calculated.     

Synthesis and piezoelectric properties of <Emphasis Type="Italic">N</Emphasis>-phthaloylglutamic acid derivatives

The (S,S)- and (R,R)-enantiomers of dimethyl 2,4-diphthalimidoglutarate were synthesized by nucleophilic substitution of bromine in dimethyl (2S,4RS)-4-bromo-N-phthaloyl-glutamate upon treatment with potassium phthalimide, followed by separation. The crystal structure of the obtained compounds was studied by X-ray diffraction. Crystals of enantiomerically pure dimethyl 4-hydroxy- and 4-phthalimido-N-phthaloylglutamates were found to possess a noticeable piezoelectric activity.     

Synthesis of amino acid conjugates of glycyrrhizic acid using <Emphasis Type="Italic">N</Emphasis>-hydroxyphthalimide and <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>'-dicyclohexylcarbodiimide

Synthesis of amino acid conjugates of glycyrrhizic acid with the use of N-hydroxyphthalimide, N,N'-dicyclohexylcarbodiimide, and tert-butyl esters of L-amino acids (valine, isoleucine, phenylalanine, and methionine) was performed followed by deprotection with trifluoroacetic acid. The target amino acid conjugates were isolated by column chromatography on silica gel in 40–45% yield. The structure of the prepared compounds was confirmed by IR and ¹³C NMR spectroscopy.     

Dissolved air flotation for treating wastewater of the nuclear industry: preliminary results

Preliminary testing of dissolved air flotation (DAF) for wastewater treatment is presented. A combined coagulation-flocculation/DAF column system is used to remove oil and ⁶⁰Co from nuclear industry wastewater. In this work, operational conditions and coagulant/flocculant concentrations are optimized by varying pH. Determinations of air-solids ratio (G/S), retention time (θ), pressure (P), volume of depressurized air–water mixture (V), turbidity and ⁶⁰Co concentrations are reported. The effect of the treatment on the efficiency of separation of oily residues is also discussed. The results establish that the coagulant/flocculant system, formed by a modified polyamine (25 mgL^?1) and a slightly cationic polyacrylamide (1.5 mgL^?1), under specific operational conditions (pH = 7, mixing intensity Im₁ = 300 s^?1 and Im₂ = 30 s^?1), allowed the destabilization of colloidal matter, resulting in resistant flocs. It was concluded that by using G/S = 0.3, θ = 15 min, P = 620 kPa and V = 0.0012 m³, the greatest percentage removals of oil, turbidity, total cobalt and ⁶⁰Co were obtained. These preliminary results then show that dissolved air flotation represents a good alternative for treatment of nuclear industry wastewater contaminated with radionuclides.     

Antifungal Lipopeptides Produced by <Emphasis Type="Italic">Bacillus</Emphasis> sp. FJAT-14262 Isolated from Rhizosphere Soil of the Medicinal Plant <Emphasis Type="Italic">Anoectochilus roxburghii</Emphasis>

This study aimed to develop biocontrol Bacillus and explore bacterial biocontrol substances. According to the blood agar test, strain FJAT-14262 was screened as a biosurfactant-producer. The biosurfactant-producing ability of FJAT-14262 was further confirmed by the oil spreading tests because of its amphipathic character. Furthermore, its fermentation supernatant could decrease the surface tension from 74.1 to 32.7 mN m^?1. Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the biosurfactant produced by the strain FJAT-14262 was a kind of lipopeptides. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that this lipopeptide contained surfactin with polar amino acids and hydrophobic fatty acid chains. Moreover, bioinformatic analysis revealed that the nonribosomal peptide synthetases genes srfAA, srfAB, and srfAC were structurally conserved in the FJAT-14262 genome. Importantly, the crude surfactant exhibited strong inhibitory activities against Fusarium oxysporum, suggesting that strain FJAT-14262 could be a potential biological control agent against Fusarium wilt.     

A Multivariate Approach to Evaluate Biomass Production,Biochemical Composition and Stress Compounds of <Emphasis Type="Italic">Spirulina platensis</Emphasis> Cultivated in Wastewater

The study was performed to investigate the effects of using cow effluent for the cultivation of Spirulina platensis on its biomass production and cell physiology. S. platensis was cultivated in three different cow effluents (CE) used as cultivation medium during 15 days. CE was prepared using dry cow manures, and it was further modified with supplement of NaNO₃ (CEN) and NaNO₃ + NaCl (CENS). High nitrate value stimulated chlorophyll-a and total protein content of the cyanobacterium and also biomass production in standards medium (SM) and CEN media. Total carbohydrate content of S. platensis grown in CE media was found to be higher (p < 0.05) than that of SM. Productions of biomass and biochemical compounds by the cyanobacterium grown on the CE and SM media were evaluated by using multivariate approach. Conductivity, oxidation reduction potential (ORP), salinity, pH, and TDS played important role (p < 0.01) in the biochemical composition. As an effective explanatory factor, ORP had a significant positive correlation with H₂O₂, whereas negatively correlated with chlorophyll-α, biomass production, filament length, and proline. Canonical correspondence analysis proposed that biochemical compounds of S. platensis were not only affected by salinity and nutrition of media but also by pH and ORP. The present study indicated that CEN as a low cost model medium had high potential for the production of biomass by S. platensis with high protein content.     

Synthesis of silver(I) and palladium(II) <Emphasis Type="Italic">N</Emphasis>-heterocyclic carbene complexes and their use as catalysts for the direct C5 arylation of heteroaromatic compounds

Silver(I) N-heterocyclic carbene complexes were synthesized in good yields by the reactions of 1,3-dialkylperhydrobenzimidazolium salts with silver(I) oxide in dichloromethane. The silver complexes were used as carbene-transfer agents to synthesize palladium(II) N-heterocyclic carbene complexes. All of the complexes were characterized by physicochemical and spectroscopic methods. The new palladium complexes were tested as catalysts in the direct C5 arylation of 2-n-butylfuran, 2-n-butylthiophene and 2-n-propylthiazole with aryl bromides at 130 °C in N,N-dimethylacetamide. The arylation reactions proceeded selectively at the C5 position of the heteroaromatic compounds, and the corresponding coupling products were obtained in moderate to good yields by using 0.5 mol% of the palladium complex.     

Alkylation of 2-(methylsulfanyl)-6-polyfluoroalkylpyrimidin-4(3<Emphasis Type="Italic">H</Emphasis>)-ones with haloalkanes

The alkylation of ambident anions of 2-(methylsulfanyl)-6-(polyfluoroalkyl)pyrimidin-4(3H)-ones with 4-bromobutyl acetate leads to concurrent formation of O- and N-(4-acetoxybutyl) derivatives. Polar aprotic solvents favor formation of the O-isomer, and weakly polar dioxane favors N-alkylation. The reaction of 2-(methylsulfanyl)-6-(trifluoromethyl)pyrimidin-4(3H)-one with an equimolar amount of 1,2-dibromoethane in polar acetonitrile gives a mixture of N,N-, O,O-, and N,O-bridged bis-pyrimidines, as well as N- and O-[2-(methylsulfanyl)ethyl] derivatives, whereas in the presence of 10 equiv of 1,2-dibromoethane the N,O-isomer is formed as the only product. The reaction in weakly polar tetrahydrofuran yields N,N- and N,O-bispyrimidines.     

Strategy for the Synthesis of pyrazolo[5,1-<Emphasis Type="Italic">d</Emphasis>][1,2,5]triazepinones,a new heterocyclic system

A preparative procedure has been developed for the synthesis of 4-oxo-7-phenyl-5,8-dihydro-4H-pyrazolo[ 5,1-d][1,2,5]triazepine-2-carbohydrazide, the first representative of a new heterocyclic system, by recyclization of methyl 4-oxo-6-phenyl-4H-pyrazolo[5,1-c][1,4]oxazine-2-carboxylate with hydrazine hydrate.     

Synthesis,antifungal and insecticidal activity of novel [1,2,4]triazolo[4,3-<Emphasis Type="Italic">a</Emphasis>]pyridine derivatives containing a sulfide substructure

A series of [1,2,4]triazolo[4,3-a]pyridine derivatives bearing a sulfide substructure was designed, synthesized and characterized via ¹H·NMR, ¹³C·NMR, IR and elemental analyses. Bioassay Results indicated some of the derivatives displayed good fungicidal activity on Rhizoctonia cerealis, moderated insecticidal activity against Plutella xylostella and good insecticidal activity on Helicoverpa armigera. The inhibitory effects of compounds 4g and 4u against Rhizotonia cerealis were 70.9% at 50 μg mL^?1; the IC₅₀ values of compounds 4d and 4s against Plutella xylostella were 43.87 and 50.75 μg mL^?1, respectively. And the IC₅₀ values of compounds 4d, 4q, and 4s on Helicoverpa armigera were 58.3, 77.14 and 65.31 μg mL^?1, respectively, which were better than that of commercial chlorpyrifos (103.77 μg mL^?1).     

New approaches to the calculation and interpretation of asymmetry factors of chromatographic peaks

The suitability of the determination of the asymmetry factor of chromatographic peaks by the ratio of areas of two components separated by a perpendicular dropped from the maximum of the peak to the base-line, A _s * = S _b /S _a , where symbol a corresponds to the leading edge of the peak and b is for its tailing slope, is discussed. It is demonstrated that this method enables the estimation of the asymmetry of even partially separated chromatographic signals, including those eluted “in the tail” of intense peaks of solvents. The concepts of the asymmetry index I(A _s *) and its increment ΔI(A _s *) = (A _s *)–I(A _s *) are introduced, which ensures the characterization of the asymmetry of peaks of polar analytes with respect to the asymmetry of nonpolar reference components, that is, the separation of the effects of the polarity of analytes and their quantities injected into the chromatographic column on this parameter. For the first time we revealed a correlation of the asymmetry factors of compounds of different chemical nature with such a characteristic of their polarity as the difference in chromatographic separation temperature and the normal boiling point of analytes.