Liquid-liquid Partition Research Articles

Estimation of descriptors for hydrogen-bonding compounds from chromatographic and liquid-liquid partition measurements

Retention factors obtained by gas chromatography and reversed-phase liquid chromatography on varied columns and partition constants in different liquid-liquid partition systems are used to estimate WSU descriptor values for 36 anilines and N-heterocyclic compounds, 13 amides and related compounds, and 45 phenols and alcohols. These compounds are suitable for use as calibration compounds to characterize separation systems covering the descriptor space E=0.2–3, S=0.4–2.1, A=0–1.5, B=0.1–1.5, L=2.5–10.0 and V=0.5–2.2. Hydrogen-bonding properties are discussed in terms of structure, the possibility of induction effects, intramolecular hydrogen bonding and steric factors for anilines, amides, phenols and alcohols. The relationship between these parameters and observed descriptor values are difficult to predict from structure but facilitate improving the general occupancy of the descriptor space by creating incremental changes in hydrogen-bonding properties. It is verified that the compounds included in this study can be merged with an existing database of compounds recommended for characterizing separation systems.

TYROSINASE INHIBITION, 2,2-DIPHENYL-1-PICRYLHYDRAZYL RADICAL SCAVENGING ACTIVITY, AND PHYTOCHEMICAL SCREENING OF FRACTIONS AND ETHANOL EXTRACT FROM LEAVES AND STEM BARK OF MATOA (POMETIA PINNATA)

Objective: This study aims to investigate the potency of matoa as a tyrosinase inhibitor and antioxidant and also to identify the chemical compounds in the most active fraction and an ethanol extract from the leaves and stem bark of matoa.Methods: The extracts were tested for their tyrosinase inhibitory activity by evaluating the formation of L-dopachrome at 490 nm. Antioxidant activity was tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The most active extract from liquid-liquid partition analysis was fractionated, and then, the assays were performed.Results: The ethanol extract of leaves and stem bark of matoa showed weak anti-tyrosinase activity (percent inhibition was 24.54±0.22% and 21.93±0.57%, respectively, final concentration 200 μg/mL), but they showed strong DPPH radical scavenging activity (IC50 values were 6.11 μg/mL and 5.47 μg/mL, respectively). The ethyl acetate fraction was the most active fraction with an IC50 value of 5.38 μg/mL. Ethanol extract from the leaves and stem bark of matoa and the ethyl acetate fraction contains flavonoids, tannins, saponins, triterpenoids, and glycosides.Conclusion: Matoa does not have potency as a tyrosinase inhibitor, but it has good antioxidant activity, although still lower than that of quercetin.

液-液二相分配クロマトグラフィーの有機化学実習への応用 I : エキストレルートカラムを用いるエーテル抽出法

液-液二相分配クロマトグラフィーの有機化学実習への応用 I : エキストレルートカラムを用いるエーテル抽出法

液・液二相分配クロマトグラフィーの有機化学実習への応用 III : エキストレルートカラムを用いる分離抽出法

液・液二相分配クロマトグラフィーの有機化学実習への応用 III : エキストレルートカラムを用いる分離抽出法

液-液二相分配クロマトグラフィーの有機化学実習への応用 II : エキストレルートカラム抽出法と分液漏斗抽出法の比較

液-液二相分配クロマトグラフィーの有機化学実習への応用 II : エキストレルートカラム抽出法と分液漏斗抽出法の比較

Antinociceptive, anti-inflammatory and anxiolytic-like effects of the ethanolic extract, fractions and Hibalactone isolated from Hydrocotyle umbellata L. (Acariçoba) – Araliaceae

Hydrocotyle umbellata Linn. (Araliaceae) is specie used in the treatment of inflammatory diseases. Crude extract (E-HU) was prepared from H. umbellata subterraneous parts and fractionated by liquid-liquid partition, resulting hexane fraction (HF-HU), dichloromethane fraction (DF-HU), ethyl acetate fraction (EAF-HU) and aqueous fraction (AF-HU). The hibalactone (HU-1) was isolated from the DF-HU and its structure was elucidated by 1H NMR and 13C NMR Spectroscopy, mass spectrometry and crystallographic x-ray analysis. The formalin-induced nociception was used to evaluate antinociceptive activity; carrageenan-induced edema and pleurisy tests to evaluate anti-inflammatory activity and light-dark box to evaluate anxiolytic-like activity in mice. The acute oral treatments with E-HU (1000mg/kg), DF-HU (150mg/kg), EAF-HU (400mg/kg) and HU-1 (33mg/kg) decreased the licking time in both phases of the formalin test. In the carrageenan-induced inflammation models, the treatment with the same doses of E-HU, DF-HU, EAF-HU and HU-1 reduced the paw edema formation and leukocytes account into pleural cavity. In silico findings suggest that hibalactone present anti-inflammatory activity by interacting with the enzymes 5-lipoxygenase and cyclooxygenase-2. In the light dark box, the treatments with DF-HU, EAF-HU and HU-1 revealed an anxiolytic like effect. Thus, the E-HU and fractions of H. umbellata showed antinociceptive, anti-inflammatory and anxiolytic like activities, as also hibalactone, a possible phytoconstituent responsible for the biological effects of this specie.

Isotope fractionation in phase-transfer processes under thermodynamic and kinetic control – Implications for diffusive fractionation in aqueous solution

Diffusive isotope fractionation of organic compounds in aqueous solution was investigated by means of liquid-liquid and liquid-gas partitioning experiments under kinetic control. The two-film model was used to describe phase-transfer kinetics. It assumes the diffusion of solutes across a stagnant water boundary layer as the rate-controlling step. For all investigated solutes (benzene-D0 and -D6, toluene-D0, -D5, and -D8, cyclohexane-D0 and -D12), there was no significant observable fractionation effect between nondeuterated and perdeuterated isotopologues, resulting in a ratio of diffusion coefficients Dlight: Dheavy=1.00±0.01. In addition, isotope fractionation due to equilibrium partitioning of solutes between water and n-octane or gas phase was measured. The deuterated compounds are more hydrophilic than their light isotopologues in all cases, giving rise to fractionation coefficients αHpart=Koctane/water,H: Koctane/water,D=1.085 to 1.15. Thus, thermodynamic fractionation effects are much larger than diffusion fractionation effects. Methodical and environmental implications of these findings are discussed.

Antibacterial and Antioxidant Activity of Green Algae Halimeda gracilis from Seribu Island District

Seaweeds have ecological functions as primary producers in marine waters. It also has an important economic value as a producer of hydrocolloids (alginate, agar and carrageenan) that is used in various industries of food and pharmaceuticals. This study aimed to determine the antibacterial and antioxidant activity of green algae Halimeda gracilis. The study was conducted in several stages, sample collection and preparation, extraction of bioactive compound, fractionation, antibacterial and antioxidant test, and phytochemical. Extraction was done by maceration method using methanol and concentrated by rotary evaporator. The methanol extracts of H. gracilis were tested against Staphylococcus aureus and Escherichia <br />coli. Methanol extract of H. gracilis formed inhibition zone against the test bacteria with diameter of inhibition zone was 10 mm and 6 mm, respectively. After liquid-liquid partition (water: ethyl acetate), inhibition zone was only seen in the ethyl acetate fraction of H. gracilis with diameter of inhibition zone was 6 mm and 7.50±1.71 mm, respectively. Antioxidant test methanol extracts and ethyl acetate fractions of H. gracilis each show IC50 value of 290.49 ppm and 375.50 ppm. Phytochemical test showed methanol extract of H. gracilis contains phenols and steroids.<br /><br />

Speciation of trace mercury impurities in fish oil supplements

Fish oil supplement is becoming increasingly popular worldwide because of beneficial long-chain omega-3 polyunsaturated fatty acids. However, mercury (Hg) impurity causes considerable concern because of its toxicity and bioaccumulation in the food chain. In this work, Hg impurities were extracted from fish oil by liquid-liquid partitioning. The sample solution was then mixed with a reductant (0.4% anthranilic acid-20% formic acid) and sequentially exposed to 311 and 254 nm UV radiation. The resulting Hg0 vapor was detected by atomic fluorescence spectrometry. Speciation was fulfilled by solving a set of two linear equations. Recovery of MeHg+ was 73%; total Hg was validated by ICP-MS. This method achieved 0.50 and 0.63 ng mL−1 limits of detection for Hg++ and MeHg+, respectively. Average Hg++ and MeHg+ contents in fish oil samples (n = 38), 0.67 ± 0.45 and 1.1 ± 1.3 ng mL−1, respectively, were 2-3 orders of magnitude lower than those in fish.

ANTIOXIDANT AND ANTICANCER PROPERTIES OF SOLVENT PARTITIONED EXTRACTS OF Phyla nodiflora L.

We previously found that the crude extracts of Phyla nodiflora L. are capable to inhibit cancer cells. In this study, we reassessed their anticancer properties after solvent partition extraction of the methanolic extracts. We also determined their total phenolic content (TPC) and total flavonoid content (TFC). Water, ethyl acetate and methanolic extracts showed higher TPC and TFC than chloroform or hexane extracts. Most of stem extracts exhibited higher antioxidant activity than the leaf extracts except for leaf methanolic extracts. Six of the extracts showed 50% growth inhibition at the concentration of 36-80 µg/ml. Both stem and leaf ethyl acetate extracts showed profound inhibition. They also caused DNA fragmentation in MCF-7 cells, indicating the occurrence of apoptosis. Our findings demonstrated that liquid-liquid partition has successfully separated methanolic extracts into several fractions with antioxidant and anticancer properties. This will facilitate the future investigation in identifying bioactive compounds for chemotherapeutic intervention.

液・液二相分配クロマトグラフィーの有機化学実習への応用 IV : カラム内ニトロ化反応を用いるニトロフェノールの合成法

液・液二相分配クロマトグラフィーの有機化学実習への応用 IV : カラム内ニトロ化反応を用いるニトロフェノールの合成法

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

BackgroundMajor drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds.MethodsFruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS).ResultsThe most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite.ConclusionsThe selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Lysergic acid amide as chemical marker for the total ergot alkaloids in rye flour – Determination by high-performance thin-layer chromatography–fluorescence detection

Ergot alkaloids are generally determined by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection, analyzing the individual compounds. However, fast and easy screening methods for the determination of the total ergot alkaloid content are more suitable, since for monitoring only the sum of the alkaloids is relevant. The herein presented screening uses lysergic acid amide (LSA) as chemical marker, formed from ergopeptine alkaloids, and ergometrine for the determination of the total ergot alkaloids in rye with high-performance thin-layer chromatography–fluorescence detection (HPTLC–FLD). An ammonium acetate buffered extraction step was followed by liquid-liquid partition for clean-up before the ergopeptine alkaloids were selectively transformed to LSA and analyzed by HPTLC–FLD on silica gel with isopropyl acetate/methanol/water/25% ammonium hydroxide solution (80:10:3.8:1.1, v/v/v/v) as the mobile phase. The enhanced native fluorescence of LSA and unaffected ergometrine was used for quantitation without any interfering matrix. Limits of detection and quantitation were 8 and 26μg LSA/kg rye, which enables the determination of the total ergot alkaloids far below the applied quality criterion limit for rye. Close to 100% recoveries for different rye flours at relevant spiking levels were obtained. Thus, reliable results were guaranteed, and the fast and efficient screening for the total ergot alkaloids in rye offers a rapid alternative to the HPLC analysis of the individual compounds.

In vitro schistosomicidal activity of the crude extract, fractions and Primin, the major active benzoquinone constituent from the leaves of Miconia willdenowii (Melastomaceae)

Eight native plant extracts from the Brazilian Atlantic Forest, Acacia polyphylla, Allophylus racemosus, Casearia arborea, Duguetia lanceolata, Metrodorea stipularis, Trichilia emarginata, Miconia willdenowii, Mollinedia widgrenii, were screened for potential schistosomicidal activity. The crude ethanolic extract of Miconia willdenowii (at a concentration of 200μg/mL) showed the most promising results, killing approximately 65% of the Schistossoma mansoni worms, in comparison to praziquantel. This extract was then submitted to a bioguided-phytochemical study. After liquid-liquid partition was performed, the hexane subfraction disclosed the most significant schistosomicidal activity of 80% against the adult worms at a concentration of 75μg/mL and 25% at 50μg/mL. Purification of this active subfraction led to the isolation of 2-methoxy-6-pentyl-benzoquinone (1, also known as Primin) as the active metabolite responsible for the observed schistosomicidal effect (IC50=7.08μg/mL). The schistosomicidal effect of M. willdenowii and Primin is the first time in which such a result has been reported.

Antiplasmodial Sesquiterpenoid Lactones from Trichospira verticillata: Structure Elucidation by Spectroscopic Methods and Comparison of Experimental and Calculated ECD Data.

A dichloromethane extract of Trichospira verticillata from the Natural Products Discovery Institute was discovered to have good antiplasmodial activity (IC50 ∼5 μg/mL). After purification by liquid-liquid partition and C18 reversed-phase HPLC, four new germacranolide-type sesquiterpenoid lactones named trichospirolides A-D (1-4) were isolated. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and MS data. The relative and absolute configurations were assigned based on a comparison of calculated and experimental ECD and UV spectra, specific rotations, internuclear distances, and coupling constants for all possible diastereomers for each compound. Among these four compounds, the conjugated dienone 1 displayed the most potent antiplasmodial activity, with an IC50 value of 1.5 μM.

Accelerated solvent extraction method for the quantification of polycyclic aromatic hydrocarbons in cocoa beans by gas chromatography–mass spectrometry

An accelerated solvent extraction (ASE) procedure for use with gas chromatography-mass spectrometry (GC-MS) was optimized for the determination of eight polycyclic aromatic hydrocarbons (PAHs) in cocoa beans. Plackett-Burman and rotatable central composite design (RCCD) indicated that three variables affected the recoveries of PAHs during the extraction and purification steps: agitation time in the second liquid-liquid partition, weight of silica gel in the column, and volume of hexane for PAH elution from the column. After obtaining the optimal conditions, a single laboratory method validation was performed. Linearity was demonstrated for benzo[a]pyrene in the concentration range from 0.5 to 8.0mgkg-1 of sample, corresponding to 1.25-20.0μgkg-1 of cocoa on a fat basis. For the other analytes, linearity was observed from 0.75 to 8.0μgkg-1 of sample (1.88-20.0μgkg-1 of cocoa on a fat basis). Significant matrix effects were found for chrysene and benzo[b]fluoranthene. The precision of the method was verified with relative standard deviations (RSDs) ranging from 2.57 to 14.13% and from 4.36 to 19.77% under repeatability and intermediate precision conditions, respectively. The average recoveries of the eight PAHs ranged from 74.99 to 109.73%. These parameters, limits and measurement uncertainties met the performance criteria established by European Union regulations, except for the theoretical limit of detection for chrysene. The method was applied to the analysis of samples of Brazilian cocoa beans, and only one sample was found to have a PAH content above the maximum limit defined by the European Union legislation. This optimized and validated method is intended to be used as part of the official Brazilian monitoring programs investigating contaminants and residues in food.

Centrifugal partition chromatography - A review of recent applications and some classic references.

Countercurrent chromatography, based on liquid-liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid-liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one-axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two-phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual-mode or elution-extrusion procedure, pH-zone-refining centrifugal partition chromatography, ion-exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).

K-targeted strategy for isolation of phenolic alkaloids of Nelumbo nucifera Gaertn by counter-current chromatography using lysine as a pH regulator

Counter-current chromatography (CCC) is an efficient liquid-liquid partition chromatography technique without support matrix. Despite there are many significant advancements in the CCC separation of natural products especially for non-ionic neutral compounds, CCC isolation of ionic compounds including alkaloids is still a challenging process guide by classical partition coefficients (K) or distribution ratio (KC) because their partition coefficient could not be equal to distribution ratio in common ionic conditions. Here, taking the extract of embryo of the seed of Nelumbo nucifera Gaertn as sample, we introduced a modified K-targeted strategy for isolation of phenolic alkaloids by use of lysine as a pH regulator. The results indicated that if the mass of basic regulators such as aqueous ammonia and lysine added into the solvent system were high enough to inhibit the ionization of the targeted alkaloids, the distribution ratio of targets with ionic and non-ionic molecular forms got stable and might not been changed as the concentration of the pH regulator. In this case, the distribution ratio of target was almost equal to the partition coefficient. Thus, the targets could be isolated by K-targeted CCC separation through adding a certain amount pH regulators into the solvent system. Further experiments also showed that the sample concentration was an important factor on the distribution ratio of targets. Meanwhile, CCC experiments indicated that lysine was more suitable than aqueous ammonia for the separation of phenolic alkaloids because the chemical property of lysine-target complex in the CCC fractions was more stable. Therefore, the preparative CCC separation was performed using 20mM lysine as a pH regulator with more than 800mg injection mass. After simple back-extraction with dichloromethane, the lysine in the CCC fraction was removed completely and pure isoliensinine and neferine were obtained. In summary, the whole results indicated that the modified K-targeted CCC strategy using lysine as the pH regulator was efficient for isolation of phenolic alkaloids from crude plant extracts. It not only provided a practical strategy for the isolation of neferine and its analogues, but also introduced a powerful method to resolve the peak skewing (leading or tailing) in CCC separation of ionic compounds.

Analytical Method for Nitroimidazoles and Their Major Metabolites in Livestock and Fishery Products Using LC-MS/MS.

A sensitive and reliable method for the simultaneous determination of four nitroimidazoles (ipronidazole (IPZ), dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ)) and three metabolites (IPZ-OH, MNZ-OH and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI)) in livestock and fishery products was developed. The analytes were extracted from samples with acetone containing acetic acid. The crude extracts were defatted by liquid-liquid partition using acetonitrile and n-hexane followed by solid-phase extraction using a cartridge column packed with divinylbenzene-N-vinylpyrolidone copolymer bearing sulfo groups. The analytes in the eluate from the cartridge column were extracted with ethyl acetate after addition of ammonium sulfate. The solvent was removed from the extract, and the residue was dissolved in 0.1 vol% formic acid. The HPLC separation was performed on a C18 column with a gradient formed from water containing 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For detection of the analytes, tandem mass spectrometry with positive ion electrospray ionization was used. The recovery tests were performed on 10 livestock and fishery products. The truenesse ranged from 74.6 to 111.1%, with repeatability of 0.5-8.3 RSD% for the entire procedure. The limit of quantification was 0.0001 mg/kg for IPZ, IPZ-OH, MNZ and MNZ-OH, and 0.0002 mg/mg for DMZ, RNZ and HMMNI.