Liquid Partition Chromatography Research Articles

Determination of experimental solute descriptor values for methyl 3-nitrobenzoate by liquid–liquid partitioning and gas chromatography

ABSTRACT In this study, methyl 3-nitrobenzoate, a popular compound used to protect crops and in the textile industry, was studied using liquid–liquid partitioning and gas chromatography and the Abraham solvation parameter descriptor values were determined for potential applications in QSPR. The solver algorithm in Excel was used to determine the descriptor values for methyl 3-nitrobenzoate, resulting in E = 1.0616, S = 1.4170, A = 0.00, L = 6.1354, B = 0.6198, and V = 1.2468 with a standard deviation of 0.076. In the situations when toxicity and exposure assessment data are scarce, the application of QSPR models, such as the Abraham solvation parameter model, can aid in the estimation of the environmental impact of chemicals. With the objective to facilitate physicochemical property estimations of methyl 3-nitrobenzoate widely used in the textile and agricultural industries, this study offers experimental solute descriptor values of the Abrham’s solvation parameter model.

Continuous Separation and Purification of Chemical Species by Continuous Liquid–Liquid Partition Chromatography Implemented with Membrane-Based Phase Separators

Continuous Separation and Purification of Chemical Species by Continuous Liquid–Liquid Partition Chromatography Implemented with Membrane-Based Phase Separators

Easy Computation of the Various Topologies and Modes of Liquid–Liquid Partition Chromatography by the Theory of Random Walks

The article revisits the discrete recurrence method to model the instruments of liquid–liquid partition chromatography as counter-current chromatography (CCC) and centrifugal partition chromatography (CPC). The purpose is to simplify the computation of the concentration profiles without supplementary approximations, rather by going back to the seminal model of binomial random walks, associated with the stochastic master equation that generates simple discrete recurrence relations. It fits the model of the prototype of liquid–liquid chromatography: the Craig’s apparatus. Three emblematic separation technique group cases are computed in batch injection, batch multiple dual mode (MDM), and continuous injection by the “True Moving Bed” (TMB) in CPC.

H2O-Induced Hydrophobic Interactions in MS-Guided Counter-Current Chromatography Separation of Anti-Cancer Mollugin from Rubia cordifolia.

Counter-current chromatography (CCC) is a unique liquid–liquid partition chromatography and largely relies on the partition interactions of solutes and solvents in two-phase solvents. Usually, the two-phase solvents used in CCC include a lipophilic organic phase and a hydrophilic aqueous phase. Although a large number of partition interactions have been found and used in the CCC separations, there are few studies that address the role of water on solvents and solutes in the two-phase partition. In this study, we presented a new insight that H2O (water) might be an efficient and sensible hydrophobic agent in the n-hexane-methanol-based two-phase partition and CCC separation of lipophilic compounds, i.e., anti-cancer component mollugin from Rubia cordifolia. Although the n-hexane-methanol-based four components solvent systems of n-hexane-ethyl acetate-methanol-water (HEMWat) is one of the most popular CCC solvent systems and widely used for natural products isolation, this is an interesting trial to investigate the water roles in the two-phase solutions. In addition, as an example, the bioactive component mollugin was targeted, separated, and purified by MS-guided CCC with hexane-methanol and minor water as a hydrophobic agent. It might be useful for isolation and purification of lipophilic mollugin and other bioactive compounds complex natural products and traditional Chinese medicines.

Advances in Separation and Purification of Bioactive Polysaccharides through High-speed Counter-Current Chromatography.

Polysaccharides, with an extensive distribution in natural products, represent a group of natural bioactive substances having widespread applications in health-care food products and as biomaterials. Devising an efficient system for the separation and purification of polysaccharides from natural sources, hence, is of utmost importance in the widespread applicability and feasibility of research for the development of polysaccharide-based products. High-speed counter-current chromatography (HSCCC) is a continuous liquid-liquid partitioning chromatography with the ability to support a high loading amount and crude material treatment. Due to its flexible two-phase solvent system, HSCCC has been successfully used in the separation of many natural products. Based on HSCCC unique advantages over general column chromatography and its enhanced superiority in this regard when coupled to aqueous two-phase system (ATPS), this review summarizes the separation and purification of various bioactive polysaccharides through HSCCC and its coupling to ATPS as an aid in future research in this direction.

Oxidative stress mediated cytotoxicity in leukemia cells induced by active phyto-constituents isolated from traditional herbal drugs of West Bengal.

In search of safe and effective therapeutic agents as alternative to synthetic chemotherapeutics for the treatment of leukemia, the herbal drugs (Leaf of Madhuca longifolia, leaf of Prosopis cineraria and bark of Flacourtia indica) with long traditional use in West Bengal have received our attention. Present work was conducted to isolate and identify the active compounds of the selected herbal drugs using bio-assay guided fractionation and also to investigate their anticancer mechanism in leukemia cell lines. Bio-assay guided fractionation was used for the isolation of active constituents such as myricitrin, vitexin and vanillin from the aqueous extracts of M. longifolia, P. cineraria and F. indica, respectively using liquid partitioning and column chromatography and the compounds were characterized by HPLC, MS and NMR. Dose and time-dependent cytotoxicity of isolated compounds were studied against leukemia cells and their anticancer mechanism such as cell wall damage, nuclear damage, ROS and NO generation, SOD level, LDH release and lipid peroxidation were investigated. Aqueous extract of M. longifolia, P. cineraria and F. indica exhibited maximum anti-proliferative activity against HL-60 (Acute myeloid leukemia, AML, 72.06%), K-562 (Chronic myeloid leukemia, CML, 42.14%) and Jurkat (Acute lymphoblastic leukemia, ALL, 51.71%) cells. Myricitrin, vitexin and vanillin exhibited dose-dependent (IC-50 values 164.4, 147 & 29.22μg/ml) and time-dependent activity with maximum cytotoxicity at 48h. All these three compounds caused apoptosis in leukemia cells by inducing free radicals such as ROS (1.33-2.65 Arbitrary units) and NO (11.17-18.53μM), cell membrane damage and nuclear condensation which were evidenced by increased release of LDH (1326-1439 U/L), improved lipid peroxidation (10.19-14.41nM/mg protein) and reduced SOD level (6.2-9.21 U/mg protein) in leukemia cells. Based on anti-proliferative activity, the isolated phyto-compounds myrcitrin, vitexin and vanillin from M. longifolia, P. cineraria and F. indica could be developed as natural drugs for treating AML, CML and ALL leukemia types, respectively.

Three new trixane glycosides obtained from the leaves of Jungia sellowii Less. using centrifugal partition chromatography.

Jungia sellowii (Asteraceae) is a shrub that grows in Southern Brazil and polar extract of its leaves presents anti-inflammatory properties. Cyperane, guaiane, nortrixane, and trixane sesquiterpene types were reported as the main metabolites in Jungia species. This work aims to describe the isolation and identification of sesquiterpenes in the leaves of J. sellowii using liquid–liquid partition and centrifugal partition chromatography. Thus, the crude extract of fresh leaves of J. sellowii was partitioned with hexane, dichloromethane, ethyl acetate and butanol, respectively. The butanol fraction was then subjected to a selected ternary system optimized for the CPC (centrifugal partition chromatography): ethyl acetate–ethanol–water (9:2:10, v/v/v). The separation was carried out isocratically at a flow rate of 25 mL/min at 1200 rpm, affording seven fractions A to G. TLC of fractions B, C and F displayed a single spot corresponding to three new glycosylated sesquiterpenoids. Their structures were established by using spectroscopic data in comparison to those reported in the literature. Furthermore, the isolates were evaluated for their leishmanicidal and cytotoxic effects. No cytotoxic effect was observed against the three cancer cell lines (HL60, JURKAT and REH), but compound 1 showed a weak antiprotozoal activity. Liquid–liquid partition and CPC turned to be a versatile technique of glycoside purification which is environmentally friendly and requires a limited amount of organic solvents.

Carotenoids and Retinoids: Nomenclature, Chemistry, and Analysis.

Carotenoids are polyenes synthesized in plants and certain microorganisms and are pigments used by plants and animals in various physiological processes. Some of the over 600 known carotenoids are capable of metabolic conversion to the essential nutrient vitamin A (retinol) in higher animals. Vitamin A also gives rise to a number of other metabolites which, along with their analogs, are known as retinoids. To facilitate discussion about these important molecules, a nomenclature is required to identify specific substances. The generally accepted rules for naming these important molecules have been agreed to by various Commissions of the International Union of Pure and Applied Chemistry and International Union of Biochemistry. These naming conventions are explained along with comparisons to more systematic naming rules that apply for these organic chemicals. Identification of the carotenoids and retinoids has been advanced by their chemical syntheses, and here, both classical and modern methods for synthesis of these molecules, as well as their analogs, are described. Because of their importance in biological systems, sensitive methods for the detection and quantification of these compounds from various sources have been essential. Early analyses that relied on liquid adsorption and partition chromatography have given way to high-performance liquid chromatography (HPLC) coupled with various detection methods. The development of HPLC coupled to mass spectrometry, particularly LC/MS-MS with Multiple Reaction Monitoring, has resulted in the greatest sensitivity and specificity in these analyses.

Validation of fatty acid predictions in milk using mid-infrared spectrometry across cattle breeds

The aim of this study was to investigate the accuracy to predict detailed fatty acid (FA) composition of bovine milk by mid-infrared spectrometry, for a cattle population that partly differed in terms of country, breed and methodology used to measure actual FA composition compared with the calibration data set. Calibration equations for predicting FA composition using mid-infrared spectrometry were developed in the European project RobustMilk and based on 1236 milk samples from multiple cattle breeds from Ireland, Scotland and the Walloon Region of Belgium. The validation data set contained 190 milk samples from cows in the Netherlands across four breeds: Dutch Friesian, Meuse-Rhine-Yssel, Groningen White Headed (GWH) and Jersey (JER). The FA measurements were performed using gas–liquid partition chromatography (GC) as the gold standard. Some FAs and groups of FAs were not considered because of differences in definition, as the capillary column of the GC was not the same as used to develop the calibration equations. Differences in performance of the calibration equations between breeds were mainly found by evaluating the standard error of validation and the average prediction error. In general, for the GWH breed the smallest differences were found between predicted and reference GC values and least variation in prediction errors, whereas for JER the largest differences were found between predicted and reference GC values and most variation in prediction errors. For the individual FAs 4:0, 6:0, 8:0, 10:0, 12:0, 14:0 and 16:0 and the groups’ saturated FAs, short-chain FAs and medium-chain FAs, predictions assessed for all breeds together were highly accurate (validation R2 > 0.80) with limited bias. For the individual FAs cis-14:1, cis-16:1 and 18:0, the calibration equations were moderately accurate (R2 in the range of 0.60 to 0.80) and for the individual FA 17:0 predictions were less accurate (R2 < 0.60) with considerable bias. FA concentrations in the validation data set of our study were generally higher than those in the calibration data. This difference in the range of FA concentrations, mainly due to breed differences in our study, can cause lower accuracy. In conclusion, the RobustMilk calibration equations can be used to predict most FAs in milk from the four breeds in the Netherlands with only a minor loss of accuracy.

Ionic liquids as novel stationary phases in gas liquid chromatography: Inverse or normal isotope effect?

The separation of deuterated and non-deuterated compounds in gas liquid partitioning chromatography (GLC) on silicone type stationary phase usually results in the inverse isotope effect. With ionic liquids (ILs) as stationary phase, however, this may show a totally different nature. The inverse isotope effect, in which heavier (deuterated) isotopic compounds (isotopologues) elute earlier, is to be expected when van der Waals (London) dispersion forces play a dominant role in the solute–stationary phase interaction. Such (apolar) interactions seem to play only a minor role when ILs are the stationary phases, leading to only a marginal inverse isotope effect, e.g. for the separation of 2,4,6-trichloroanisole and its [2H5]-isotopologue on 1,12-di(tripropylphosphonium) dodecane bis(trifluoromethansulfonyl) amide (commercialized as SLB-IL59, Supelco). Indeed, with the most polar stationary phase available (commercialized as SLB-IL111; Supelco), this separation showed a normal isotope effect. Further examples are presented and the nature of the isotope effect observed is discussed.

Analysis of Camellia sinensis green and black teas via ultra high performance liquid chromatography assisted by liquid–liquid partition and two-dimensional liquid chromatography (size exclusion × reversed phase)

Green and black teas ( Camellia sinensis) contain compounds ranging from simple phenolics to complex glycosides, many of which have well-recognized health benefits. Here, we describe two methodologies aiming to achieve a comprehensive analysis of hydro-alcoholic extracts of C. sinensis. In the first step, the extracts were partitioned in water, n-butanol, ethyl acetate and chloroform to separate the compounds according to their polarity, yielding less complex samples to be analyzed by ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC–MS). Additionally, a comprehensive two dimensional liquid chromatography (2D-LC) technique, employing size exclusion chromatography (SEC) × reversed phase (BEH-C18) was developed. The following compounds were identified on the basis of retention time, UV-spectra and MS fragmentation patterns: catechins, theaflavins and their gallate derivatives; kaempferol, quercetin and myricetin mono-, di-, tri- and tetraglycosides; esters of quinic acid and gallic or hydroxycinnamic acids; purine alkaloids, such as caffeine and theobromine and many lipids. Additionally, there were many novel compounds that were previously undescribed, such as saponin isomers and gallic acid esters of four glycosides of myricetin, quercetin and kaempferol.

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid–liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane–ethyl acetate–methanol–water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol–potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.

Progress in quantitative analysis of plant hormones

Plant hormones are small molecular natural products that regulate all plant developmental processes at low concentrations. Quantitative analysis of plant hormones is increasingly important for in-depth study of their biosynthesis, transport, metabolism and molecular regulatory mechanisms. Although plant hormone analysis remains a bottleneck in plant scientific research owing to the trace concentrations and complex components in plant crude extracts, much progress has been achieved in the development of extraction, purification and detection techniques in recent years. Solid phase extraction and chromatography/mass spectrometry have been applied widely for purification and quantitative analysis of plant hormones owing to their high selectivity and sensitivity. Purification methods such as liquid partition and immunoaffinity chromatography, and detection methods including immunoassay and electrochemical analysis, are employed. The advantages and disadvantages of these methods are discussed. In situ, real-time and multi-plant hormone profiling will comprise mainstream techniques for quantitative analyses in future studies on the regulatory mechanisms and crosstalk of plant hormones.

Determination of sugars in honey by liquid chromatography

Honey is a rich conventional natural resource of sweetness and energy for human beings. A protocol for the determination of two important monosaccharide sugars (fructose and glucose) in honey was established in the current study by using normal phase partition liquid chromatography and 1–5% combined working standard of glucose, fructose and sucrose.

Potencial alelopático do extrato foliar de Annona glabra L. (Annonaceae)

A. glabra cresce em áreas alagadas, formando aglomerados. Este comportamento pode indicar ocorrência de competição com outras espécies ou que existam processos alelopáticos. Neste estudo, os objetivos foram: a) avaliação do potencial alelopático de extratos foliares de A. glabra sobre a germinação e crescimento de outras espécies, e sobre o crescimento de coleóptilos de trigo e b) verificação da presença de grupos de substâncias nos extratos. Os testes qualitativos detectaram triterpenos, taninos e flavonóides. Foi realizada cromatografia de partição líquido:líquido com hexano e acetato de etila. A atividade das frações obtidas foi testada sobre de Lactuca sativa, Echinochloa crus-galli, Euphorbia heterophylla e Ipomoea grandifolia. A fração acetato de etila reduziu a porcentagem de germinação de L. sativa e atrasou o tempo médio de germinação de E. crus-galli além de afetar o crescimento de todas as espécies. Esta fração foi purificada em coluna de sílica, obtendo-se cinco frações (A, B, C, D e E). Para cada uma delas foram preparadas quatro concentrações (1000; 158; 79; 39,5 ppm). Os efeitos causados pelas frações foram avaliados com o teste de coleóptilo de trigo. A fração A estimulou o alongamento destes nas três menores concentrações, na maior foi inibitória, B inibiu nas três maiores, C inibiu apenas na maior e E não provocou efeito significativo.

Dynamic covalent hydrazine chemistry as a selective extraction and cleanup technique for the quantification of the Fusarium mycotoxin zearalenone in edible oils

A novel, cost-efficient method for the analytical extraction of the Fusarium mycotoxin zearalenone (ZON) from edible oils by dynamic covalent hydrazine chemistry (DCHC) was developed and validated for its application with high performance liquid chromatography-fluorescence detection (HPLC-FLD). ZON is extracted from the edible oil by hydrazone formation on a polymer resin functionalised with hydrazine groups and subsequently released by hydrolysis. Specifity and precision of this approach are superior to liquid partitioning or gel permeation chromatography (GPC). DCHC also extracts zearalanone (ZAN) but not α-/β-zearalenol or -zearalanol. The hydrodynamic properties of ZON, which were estimated using molecular simulation data, indicate that the compound is unaffected by nanofiltration through the resin pores and thus selectively extracted. The method's levels of detection and quantification are 10 and 30 μg/kg, using 0.2 g of sample. Linearity is given in the range of 10–20,000 μg/kg, the average recovery being 89%. Bias and relative standard deviations do not exceed 7%. In a sample survey of 44 commercial edible oils based on various agricultural commodities (maize, olives, nuts, seeds, etc.) ZON was detected in four maize oil samples, the average content in the positive samples being 99 μg/kg. The HPLC-FLD results were confirmed by HPLC–tandem mass spectrometry and compared to those obtained by a liquid partitioning based sample preparation procedure.

Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography

Flavonoids form a large class of important naturally occurring bioactive compounds. Their isolation and purification from natural sources can sometimes be very difficult and time-consuming when traditional phytochemical techniques are used. Countercurrent chromatography (CCC), a support-free liquid-liquid partition chromatography technique, is very useful for the isolation of polar compounds and its use is increasing in the natural products field. In this paper, we propose strategies of solvent system selection for the isolation of flavonoids by CCC, based on data from the literature, plus incorporation of own practical experiences. The selected references report the isolation of over 300 different flavonoid compounds from more than 100 plant species, using 40 different solvent systems, showing the versatility of this technique. The solvent system hexane-ethylacetate-methanol-water is proposed as a starting point for the separation of samples containing free flavonoids, as it was cited in more than 60% of the papers. A "fine tuning" step is proposed at each level of this solvent family. Other modifications include exchanging the alcohol in the system as well as introducing a fifth solvent. The solvent system ethyl-acetate-butanol-water is proposed as the starting point for glycosylated flavonoids. Other solvent systems are also discussed. The use of gradients is proposed for samples containing both free and glycosylated flavonoids, as the polarity window is larger in these cases. High-speed countercurrent chromatography was used in 89% of the reviewed data.

Antioxidant power, bacteriostatic activity, and characterization of white grape pomace extracts by HPLC–ESI–MS

Polyphenolic fractions obtained from white grape pomace by aqueous extraction were analyzed to determine their antioxidant power (DPPH and TBARS methods), bacteriostatic activity, the effect of particle size on total extractable polyphenols (expressed as gallic acid equivalents (GAE)), and to characterize by HPLC–ESI–MS. The extraction of polyphenols from Albarino variety grape pomace was performed both on lab-scale and on pilot-plant scale using particles sizes of <1 mm, 3–5 mm and entire particle. Lab-scale assays showed that the lower the particle size the higher the raw extract (RE) percentage (%w/w), which values were 21.3 ± 0.37% (SD), 12.6 ± 0.38%, and 6.6 ± 0.44%, respectively. Several fractions were obtained from RE, either as result of applying liquid–liquid partition or gel permeation chromatography. The antiradical activities of the fractions were lower than that of gallic acid (17.8 ± 0.1 mg GAE/mol DPPH) but similar to that of catechin or procyanidin B2 (46.0 ± 2.3 mg GAE/mol DPPH and 52.3 ± 6.3 mg GAE/mol DPPH). Several fractions showed bacteriostatic activity both at 50 and at 100 mg/L. Total aerobic bacterial enumeration (TABE, log UFC/cm2) of treated samples revealed a significant lower TABE than that for control. HPLC–ESI–MS studies demonstrated that fractions V and VI included monomers, dimers, and trimers of procyanidins, few of them galloylated. Fraction VII contained several oligomers and galloylated procyanidins.

Synthesis of boronic acid derivatives of tyropeptin: Proteasome inhibitors

Boronic acid derivatives of tyropeptin were synthesized with TP-110 as the lead compound. Due to the lability of the aminoboronic acid moiety, careful design of the deprotection and coupling sequence was required. Liquid–liquid partition chromatography was found to be a powerful tool for purification of compounds of this class. The obtained derivatives showed potent inhibitory activities against the human 20S proteasome in vitro.

Discovery of Incednine as a Potent Modulator of the Anti-apoptotic Function of Bcl-xL from Microbial Origin

Anti-apoptotic oncoproteins Bcl-2 and Bcl-xL are overexpressed in many cancers and play a crucial role in cancer initiation, progression, and resistance to chemotherapy. Therefore, the discovery of a functional inhibitor for these proteins and improved understanding of the molecular mechanisms of these proteins will be an aid to novel anti-tumor therapies. Here, using chemical−genetic cell-based screening, we have discovered a chemically and biologically unique substance, incednine, as a novel functional modulator of Bcl-2/Bcl-xL from the fermentation broth of Streptomyces sp. ML693-90F3. This compound was isolated as a HCl salt by solvent extraction and using centrifugal liquid−liquid partition chromatography. Its structure was elucidated by spectroscopic analysis, X-ray crystallographic analysis, and computational studies. Incednine has a molecular formula, C42H63N3O8, and consists of a novel skeletal structure, enol-ether amide in a 24-membered macrolactam core, with two aminosugars. Bcl-xL-overexpress...