Liquid-liquid Partition Chromatography Research Articles

An Unprecedented 4,8-Cycloeudesmane, Further New Sesquiterpenoids, a Triterpene, Steroids, and a Lignan from the Resin of Commiphora myrrha and Their Anti-Inflammatory Activity In Vitro.

Myrrh has a long tradition in the treatment of inflammatory diseases. However, many of its (active) constituents are still unknown. In the present study, secondary metabolites were isolated from an ethanolic extract by various separation methods (liquid-liquid partition, silica and RP18 flash chromatography, CPC, and preparative HPLC), their structures were elucidated with NMR spectroscopy and mass spectrometry, and the selected compounds were tested for their effect on LPS-induced NO production by RAW 264.7 murine macrophages. Among the isolated substances are 17 sesquiterpenes (1-17) including the first 4,8-cycloeudesmane (1), a triterpene (38), two phytosterols (39, 40) and one lignan (43), which were previously unknown as natural products. Numerous compounds are described for the first time for the genus Commiphora. Eight of the eleven compounds tested (1, 29, 31, 32, 34-37) showed a statistically significant, concentration-dependent weak to moderate anti-inflammatory effect on NO production in the LPS-stimulated RAW 264.7 macrophages in vitro. For the reference substance, furanoeudesma-1,3-diene, an IC50 of 46.0 µM was determined. These sesquiterpenes might therefore be part of the multi-target molecular principles behind the efficacy of myrrh in inflammatory diseases.

Determination of experimental solute descriptor values for safrole by liquid-liquid partitioning and gas chromatography

ABSTRACT Abraham’s solvation parameter model was employed as a Quantitative-Structure Property Relationship in the present study to define safrole descriptor values. Safrole has applications in a wide range of fields, including pharmaceuticals and fragrances, as a flavouring ingredient, a fragrant agent, and a raw material. Safrole, on the other hand, can behave as a toxic organic substance. Safrole descriptor values were calculated using gas-liquid chromatographic retention factors in combination with liquid-liquid partition coefficients. Experimentally determined descriptor values for safrole using gas chromatography and liquid-liquid partition systems are E = 1.108, S = 1.123, A = 0.000, L = 5.340, B = 0.174, and V = 1.246. Proposed descriptor values will be useful in determining and estimating physicochemical properties of Safrole.

Evaluation of uranium and plutonium isotopes in marine samples from Veracruz coastline (Mexico)

Activity ratios (A.R.) of 234U/238U and activity concentration of 238+234U and 239+240Pu were measured in collected seawaters and sand beach samples from various locations along of littoral of Mexican state of Veracruz. Uranium and plutonium were separated and concentrated in a liquid-liquid partition chromatography, afterwards, detected and analyzed by means of alpha spectrometric technique. The 234U/238U activity ratio (AR) ranges from 0.72 to 1.11 in sand beach and from 0.77 to 1.22 in seawater. The activity concentration was found in sea water from 0.31 to 1.94 Bq/L for 234+238U and from 15 to 137 μBq/L for 239+240Pu, in sand beach samples was found to be from 0.64 to 3.86 Bq/kg for 234+238U and from 33 to 249 μBq/kg for 239+240Pu

In vitro Cytotoxic Potential of Alkaloid and Flavonoid Rich Fractions of Alseodaphne semecarpifolia Against MCF-7 Cells

The stem bark and leaves of A. semecarpifolia (Lauraceae) have been employed by traditional healers in Western Ghats region of Karnataka, India to treat human breast cancer. The present study was initiated to explore the cytotoxic properties of A. semecarpifolia. The secondary metabolites were extracted from stem bark and leaves. The stem bark methanol extract (SBME) and leaf methanol extract (LME) were subjected to liquid-liquid partition chromatography, followed by evaluating the presence of phytochemical constituents in liquid fractions and their cytotoxic potential against MCF-7 and L6 cells by MTT assay. The qualitative phytochemical screening of theliquid fractions revealed the presence of different secondary metabolites. The quantitative analysis revealed that the liquid fractions were rich in alkaloids, flavonoids and phenolic compounds. Stem bark methanol fraction (SBMF) and leaf methanol fraction(LMF) showed potential cytotoxicity on MCF-7 cells with an IC50 of 47.11±3.53µg/ml and 48.62±2.40µg/ml respectively. Whereas, stem bark chloroform fraction(SBCF) and leaf chloroform fraction(LCF) showed moderate activity on MCF-7 cells. Vinblastine sulphate was used as a reference standard and it showed potent cytotoxic activity against MCF-7 cells with an IC50 of 24.03±2.12µg/ml. Even though Vinblastine is a potentchemotherapeutic drug it affected the viability of normal cells. In comparison with Vinblastine, the liquid fractions showed very less toxicity on normal cells. Hence, the present study suggested that A. semecarpifolia stem bark and leaves are the potent cytotoxic agents against MCF-7 cells.

Recent progress in separation prediction of counter‐current chromatography

As a liquid-liquid partition chromatography, counter-current chromatography has advantages in large sample loading capacity without irreversible adsorption, which has been widely applied in separation and purification fields. The main factors, including partition coefficient, two-phase solvent systems, apparatus, and operating parameters greatly affect the separation process of counter-current chromatography. To promote the applications of counter-current chromatography, it is essential to develop theoretical research to master the principles of counter-current chromatographic separations so as to achieve predictions before laborious trials. In this article, recent progress about separation prediction methods are reviewed from a point of the steady and unsteady state of the mass transfer process of counter-current chromatography and its mass transfer characteristics, and then it is divided into three aspects: prediction of partition coefficient, modeling the thermodynamic process of counter-current chromatography, and modeling the dynamic process of counter-current chromatography.

A rapid and practical prediction method for the Arizona solvent system family used in high speed countercurrent chromatography.

Selecting the appropriate solvent system is the key to the successful separation of samples by using countercurrent chromatography. Although high-speed countercurrent chromatography has been widely used in the separation and preparation of natural products, the selection of a solvent system has always been a stumbling block to the application of high-speed countercurrent chromatography. In order to explore a rapid and practical prediction method to select countercurrent chromatography solvent system, five linear prediction models of the Arizona solvent system family (HEMW) was established by using fourteen compounds with different structures and five HPLC columns of different brands. And two different solvent system selection methods (The partition coefficient K of the target compound in the solvent system was in the range of 0.25<K<2.5) were proposed for targeted separation of compounds and multi-component separation in a complex sample respectively. The appropriate HSCCC solvent system of five known compounds was determined by a HPLC analysis and a shake flask test and the appropriate HSCCC solvent system of two Chinese herbal extracts was determined by a HPLC analysis to verify the prediction method. In this study, solid-liquid partition chromatography (HPLC) and liquid-liquid partition chromatography (HSCCC) were linked by polarity to simplify the screening process of solvent system. This method reduced the difficulty and workload of solvent system selection, which provided methods and ideas for more solvent system prediction models.

Effect of Cyanobacteria (Nostoc species) Extracts on Osteogenesis Activities

This study focused on osteogenesis activities from Cyanobacteria (Nostoc species), including cytotoxicity, cell proliferation, cell differentiation and cell mineralization with osteoblast cell (MC3T3-E1). The four Nostoc species; N. parmelioides, N punctiforme, N. muscorum and N. paludosum were made axenic and cultured for a month. The ethanolic crude extracts were obtained by maceration extraction; the yield was in the range of 7.00 - 10.00 % (w/w). By using the TLC technique, it was observed that in all of the samples, there were phytochemical bands corresponding with β-carotene and chlorophyll a which were used as standard compounds. As a result, all crude extracts had no toxic effect on osteoblast cells after being incubated for 24 and 48 h with various concentrations (1 - 1,000 µg/mL), although they did not significantly promote osteoblast proliferation. In addition, it was found that 3 crude extracts (N. parmelioides, N punctiforme, and N. paludosum) also promoted osteoblast differentiation significantly after being induced for 9 days. Similarly, there was no difference between the relative cellular mineralization among treatment groups after being induced for 25 days. The potential ethanolic extracts on osteoblast cell differentiation were further partitioned by liquid-liquid partition chromatography (hexane, dichloromethane and aqueous fractions), and were analyzed for osteoblast differentiation with alkaline phosphatase (ALP) activity. All of hexane and dichloromethane fractions from N. parmelioides (181.72±6.75 % and 195.11±3.27 %), N punctiforme (194.34±2.36 % and 168.69±1.08 %), and N. paludosum (191.54±7.66 % and 167.12±9.46 %) had a significantly higher effect of ALP activity than their aqueous fractions. In conclusion, our data suggested that the nonpolar potential major compound, which might correspond with carotenoids (β-carotene, xanthophyll and chlorophyll a) from Nostoc species, exhibited positive effects on osteoblast differentiation by increasing the alkaline phosphatase enzyme.

The combination of partition, size exclusion, and hydrodynamic models in chromatography, and application to bonded phases on porous supports

Liquid-liquid partition chromatography has been used for many years as a model and teaching introduction to column chromatography. However, the partition model does not describe separations on bonded phases with porous supports particularly well, especially regarding the thermodynamics controlling solute distribution. Further difficulties arise when more than one mechanism is involved in solute retention. Nomenclature is not perfectly aligned with the underlying thermodynamic descriptors and is inconsistently applied over various chromatographic techniques. Presented here is a general description of retention that spans partition, size exclusion, and hydrodynamic separation processes, and is then applied to bonded-phase separations on porous supports. The model provides a general description applicable to adsorption, reversed-phase, hydrophilic interaction, size-exclusion, hydrodynamic chromatography, and any combination of these techniques including liquid chromatography at the critical condition. Further expansion to include retention by ion-exchange and field-flow fractionation appears to be possible. Recommendations on retention factor definition and evaluation are given.

Hydrophobic and hydrophilic interactions in countercurrent chromatography

Countercurrent chromatography (or counter-current chromatography, CCC) is a unique support-free liquid-liquid partition chromatography. Since it was invented by Y. Ito in 1960s, CCC has been widely accepted and applied as popular separation and purification technique for natural and synthetic complex. However, up to date there is little study to address on hydrophobic and hydrophilic interactions in CCC process, although hydrophobic interaction chromatography (HIC) and hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) as solid-support chromatographic techniques are widely applied at different stages of downstream processing. In fact, hydrophobic and hydrophilic interactions might be more popular in CCC separation than that in the liquid chromatography. For example, adding small solvents or additives in two-phase solvent systems may change significantly hydrophobic or hydrophilic interactions between solvents and solutes. Normally, CCC separation employs a light and hydrophobic organic phase as the stationary phase, and a heavy and hydrophilic aqueous phase as the mobile phase. Hydrophobic interactions between the solvent system and solutes (targets) will increase the partition coefficients (K values) of solutes and lengthen retention time, while hydrophilic interactions will reduce the K values and separation time. In this work, we aim to provide a general insight on the hydrophobic and hydrophilic interactions in CCC separation. We also highlight the current advances in utilizing the hydrophobic and hydrophilic interactions for K-targeted CCC separation and purification.

Methanol linear gradient counter-current chromatography for the separation of natural products: Sinopodophyllum hexandrum as samples

Counter-current chromatography (CCC) is a unique, liquid-liquid partition chromatography process. Both the mobile and stationary phases are liquids, so no solid support matrix is used. CCC has gained wide acceptance as a preparative technique in a variety of fields. Because the mobile and stationary phases are both liquids, gradient elution is difficult to perform with CCC. Phase equilibrium must be maintained, so any change in the composition of one phase may induce a compositional change in the other. In this work, a new linear gradient elution method was developed for CCC. Biphasic solvent systems containing heptane, ethyl acetate, methanol, and water (HepEMWat) in various ratios were prepared and used to optimize both isocratic and linear gradient CCC separation with methanol. We first separated a test mixture of four standard compounds with partition coefficients ranging from 0.8 to 7.8. The separation resembled a reversed-phase process, and elution was performed while progressively decreasing the polarity of the mobile phase. Target molecules with small partition coefficients eluted first in the lower phase of the optimized HepEMWat solvent system. Elution of constituents with large partition coefficients was quite slow under isocratic conditions. Separation time was significantly reduced when elution was performed with a linear gradient using methanol and the optimal HepEMWat system. Elution with a 3:7:4:6 (v/v/v/v) HepEMWat system took approximately 200 min. This included an 80-min isocratic step, followed by gradient elution with methanol from 0% to 30%. The optimized methanol linear gradient CCC method was then used to separate a complex mixture of natural products isolated from Sinopodophyllum hexandrum (Royle) Ying roots. Twelve compounds with a wide range of polarities were well-resolved in a single separation. We have developed a convenient and cost-effective strategy for the separation of complex mixtures. No tedious mobile phase preparation step is required. The volume of unused mobile phase is minimal, so little solvent is wasted. The method is an important advance for the separation of mixtures that contain many compounds with a large range of polarities and partition coefficients, which are common features of natural products.

Distribution of Saponins in the Sea Cucumber Holothuria lessoni; the Body Wall Versus the Viscera, and Their Biological Activities.

Sea cucumbers are an important ingredient of traditional folk medicine in many Asian countries, which are well-known for their medicinal, nutraceutical, and food values due to producing an impressive range of distinctive natural bioactive compounds. Triterpene glycosides are the most abundant and prime secondary metabolites reported in this species. They possess numerous biological activities ranging from anti-tumour, wound healing, hypolipidemia, pain relieving, the improvement of nonalcoholic fatty livers, anti-hyperuricemia, the induction of bone marrow hematopoiesis, anti-hypertension, and cosmetics and anti-ageing properties. This study was designed to purify and elucidate the structure of saponin contents of the body wall of sea cucumber Holothuria lessoni and to compare the distribution of saponins of the body wall with that of the viscera. The body wall was extracted with 70% ethanol, and purified by a liquid-liquid partition chromatography, followed by isobutanol extraction. A high-performance centrifugal partition chromatography (HPCPC) was conducted on the saponin-enriched mixture to obtain saponins with a high purity. The resultant purified saponins were analyzed using MALDI-MS/MS and ESI-MS/MS. The integrated and hyphenated MS and HPCPC analyses revealed the presence of 89 saponin congeners, including 35 new and 54 known saponins, in the body wall in which the majority of glycosides are of the holostane type. As a result, and in conjunction with existing literature, the structure of four novel acetylated saponins, namely lessoniosides H, I, J, and K were characterized. The identified triterpene glycosides showed potent antifungal activities against tested fungi, but had no antibacterial effects on the bacterium Staphylococcus aureus. The presence of a wide range of saponins with potential applications is promising for cosmeceutical, medicinal, and pharmaceutical products to improve human health.

Preparative separation of flavone dimers from Dysosma versipellis by counter-current chromatography: Trifluoroacetic acid as a solvent system modifier.

The separation of natural products is grueling and time-consuming work with repeated isolations needed to obtain purified compounds. However, using counter-current chromatography, a unique liquid-liquid partition chromatography, constituents can usually be purified efficiently. During the separation of flavone dimers from Dysosma versipellis (Hance) by counter-current chromatography, the separation resolution and sample loading was impeded by the emulsification of the sample. By screening, trifluoroacetic acid was selected as the solvent modifier to eliminate the emulsification. Then, a quaternary solvent system of hexane/ethyl acetate/methanol/water (4:6:5:5 v/v/v/v) with trifluoroacetic acid at a low concentration of 0.5% v/v was used to purify the components from D. versipellis. Compared to that without trifluoroacetic acid, the separation resolution as well as the sample loading both increased greatly. In addition, flavone dimers in low concentrations could be enriched and purified at high sample loading. As a result, five podophyllotoxins and 11 flavonoids were purified and characterized by interpretation of spectroscopic data, in which two of eight flavone dimers were new and a known flavone dimer was first separated from this species.

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

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

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.

Room temperature ionic liquids-based salting-in strategy for counter-current chromatography in the separation of arctiin

Counter-current chromatography (CCC) is a solid support-free liquid-liquid partition chromatography and has wide applications. However, CCC separation is still a challenging process and the selection of appropriate solvent system for separation of target compound(s) is still relatively time-consuming. In this work, we introduced a room temperature ionic liquids-based salting-in strategy for the rapid selection of suitable solvent systems for CCC separation. In the randomly selected solvent systems, such as ethyl acetate-water, n-butanol-water, n-pentanol-water, n-hexanol-water, and n-octanol-water, several ionic liquids such as [AMIM]Cl, [MAMIM]Cl, and [BMIM]Cl can increase the solubility of the solutes in the lower phase, which made a dose-dependent decreasing of partition coefficient of solute in the two-phase solvent system. Thus, it is possible to get a suitable solvent system with sweet K spot such as K=1 only by adding some ionic liquids into the systems. As an example, arctiin, a bioactive lignin component of the fruit of Arctium lappa. L. (Niubangzi in Chinese), was selected and successfully separated by CCC with room temperature ionic liquids-based n-butanol-water systems. It seems a very efficient alternative strategy for the optimization of solvent systems for CCC separation of natural products.

Advances in Technology of Countercurrent Chromatography for Separation of Protein and Peptide

Abstract Countercurrent chromatography is a kind of continuous and effective liquid-liquid partition chromatography with many unique characteristics such as large load capacity, no irreversible adsorption, high recovery rate, low risk of sample denaturation and so on, which has irreplaceable advantages in the separation of proteins and peptides. This review presents the advances of several kinds of new technology of countercurrent chromatography in the separation of proteins and peptides. The development prospect of this field is also discussed.

Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns.

Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of partition efficiency and shortening theseparation time. The locular multilayer coils modified with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl (MU) sugar derivatives using the small-scale cross-axis coil planet centrifuge. Proteins were well separated from each other and the separation was improved at a low flow rate of the mobile phase. On the other hand, 4-MU sugar derivatives were sufficiently resolved with short separation time at a highflow rate of the mobile phase under satisfactory stationary phase retention. Effective separations were achieved using the locular multilayer coil for proteins with aqueous-aqueous polymer phase systems and for 4-MU sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule.