Partition Chromatographic Method Research Articles

Efficient Isolation of Mycosporine-Like Amino Acids from Marine Red Algae by Fast Centrifugal Partition Chromatography.

Marine rhodophyta are known to synthesize specific secondary metabolites, mycosporine-like amino acids (MAAs), to protect themselves from harmful UV-radiation. Shinorine and porphyra-334 are among the most abundant representatives of this compound class. In the present work, a novel approach for their isolation is described. As a first step, a fast centrifugal partition chromatography method, with an aqueous two-phase system comprising water, ethanol, ammonium sulfate and methanol in ascending mode, was developed to isolate the two MAAs from crude aqueous-methanolic extracts of three algal species within 90 min. The compounds could be isolated when just one of them was present in a sample or also both at the same time. By employing solid phase extraction as a second purification step, the individual MAAs were obtained in high purity and good quantity within a much shorter time frame than the established purification protocols, e.g., semi-preparative HPLC. For example, from 4 g Porphyra sp. (Nori) crude extract, 15.7 mg shinorine and 36.2 mg porphyra-334 were isolated. Both were highly pure, as confirmed by TLC, HPLC-MS and NMR analyses.

Melodamide A from Melodorum fruticosum - Quantification using HPLC and one-step-isolation by centrifugal partition chromatography.

Melodamide A, a phenolic amide from the leaves of Melodorum fruticosum Lour., has previously shown pronounced anti‐inflammatory activity. In order to rapidly isolate larger quantities for biological testing, a fast, one‐step isolation method by centrifugal partition chromatography was developed within this study. Fractionation of the dichloromethane extract was performed with a two‐phase solvent system consisting of n‐hexane, ethyl acetate, methanol, and water (3:7:5:5, v/v), leading to the isolation of melodamide A with a purity of >90% and a yield of 6.7 w% within 32 min. The developed method can also be used in dual mode for the enrichment of further constituents like flavonoids or chalcones. In order to support the centrifugal partition chromatography method development, additionally, a high‐performance liquid chromatography method was established and validated to determine quantities of melodamide A in plant material and crude extracts. Analysis of M. fruticosum leaves and a dichloromethane extract obtained from this plant material showed a total melodamide A content of 0.19 ± 0.008 and 8.9 ± 0.249 w%, respectively.

Semi-synthetic preparation, structural elucidation and pharmacological study of nitrogen-containing ecdysteroid analogs

Ecdysteroids are specific hydroxysteroids that exert a wide a range of biological functions in the flora and the fauna. Our group has recently described that less polar ecdysteroid derivatives can strongly sensitize various cancer cell lines to certain chemotherapeutics, which can be observed both in multi-drug resistant (MDR) and drug susceptible cancer cells [1 – 2]. Recent studies also revealed that oxime groups can increase the antitumor activity of certain steroid derivatives [3], but ecdysteroid oximes have previously not been studied from this point of view. In our current work, we aimed to synthesize C6 ecdysteroid oximes of 20-hydroxyecdysone 2,3;20,22-diacetonide, optimizing the previously available methods [4]. Rotational planar chromatographic, analytical and preparative RP-HPLC and centrifugal partition chromatographic methods were developed for the purification. The diacetonides of two 14,15-anhydro ecdysteroid oxime isomers (E/Z-oximes) were obtained, and Beckmann rearrangement of the new 14,15-anhydro E-oxime yielded another new derivative containing a seven-membered lactam B-ring [5]. The ecdysteroid oximes were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their sub-cell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). Each oxime was found to be a promising modulator of resistance on the MDR cell line, and the E-oxime could also sensitize the non-MDR cells to doxorubicin. Our preliminary SAR study also revealed, that the antitumor activity of ecdysteroid diacetonides follow the ecdysteroid < Z-oxime < E-oxime order. Acknowledgement: Szeged Foundation for Cancer Research.

The Generally Useful Estimate of Solvent Systems (GUESS) method enables the rapid purification of methylpyridoxine regioisomers by countercurrent chromatography

The TLC-based Generally Useful Estimate of Solvent Systems (GUESS) method was employed for countercurrent chromatography solvent system selection, in order to separate the three synthetic isomers: 3-O-methylpyridoxine, 4′-O-methylpyridoxine (ginkgotoxin), and 5′-O-methylpyridoxine. The Rf values of the three isomers indicated that ChMWat+2 (chloroform-methanol-water 10:5:5, v/v/v) was appropriate for the countercurrent separation. The isomer separation was highly selective and demonstrated that the TLC-based GUESS method can accelerate solvent system selection for countercurrent separation. Accordingly, the study re-emphasizes the practicality of TLC as a tool to facilitate the rapid development of new countercurrent and centrifugal partition chromatography methods for this solvent system. Purity and structure characterization of all samples was performed by quantitative 1H NMR.

Chemical and Biological Investigation of Olive Mill Waste Water - OMWW Secoiridoid Lactones.

Olive mill waste water is the major byproduct of the olive oil industry containing a range of compounds related to Olea europaea and olive oil constituents. Olive mill waste water comprises an important environmental problem in olive oil producing countries, but it is also a valuable material for the isolation of high added value compounds. In this study, an attempt to investigate the secoiridoid content of olive mill waste water is described with the aid of ultrahigh-performance liquid chromatography-electrospray ionization (±)-high-resolution mass spectrometry and centrifugal partition chromatography methods. In total, seven secoiridoid lactones were isolated, four of which are new natural products. This is the first time that a conjugate of hydroxytyrosol and a secoiridoid lactone has been isolated from olive mill waste water and structurally characterized. Furthermore, the range of isolated compounds allowed for the proposal of a hypothesis for the biotransformation of olive secoiridoids during the production of olive mill waste water. Finally, the ability of the representative compounds to reduce the intracellular reactive oxygen species was assessed with the dichlorofluorescein assay in conjunction with the known antioxidant agent hydroxytyrosol.

Gradient elution method in centrifugal partition chromatography for the separation of a complex sophorolipid mixture obtained from Candida bombicola yeasts

Sophorolipids represent an important class of natural surfactants with a variety of environmental, cosmetic, and pharmaceutical applications. Despite their promising physicochemical and biological properties, the use of sophorolipids is hampered by the lack of information regarding their individual structure-activity relationships. The major difficulty in isolating pure sophorolipids arises from the high complexity of crude fermentation media composition and from their strong structural similarities. In this work, a centrifugal partition chromatography method was developed in an original gradient elution mode for the separation of sophorolipids produced by the yeast Candida bombicola. Experiments were realized by using three sets of solvent systems composed of n-heptane, ethyl acetate, n-butanol, methanol, and water in different proportions. The separation was performed at 5 mL/min in the ascending mode by increasing progressively the polarity of the organic mobile phase. In these conditions, more than 80% of the sophorolipids present in the initial crude fermentation extract were eluted successively from the most hydrophobic lactone forms to the most hydrophilic acid forms. The structures of the isolated sophorolipids were further elucidated by HPLC and NMR analyses.

Natural product isolation – how to get from biological material to pure compounds

Since the last comprehensive review by Otto Sticher on natural product isolation in NPR (O. Sticher, Nat. Prod. Rep., 2008, 25, 517), a plethora of new reports on isolation of secondary compounds from higher plants, marine organisms and microorganisms has been published. Although methods described earlier like the liquid-solid chromatographic techniques (VLC, FC, MPLC, HPLC) or partition chromatographic methods are still the major tools for isolating pure compounds, some developments like hydrophilic interaction chromatography (HILIC) have not been fully covered in previous reviews. Furthermore, examples of using different preparative solid-phase extraction (SPE) columns including molecular imprinting technology have been included. Special attention is given to chiral stationary phases in isolation of natural products. Methods for proper identification of plant material, problems of post-harvest changes in plant material, extraction methods including application of ionic liquids, de-replication procedures during natural product isolation are further issues to be discussed by the review. Selected work published between 2008 and mid-2012 is covered.

Multiple dual-mode centrifugal partition chromatography as an efficient method for the purification of a mycosporine from a crude methanolic extract of Lichina pygmaea

Centrifugal partition chromatography method was applied to the separation and purification of a crude methanolic extract of a cyanobacterial lichen, Lichina pygmaea. A multiple dual-mode was used to separate two compounds of interest, namely mycosporine-serinol and a glutamic acid derivative. These compounds are described here for the first time in a lichen. Their structures were identified by UV, IR, ESI-MS, 1H NMR, 13C NMR, and 2D NMR.

The Liquid-liquid InterfaceUnder Planetary Rotation

Countercurrent chromatography is a liquid-liquid partition chromatography method allowing high-resolution purifications. It utilises the hydrodynamic behaviour of two immiscible liquid phases in coil columns undergoing a planetary motion generated by a synchronous coil planet centrifuge. In this study planetary rotation is applied to liquid-liquid flows in two types of ring geometries. The influence of the epicyclic force field is studied by applying a variety of different operating conditions, namely rotational speed and the ratio of the planet and sun radii known as the beta value. The results show that variation of the beta value has far greater influence on phase distribution than variation of the rotational speed.

Determination of citrate, inositol and gentisic acid in a pharmaceutical diagnostic formulation by ion-moderated partition chromatography

An ion-moderated partition chromatographic method has been developed for rapid determination of excipients in a pharmaceutical diagnostic formulation. Citrate, inositol, and gentisic acid (2,5-dihydroxybenzoic acid) are separated and analyzed using an 0.005 M sulfuric acid-acetonitrile (80:20) mobile phase with a 100 × 7.8 mm strong cation-exchange column consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the hydrogen form. Detection is by refractive index. The method has proven to be rugged for more than 100 injections over a one-year period on the same column.

Separation Studies of Metal Ions in Sodium Thioglycolate Medium by Thin Layer Partition Chromatography

Abstract A thin layer partition chromatographic method has been developed for separation of Fe(III), Ni(II), Zn(II), Cu(II), Pb(II) and Mn(II) on thin layers of silica gel-G as an adsorbent. The Rf values were determined using 0.01–0.2 aqueous solution of sodium thioglycolate as a mobile phase. The dependence of Rf values on the migration time, pH and concentration of mobile phase has been studied. The optimum conditions for possible 3-component separation have been determined. Metal ions have been separated, detected, eluted and quantitatively determined by atomic absorption spectroscopy. The present method was applied to the separation and determination of zinc in forensic samples.

Infinite-dilution activity coefficients of water in TEG, PEG, glycerol and their mixtures in the temperature range 50 to 140 °C

Infinite-dilution activity coefficients of water, γ ∞ H 2 O, in triethylene glycol (TEG) polyethylene glycol (PEG) of various molecular weights, glycerol and their mixtures are measured using the gas—liquid partition chromatography method. The data are used to provide a preliminary comparison of the relative merit of these solvents as dehydrating agents at elevated temperatures. The mixed solvents are less volatile than the lower boiling pure solvents and therefore will suffer less loss in the solvent regeneration process. The highest molecular weight PEG was found to have the largest drying capacity on a molar basis, but the lowest capacity on a mass basis. The converse is true for glycerol, the lowest molecular weight dehydrating agent studied. It was found that over the temperature range covered, ln γ ∞ H 2 O is linear in 1/ T. γ ∞ H 2 O for TEG is least sensitive to temperature while γ ∞ H 2 O for the highest molecular weight PEG is the most sensitive. For mixed dehydrating agents, the results are very well correlated by a weight fraction weighted average of the data for the constituent pure solvents with a single small temperature independent empirical constant.

Hepatic microsomal N-demethylation of N-methylbenzamidine. N-dealkylation vs N-oxygenation of amidines.

The microsomal oxidative N-demethylation of N-methylbenzamidine, a model compound for active substances containing the basic amidine function, was investigated. N-Methylbenzamidine was converted into benzamidine and formaldehyde by aerobic incubation with non-induced microsomal fractions of rabbit liver homogenates and NADPH. The formation of benzamidine in the incubation mixtures under widely differing conditions was assayed using a newly-developed, high-performance, ion pair, reverse-phase partition chromatographic method. Optimal reaction conditions were determined. The benzamidine formation in the incubation mixture followed Michaelis-Menten kinetics and required the presence of molecular oxygen and NADPH. The effects of the inducer phenobarbital, methylcholanthrene, ethanol and N-methylbenzamidine itself on the activity were studied. Neither superoxide anion nor hydrogen peroxide was directly involved in the demethylation reaction. The direct involvement of cytochrome P-450 in this reaction is supported by the observation that the presence of inhibitors of cytochrome P-450, in particular of carbon monoxide, markedly decreased the rate of N-demethylation. This N-demethylation of N-methylbenzamidine proves the hypothesis that benzamidines with hydrogen atoms in the alpha-position to the amidine nitrogen atoms are N-dealkylated instead of N-oxygenated by the microsomal mixed function oxidase system.

Ｇｒａｙａｎａｎｅ系化合物の遠心液液分配クロマトグラフィー

A centrifugal partition chromatographic method was developed for separation of grayanotoxin (G)-I, G-II and G-III, which are toxic diterpenoids of Leucothoe grayana MAX. The optimum conditions for separating these grayanotoxins from each other utilized the following: solvent composition, CHCl3:MeOH:H2O=45:60:40 (mobile phase, upper layer; stationary phase, lower layer); rotational speed, 700 rpm; flow rate, 1.0ml/min. The constituents in the eluates were checked by TLC and quantitatively determined by spectrophotometry (at 460 nm) after being treated with vanillin in a solution of perchloric acid.

Reversed-Phase Ion-Pair Chromatography Of Antihistamine Drugs

Abstract A reversed-phase ion-pair chromatographic separation of some antihistamine drugs is presented. The stationary phase consists of 1-pentanol or methylene-chloride adsorbed on a CH-10 column and the mobile phase of a phosphate buffer containing cyclohexylsulphamate and dimethyl-octylamine. It is concluded that this method is a valuable complement to a partition chromatographic method reported in an earlier publication.

Column Chromatographic Analysis of Barbiturates in Their Dosage Forms. IV. Combinations with Parabens

The nature of the alkaline hydrolysis of some barbiturates in combinations with parabens (p-hydroxybenzoates) was studied with controlled variables, including temperature, viscosity, and concentrations of sodium hydroxide, barbiturate, and parabens. The kinetic studies showed that parabens could be completely hydrolyzed in strong base at 40 degrees C in 1 hr, while the barbiturate remained intact and was readily isolated by partition chromatography, Based on the theoretical results, a partition chromatographic procedure for butabarbital with parabens was devised. Standard recoveries averaged 100.7% with a standard deviation of 0.89. Kinetic data indicate that the hydrolysis of parabens could also be applied to analyze combinations with amo-, seco-, and pentobarbitals. Phenobarbital and parabens are readily separated by partition chromatographic methods without prior hydrolysis of the parabens. The low extraction constant for phenobarbital allowed its retention on a column against relatively strong solvents while the intact parabens are eluted. A slightly modified method was applied to the separation of phenobarbital from parabens. Standard recoveries average 99.9% with a standard deviation of 0.69.

An improved separation procedure for nucleoside monophosphates on polyethyleneimine- (PEI-)cellulose thin layers

A procedure is described for the two-dimensional separation of the 4 major and 16 modified nucleoside-(5') monophosphates on anion-exchange thin layers of polyethyleneimine- (PEI-)cellulose. The method, which is simple and less time-consuming than existing partition chromatographic methods, may be used for the identification of 5'-termini of RNA and RNA fragments.

Determination of Conjugated and Esterified Estrogens in Pharmaceutical Tablet Dosage Forms by High-Pressure, Normal-Phase Partition Chromatography

A high-pressure, normal-phase partition chromatographic method for the identification and determination of conjugated and esterified estrogens in pharmaceutical tablet dosage forms is described. The method is based on the separation of the estrogen sulfate esters on a conventional diatomaceous earth-H20 column, followed by HCI-methanol hydrolysis, and finally a chromatographic separation on a chemically bonded ether (ETH-Permaphase) column and measurement. Mobile phases found useful were combinations of 2-propanol and n-heptane. Equillin and -dehydroestrone, a structurally related isomer, not separately determined in the proposed method, are determined as a sum by performing a chromatographic study of their corresponding 2, 4-dinitrophenylhydrazine derivatives. Studies have indicated several estrogen derivatives can be rapidly formed and then separated on the same column, providing a useful qualitative analysis scheme. Commercial tablet dosage forms were analyzed for conjugated and esterified estrogens and the results are presented.

Combined partition-chromatographic methods

The possiblities of partition—chromatographic methods in the analysis of organic compounds are discussed and some new versions of the methods are mentioned.

Separation and Quantitative Determination of Impurities in Tetracycline

Anhydrotetracycline, epianhydrotetracycline, epitetracycline, and chlortetracycline are generally found as impurities in tetracycline. A new thin-layer partition chromatographic method is described in which the impurities are separated on a kieselguhr layer impregnated with ethylene glycol-water-acetone-ethyl acetate (2:2:15:15). Compared to previous methods, this determination is easier to perform and more versatile and it prevents rapid epimerization. The quantitative determination of the tetracyclines utilizes spectrophotometry or direct TLC fluorometry.