One Step Large-Scale Separation and Purification of Rutin from Boenninghausenia sessilicarpa by Countercurrent Chromatography

In this study, a preparative countercurrent chromatography (CCC) method for isolation and purification of the bioactive component rutin directly from the ethanol extract of Boenninghausenia sessilicarpa was successfully established by using n-butanol-ethyl acetate-water as the two-phase solvent system. The upper phase of n-butanol-ethyl acetate-water (4:1:5, v/v) was used as the stationary phase of CCC. Under the optimum conditions, 112 mg of rutin at 98.6% purity was obtained from 2.0 g of the crude extract in a single CCC separation. The peak fraction of CCC was identified by negative ESI, ¹H NMR, and ¹³C NMR.     

One Step Purification of Piperine Directly from Piper nigrum L. by High Performance Centrifugal Partition Chromatography

Abstract

In this study, a preparative high performance centrifugal partition chromatography (HPCPC) method for isolation and purification of the bioactive component piperine directly from the ethanol extract of Piper nigrum L. was successfully established by using n-hexane-ethyl acetate-methanol-water as the two-phase solvent system. The upper phase of n-hexane-ethyl acetate-methanol-water (6:5:6:5, v/v) was used as the stationary phase of CPC. Under the optimum conditions, 40 mg of piperine at 98.5% purity, as determined by HPLC, was yielded from 300 mg of the crude extract in a single CPC separation. The peak fraction of CPC was identified by ¹H NMR and ¹³C NMR.     

Separation of Four Compounds from Forsythia suspensa by Counter-Current Chromatography with Stepwise Elution

High speed counter-current chromatography technique in preparative scale has been successfully applied to separate and purify main compounds from the ethyl acetate extract of Forsythia suspense using stepwise elution with two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:4:1:4,v/v) and (1:4:2:3,v/v). Under the optimized conditions, the preparative high-speed counter-current chromatography was performed on 350 mg of the ethyl acetate yielding phillyrin (12.8 mg), isolariciresinol-9’-O-β-D-glucopyranoside (5.3 mg), pinoresinol (21.2 mg), and phillygenin (8.3 mg) in a one-step separation, with purities over 90% as determined by HPLC. The structures of the separated compounds were identified by HPLC-MS and ¹H NMR.     

Application of High-Speed Counter-Current Chromatography Preparative Separation of Flavone C-Glycosides From Lophatherum gracile

Preparative high-speed counter-current chromatography (HSCCC) was used to separate and purify bioactive constituents from the stems and leaves of Lophatherum gracile Brongn. Six flavone C-glycosides each at over 95% purity including two new compounds were obtained in one-step separation by HSCCC with an optimized two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water at volume ratio of 4:2:1.5:8.5 (v/v/v/v). The experiment yielded 19.9 mg of luteolin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (1), 28.5 mg of luteolin 6-C-α-L-arabinopyranosyl-7-O-β-D-glucopyranoside (2), 31.5 mg of isoorientin (3), 44.8 mg of orientin (4), 25.3 mg of swertiajaponin (5) and 12.1 mg of apigenin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (6) from 500 mg of crude extracts. The purity of these compounds was determined by high-performance liquid chromatography (HPLC). Their chemical structures were identified by electron spray ionization mass spectroscopy (ESI-MS), ¹H and ¹³C nuclear magnetic resonance spectroscopy (NMR).     

Investigation of the Effects of Virgin Olive Oil Cleaning Systems on the Secoiridoid Aglycone Content Using High Performance Liquid Chromatography–Mass Spectrometry

Phenolic compounds are useful markers to control olive oil technological processes, including the virgin olive oil (VOO)/water separation after olive oil extraction. In this investigation, VOO extracted from olives of cv. Coratina using a mild oil/water separator called the hydrocyclone sedimentation system (Hydroil) was compared with VOO obtained using a conventional vertical centrifuge separator (Cenoil), which is mostly used in the modern olive oil industry. Secoiridoid aglycones were selected, among phenolic compounds, as markers and analyzed using reversed‐phase liquid chromatography coupled to linear quadrupole ion‐trap mass spectrometry with electrospray ionization in the negative mode. VOO samples obtained using the Hydroil system were found to contain significantly higher levels of secoiridoid aglycones, compared to the Cenoyl‐type samples. In particular, the total content of the aglycones of decarboxymethyl oleuropein, decarboxymethyl ligstroside, ligstroside, and oleuropein, expressed in terms of oleuropein, was estimated as 35.40 ± 0.80 mg kg^?1, compared to 8.06 ± 0.41 mg kg^?1 in the Cenoil samples (n = 3). Since no significant difference in residual water (P < 0.05) was found between the two types of VOO samples, the higher amount of secoiridoids obtained for Hydroil‐type ones was explained by the lower extent of oxidation occurring during the mild oil/water separation achieved using the Hydroil system.     

Effective and Preparative Separation of Bioactive Flavonoids From Gynostemma Pentaphyllum Tea Using Elution-Extrusion Counter-Current Chromatography

Elution-extrusion counter-current chromatography (EECCC) was successfully applied for screen and separation of four flavonoids from Gynostemma pentaphyllum tea, a popular herbal tea extract in China. With the hexane/ethyl acetate/methanol/water 5/6/5/6 (v/v) system, 300 mg of G. pentaphyllum tea extract were fractionated on a 180 mL-capacity preparative hydrodynamic CCC column. Satisfactory separation efficiency was achieved, producing milligram-amounts of quercetin, isorhamnetin, and cirsiliol over 90% pure in one EECCC process. Due to the hydrophilic property, the major flavonoid glycoside, rutin, was co-eluted with the solvent front as a mixture. Therefore, another carefully selected biphasic liquid system composed of ethyl acetate/n-butanol/water (4/1/5, v/v) was employed, yielding 35 mg of rutin with 97.1% purity. Structures of all separated compounds were identified by ESI-MS, ¹H NMR, and ¹³C NMR.     

Effect of Accelerated Storage on Microencapsulated Kenaf Seed Oil

In order to improve the quality and protect against degradation, kenaf (Hibiscus cannabinus L.) seed oil was microencapsulated by using spray drying. The microencapsulated kenaf seed oil (MKSO) was then stored at 65 °C for 24 days, the changes of fatty acids and bioactive compounds were examined every six days. Bulk (unencapsulated) kenaf seed oil was used as a control and was compared to the MKSO. The fatty acids and phytosterols compositions were determined by using gas chromatography, while tocopherols and phenolic acids of microencapsulated kenaf seed oil were determined by using high performance liquid chromatography. The results showed that there was a significant decrease (p < 0.05) in bioactive compounds in kenaf seed oil while the bioactive compounds in MKSO were maintained in a stable condition upon accelerated storage. Microencapsulation was shown to protect kenaf seed oil against oxidation, as well as preventing the degradation and/or loss of bioactive compounds in kenaf seed oil.     

Preparative Isolation of Bioactive Nitrile Glycoside “Niazirin” from the Fruits of Moringa oleifera Using Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography was successfully applied in the separation of bioactive constituent niazirin directly from the chloroform extract of Moringa oleifera. The experiment was performed with a two-phase solvent system composed of ethyl acetate/BuOH/water (6:0.5:4 v/v/v) in which the upper organic layer was used as stationary phase and lower aqueous phase was used as mobile phase. From 1 g of crude extract, 70 mg of niazirin was obtained in 94.8% purity as determined by HPLC. The total yield recovery was >94%. The isolated nitrile glycoside (niazirin) was characterized on the basis of its ¹H, ¹³C-NMR and ESI-MS data.     

COMPARISON OF TWO ANALYTICAL METHODS FOR THE DETERMINATION OF AZAARENES IN ATMOSPHERIC PARTICULATE MATTER

In this paper, the development of an analytical method for the separation and quantification of 20 azaarenes is described. Two methods are compared: high performance liquid chromatography with fluorescence detection (HPLC-fluorescence) and gas chromatography with mass spectrometry detection (GC-MS).

Although HPLC-fluorescence was proven to be the most sensitive method, GC-MS was selected in particular for the efficiency of the separation of the 20 azaarenes. The detection limits of the HPLC-fluorescence and GC-MS methods varied between 0.04 μ g.L^?1 (dibenz[a,c]acridine) and 1.30 μ g.L ^?1 (acridine) and between 1.50 μ g.L ^?1 (benz[c]acridine) and 2.56 μ g.L ^?1 (dibenz[a,c]acridine) respectively.

The GC-MS method was applied to particulate matter (PM ₁₀ ) samples collected over 48–72 h periods between April 2006 and February 2007 in Strasbourg (East of France). Before analysis aerosol samples were Soxhlet extracted and concentrated to a final volume of about 1 mL of hexane.

The seasonally mean concentrations of all azaarenes for this urban site have shown a seasonal variation in which the maximum concentration occurred in the winter (6.0 ng.m ³ ) and the minimum in the summer (0.90 ng.m³). For all the seasons the 2 rings species were the predominant azaarenes while the > 4 rings species were the less abundant.     

Separation of five bioactive compounds from Glycyrrhiza uralensis Fisch using a general three-liquid-phase flotation followed by preparative high-performance liquid chromatography

ABSTRACT

Glycyrrhiza uralensis

Fisch is homologous medicine for food plant and health-protective affection in the human body system. To further understand the functions of G. uralensis Fisch, separation of the bioactive compounds from this natural product is very important. A general three-liquid-phase flotation (TLPF) technique was established to separate and concentrate five bioactive compounds from G. uralensis Fisch. The three-liquid-phase system, pH of initial aqueous phase, (NH₄)₂SO₄ concentration, nitrogen flow rate, and flotation time were investigated. The flotation efficiency of liquiritin apioside, liquiritin, and glycyrrhizic acid in the middle phase and semilicoisoflavone B and licoisoflavone B in the upper phase could reach 91.24%, 94.23%, 88.04%, 94.67%, and 96.44%, respectively. Furthermore, the established TLPF was used for separation of four kinds of anthraquinones from Cassiae Semen. The results showed that TLPF is feasible for separating the compounds with quite a different polarity from natural products.     

Preparative separation of four sesquiterpenoids from Curcuma longa by high-speed counter-current chromatography

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation of four major sesquiterpenoids with similar structures, namely ar-turmerone, β-turmerone, α-turmerone, and E-α-atlantone, from the essential oil of Curcuma longa Linn. using a two-phase solvent system composed of n-heptane-ethyl acetate–acetonitrile–water (9.5/0.5/9/1, v/v). From a single HSCCC run, 1.7861 g of ar-turmerone, 0.4708 g of β-turmerone, 1.3427 g of α-turmerone, and 0.1650 g of E-α-atlantone were obtained. The purity of each compound was over 98% as determined by HPLC. The chemical structures of these compounds were determined by ¹H NMR and ¹³C NMR.     

One-Step Isolation and Purification of Four Xanthone Glycosides from Tibetan Medicinal Plant Halenia elliptica by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC) was successfully applied to the isolation and purification of four xanthone glycosides from Halenia elliptica, a plant widely used in traditional Tibetan medicine. The introduction of HSCCC greatly improved the efficiency of compounds preparation from Halenia elliptica. The following were obtained from 100 mg of crude sample in one-step separation: 2.5 mg of 1-O-primeverosyl-2,3,4,5,7-pentamethoxyxanthone, 7.0 mg of 1-O-primeverosyl-2,3,4,7- tetramethoxyxanthone, 10.0 mg of 1-O-primeverosyl-2,3,5-trimethoxyxanthone (demethyoxyhaleniaside), and 8.5 mg of 1-O-primeverosyl-2,3,4,5-tetramethoxyxanthone. HPLC analysis showed that each target compound had a purity of over 98%, and UV, ¹H NMR, and ¹³C NMR data confirmed the component chemical structures.     

Isolation and Purification of Kaempferol-3,7-O-α-L-Dirhamnopyranoside from Siraitia grosvenori Leaves by High-Speed Counter-Current Chromatograph and Its Free Radical Scavenging Activity

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of flavonoid glycoside from the leaves of Siraitia grosvenori by using a two-phase-solvent system composed of ethyl acetate–n-butanol–water (4:1:5, v/v/v). kaempferol-3,7-O-α-L-dirhamnopyranoside was obtained in one-step separation and less than 5.5 h from 90 mg of crude extract from the S. grosvenori leaves. The chemical structure of this compound was identified by MS, ¹H NMR, and ¹³C NMR. Free radical scavenging activity of kaempferol-3,7-O-α-L-dirhamnopyranoside was also evaluated and the results showed that it had good free radical scavenging activity with its IC₅₀ value being 3.97 mg/ml.     

Enrichment and Separation of Sinomenine and Acutumine from Sinomenium acutum by pH-Zone-Refining Counter-Current Chromatography

Enrichment and separation of alkaloids from a chloroform extract of Sinomenium acutum has been successfully performed for the first time using pH-zone-refining counter-current chromatography. The two-phase solvent system used for enrichment was composed of Methyl tert-butyl ether (MtBE)–acetonitrile (CH₃CN)–water (4:1:5, v/v), where 10 mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10 mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter, which could enrich the alkaloids from the crude extract well. For the preparative separation, the solvent system consisted of MtBE–CH₃CN–water (4:0.5:5, v/v) with 10 mM TEA in organic stationary phase and 5 mM HCl in the aqueous mobile phase, which could separate and purify the enriched crude alkaloids successfully. 0.82 g of crude alkaloids was enriched from 1.60 g of chloroform extract in the first step separation. From the enriched crude alkaloids, 376 mg of sinomenine and 85 mg of acutumine were obtained in the second step separation with the purity of 98.1% and 98.7%, respectively. The chemical structures of the isolated compounds were identified by UV, ESI-MS and ¹H NMR.     

Separation of 4’-demethyldeoxypodophyllotoxin from Sinopodophyllum emodi by medium-pressure LC and high-speed counter-current chromatography guided by HPLC-MS

A minor lignan, 4ˊ-demethyldeoxypodophyllotoxin, was isolated from Sinopodophyllum emodi. First, a crude extract of S. emodi was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to confirm the presence of the target compound. Then, the target lignan was successfully enriched in the crude extract precipitate by medium-pressure LC (MPLC) with high recovery (85%). Finally, an efficient separation was achieved by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system of n-hexane-ethyl acetate–methanol–water (1:1:1:1, v/v/v/v). These results clearly demonstrate that the combination of HPLC-MS, MPLC, and HSCCC could be a powerful technique for the rapid screening, enrichment, and separation of minor compounds from complex natural products.     

Hyphenated Flash Chromatographic Separation and Isolation of Coumarins and Polymethoxyflavones from Byproduct of Citrus Juice Processing Industry

The present study describes an efficient hyphenated flash chromatographic (FC) for the purification of coumarins and polymethoxy flavones (PMF's) from cold pressed grapefruit peel oil (GFO), a byproduct from citrus juice processing plant. GFO was subjected to silica column chromatography using sequential isocratic elution by hexane and chloroform to separate the waxes and phytochemicals enriched fractions, respectively. The chloroform extract was used for hyphenated FC separation using a gradient mobile phase of hexane and acetone. The peak separations were monitored at dual wavelengths of λ₂₁₀ nm and λ₃₄₀ nm. The individual fractions were analyzed by HPLC, pooled and concentrated resulting in isolation of two coumarins (auraptene and bergapten) and three PMF's (tangeretin, heptamethoxyflavone, and nobiletin). The purity and identity of the isolated compounds was confirmed by spectral analysis using high performance liquid chromatography, matrix-assisted laser desorption/Ionization-time of flight-mass spectrometry, and nuclear magnetic resonance. The hyphenated FC separation method was further validated by test of repeatability resulting in low RSD for the yield of the phytochemicals. Utilization of the developed separation method for isolation of value added products from grapefruit byproducts could be beneficial to the citrus processing industry. Supplemental materials are available for this article. Go to the publisher's online edition of Separation Science & Technology to view the supplemental file.     

Chemical characterization and origin of dyes used in the manufacture of Beninese cultural heritage objects

Six objects of Beninese cultural heritage provided by African and Confluences museums of Lyon (France) were the focus of this study. The characterization of colored compounds was achieved using: microchemical tests, Fourier transform infrared spectroscopy and liquid chromatography coupled to a photodiode array detector. The main results reflect the presence of organic compounds like indigotin, 2‐hydroxynaphthoquinone, and mineral ions such as Al³⁺, S^2?, Na⁺, and Fe³⁺. Dyes were identified from Philenoptera cyanescens (Yoruba indigo) and Lawsonia inermis L. (henna); pigments were identified as laundry blue, Prussian blue, and iron oxides. All of these data therefore make possible the conservation and the restoration of these objects while maintaining their visual and functional integrity.     

Preparative Isolation of Bioenhancer Loganetin from sf Alstonia scholaris by Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography (FCPC) was successfully applied in the separation of close R f complex bioactive iridoid, loganetin directly from the ethyl acetate extract of Alstonia scholaris . The experiment was performed with a two-phase solvent system composed of methyl tert- butyl ether (MtBE)/ACN/Water (3:1.5:3 v/v/v) where the lower phase of the biphasic system, the aqueous layer containing 8 mM HCl, was the stationary phase, while the upper organic layer supplemented with 15 mM triethylamine TEA was designated as the mobile phase. From 1.5 g of EtOAc extract, 48 mg of loganetin (1) was obtained in 94.4% purity as determined by HPLC. The isolated compound (1) was characterized on the basis of its ¹ H, ¹³ C–NMR, and ESI-MS spectroscopic data. Although loganetin does not possess antibacterial activity of its own, but in combination, it appreciably reduces the minimum inhibitory concentration (MIC) of nalidixic acid (NA) against nalidixic acid resistant (NAREC) and nalidixic acid sensitive (NASEC) strains of Escherichia coli . Loganetin, a very common, inexpensive, and non-toxic natural product may finds its application in the antibacterial drug development for treating multidrug-resistant Gram negative infections.     

Dielectric and Chromatographic Characterization of Polyethylene Glycols as Stationary Phases

Abstract

Three types of polyethylene glycols of different molecular weights namely, 600, 4000 and 20000, were evaluated as liquid stationary phases in gas liquid chromatography. Thus, the retention mechanism for the studied polymer stationary phases, 15% by weight on chromosorb PAW and their thermodynamic parameters have been investigated via inverse gas chromatography. The effect of polymer molecular weight on their efficiency as liquid stationary phases for the gas chromatographic separation of different types of hydrocarbons is also studied. The dielectric constant of dilute solutions for the studied polymers as dissolved in solvent chloroform was studied corresponding to their polarities. Two different modes of clusters were determined due to the solute-solute and solute-solvent interaction through hydrogen bonds.

All studied polymers have higher performance for separation of cyclic and aromatic compounds. Good chromatographic separation of n-alcohols is obtained toward polyethylene glycol (PEG20000) of relatively higher molecular weight. The saturated hydrocarbons can be separated very efficiently using low molecular weight polyethylene glycol (PEG600).     

Isolation and Purification of Geniposide,Crocin-1, and Geniposidic Acid from the Fruit of Gardenia jasminoides Ellis by High-Speed Counter-Current Chromatography

Geniposide, crocin-1, and geniposidic acid were simultaneously separated from the fruit of Gardenia jasminoides Ellis by one step of high-speed counter-current chromatography with the solvent system composed of ethyl acetate-n-butanol-water(1:4:5, v/v/v) within 130 min. The purities of the three compounds were 98.7%, 97.1%, and 90.4%, respectively, as determined by HPLC. Their structures were confirmed by MS, UV, ¹H NMR, and¹³C NMR analysis.