Use Of Countercurrent Chromatography Research Articles

Separation of Dihydro-Isocoumarins and Dihydro-Stilbenoids from Hydrangea macrophylla ssp. serrata by Use of Counter-Current Chromatography.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar ‘Yae-no-amacha’ using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.

Isolation of Phytochemicals from Salvia plebeia Using Countercurrent Chromatography Coupled with Reversed-phase HPLC

Salvia plebeia R. Br. is a plant which has been used as an edible crop and traditional medicine in Asian countries. In this study, HPLC-PDA analysis and countercurrent chromatography (CCC) coupled with reversed-phase (RP) HPLC method were applied to isolate ten isolates from 3.3 g of n-butanol soluble extract from hot-water extract of S. plebeia. The use of CCC enabled us to efficiently fractionate the starting material with less sample loss and facilitate the isolation of compounds from S. plebeia extract using RP-HPLC. The isolates were determined to be caffeic acid (1), 6-hydroxyluteolin 7-O-β-D-glucoside (2), eudebeiolide B (3), (R)- rosmarinic acid (4), homoplantaginin (5), eudebeiolide D (6), plebeiolide C (7), salpleflavone (8), eupafolin (9) and hispidulin (10) based on the spectroscopic evidence.

Use of counter-current chromatography as a selective extractor for the diterpenequinone 7α-hydroxyroyleanone from Tetradenia riparia

A solvent system was developed for selective isolation by high-speed counter-current chromatography (HSCCC) of the benzoquinone 7α-hydroxyroileanone, 1, a bioactive diterpene from a dichloromethane extract of Tetradenia riparia leaves. Several solvent systems were initially studied, including hexane-ethyl acetate-methanol-water in several ratios, hexane-acetone-methanol-water, hexane-ethanol-water and hexane-acetonitrile-methanol, which gave recovery rates for the target compound between 13.4 and 35.9%. The new solvent system hexane-5% aqueous Na2CO3 (1:1) was developed based on the chemical ionization reaction of the benzoquinone hydroxyl group in the basic pH of the carbonate solution, prompted by the extraction procedure used for the extraction of lapachol (a natural naphtoquinone) from a Tabebuia species wood. By using the HSCCC chromatograph as a liquid–liquid extractor with the above mentioned solvent system the recovery rate of 1 increased to 81.8%, affording the quinone with 97% purity.

The use of countercurrent chromatography in the separation of nonpolar lipid compounds

Isolation of lipophilic compounds by countercurrent chromatography (CCC) is a challenge because biphasic solvent systems in which these compounds distribute evenly are difficult to obtain. In this article we present novel applications of lipid compound isolation from natural sources. Conjugated linolenic acids (CLnAs, log KOW ∼7) were isolated from pomegranate oil using a solvent system consisting of n-heptane/methanol/water 100:91:9 (v/v/v). The CLnA fraction was free of other fatty acids but consisted of different isomers which were not resolved from each other. In the less polar range (log KOW ∼12), three tocotrienols (α-, γ- and δ-tocotrienol) were isolated from a vitamin E capsule produced from palm oil by using the solvent system n-hexane/acetonitrile/benzotrifluoride (BTF) at a ratio of 10:6.5:3.5 (v/v/v). Between 36 and 65mg of each of the three tocotrienols were obtained in one injection with purities >97%. Advantages and disadvantages of the “BTF system” are discussed by comparing the phase composition with the simple n-hexane/acetonitrile system and by the fractionation of phytosterols (log KOW ∼9.5) from rapeseed oil.

Facilitating the use of counter-current chromatography in pharmaceutical purification through use of organic solvent nanofiltration

This paper demonstrates a combined approach for separating an active pharmaceutical ingredient (API) from a heavily contaminated waste stream. The approach uses organic solvent nanofiltration (OSN) to improve the application of counter-current chromatography (CCC) in an industrial process. OSN provides an efficient route for exchanging solutes from the process solvent into the desired mobile phase for CCC, generating a CCC feed containing less than 0.01% (area % by GC) of the original process solvents. The high solvent burden of CCC was additionally reduced through recovery of mobile phase using OSN, with the recovered solvent containing less than 1% (area % by HPLC) impurities. The recovered solvent was then successfully recycled into a subsequent CCC run with no indication of impurity build-up. Coupling OSN with CCC improved the mass-intensity of the CCC process, reducing the solvent use by 56%. OSN can be a useful tool in facilitating the application of CCC to pharmaceutical process streams.

Use of countercurrent chromatography during isolation of 6-hydroxyluteolin-7-O-β-glucoside, a major antioxidant of Athrixia phylicoides

Athrixia phylicoides, an indigenous South African herbal tea, has potential as a source of nutraceutical antioxidant extracts. Countercurrent chromatography (CCC) was employed as part of a multi-step process to isolate one of the major antioxidant compounds in A. phylicoides extracts. Antioxidant activity of the extracts was comparable to commercial nutraceutical extracts from Aspalathus linearis and Cyclopia spp. in a range of assays. The extracts were tested for radical scavenging (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) di-ammonium radical cation (ABTS+) scavenging, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging and oxygen radical absorbance capacity (ORAC)), ferric reducing antioxidant potential (FRAP) and iron chelating activity, as well as inhibition of microsomal lipid and linoleic acid emulsion oxidation. After extraction optimisation, the antioxidant activity of the major phenolic compounds in an A. phylicoides extract was determined using the on-line HPLC-diode-array-DPPH and -ABTS+ radical scavenging assays. Major compounds reported for the first time included chlorogenic acid, 1,3-dicaffeoylquinic acid, several hydroxycinnamic acid derivatives, including dicaffeoyl quinic acids, and an unidentified flavone–hexose. Finally, CCC was used in conjunction with liquid–liquid partitioning and semi-preparative reversed-phase HPLC to isolate 6-hydroxyluteolin-7-O-β-glucoside (a major antioxidant) and quercetagetin-7-O-β-glucoside (a minor compound present in CCC fraction containing 6-hydroxyluteolin-7-O-β-glucoside) from an A. phylicoides extract. The chemical structures of the isolated compounds were confirmed by LC high-resolution electrospray ionisation MS, as well as 1H, 13C and 2D NMR spectroscopy. This is the first report of the isolation of these compounds from A. phylicoides.

Purification of high-throughput organic synthesis libraries by counter-current chromatography

Experiments were performed to evaluate whether counter-current chromatography (CCC) could function as an alternative purification method to reversed-phase high-performance liquid chromatography (RP-HPLC) and normal-phase supercritical fluid chromatography (SFC). RP-HPLC and SFC are the routine methods currently used in our high-throughput purification (HTP) facility for the purification of high-throughput organic synthesis (HTOS) libraries and medicinal chemistry reaction mixtures. Pre-equilibration of the solvent mixture layers was not mandatory for effective chromatography when hexanes–ethyl acetate–methanol–water (HEMW) solvent mixtures were used. Key to the use of CCC for high-throughput applications is the ability to effectively select a solvent system appropriate to each library member. Pilot-scale CCC elution time was used to estimate a starting solvent ratio and RP-HPLC retention time was then used to adjust solvent ratios within a particular library. It was also found that dimethyl sulfoxide (DMSO) and DMSO–methanol were suitable as sample injection solvents when using the HEMW solvent systems.

Practical solvent system selection for counter-current separation of pharmaceutical compounds

Counter-current chromatography (CCC) is a technique that shows a lot of potential for large scale purification. Its usefulness in a “research and development” pharmaceutical environment has been investigated, and the conclusions are shown in this article. The use of CCC requires the development of an appropriate solvent system (a parameter of critical importance), a process which can be tedious. This article presents a novel strategy, combining a statistical approach and fast HPLC to generate a three-dimensional partition coefficient map and rapidly predict an optimal solvent system. This screen is performed in half a day and involves 9 experiments per solvent mixture. Test separations were performed using that screen to ensure the validity of the method.

Separation of U and Pu by countercurrent chromatography with support-free liquid stationary phase in the TBP white spirit nitric acid system

The article deals with the use of countercurrent chromatography (CCC), support-free partition chromatography, for separation of U and Pu from the organic extract obtained directly by the dissolution of MOX fuel in supercritical CO2 containing the complex TBP · nHNO3. White spirit solutions with various TBP concentrations were used as a stationary phase. The effects of the compositions of the stationary and mobile phases on the U/Pu partition efficiency are studied. The CCC method allows the separation of U and Pu under conditions of a TBP concentration gradient in the stationary phase and also of an HNO3 concentration gradient in the mobile phase. Chromatographic separation first gives the Pu fraction containing 98.9% of Pu and 0.07% of U, and then the U fraction (99.93% of U and 1.1% of Pu). The separation time is 50 min.

Chromatographie à contre courant et micelles inverses pour la séparation et l'extraction de cations métalliques

Countercurrent chromatography (CCC) is a separation technique in which the stationary phase is a liquid. Diethylhexyl phosphoric acid (DEHPA) forms reverse micelles in heptane. Metallic ions, located in an aqueous phase, can be extracted into the aqueous core of the reverse micelles in the heptane phase. A CCC apparatus can be considered as a powerful mixing and extracting machine with efficiency above several hundreds of theoretical plates. La3+, Ce3+, Pr3+, and Nd3+ lanthanide cations were separated using CCC with a DEHPA-containing heptane stationary phase. Studying the retention variations with aqueous mobile phase pH, it was possible to determine the lanthanide extraction constants and separation coefficients. Overloading conditions are described. Frontal chromatography was performed using a Co2+ and Ni2+ solution. The Co2+ ions were concentrated in the heptane + DEHPA stationary phase, a part of the solution was deionized, and another part was enriched in only Ni2+ ions. This method also produced the extraction constants and separation coefficients. The use of CCC with a complexing stationary phase can be applied to any cation for ion filtering and concentration, or for deionization of aqueous phases. Key words: countercurrent chromatography, CCC; ion extraction, ion filtering, deionization, lanthanides, transition metals.

Complementary use of counter-current chromatography and hydroxyapatite chromatography for the separation of three main classes of lipoproteins from human serum.

High-density, low-density and very-low-density lipoproteins (HDLs, LDLs and VLDLs) were purified from human serum by the combined use of counter-current chromatography (CCC) and hydroxyapatite chromatography. Polymer-phase CCC of human serum using the cross-axis coil planet centrifuge yielded two lipoprotein fractions, one containing HDLs and LDLs and the other VLDLs and serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions, all free from serum proteins. Each lipoprotein was confirmed by agarose gel electrophoresis.

Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins

A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofluoresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4′,5′-dibromo-4,5,6,7-tetrachlorofluorescein and 2′,4′,5′-tribromo-4,5,6,7-tetrachlorofluorescein, were isolated and characterized by ~H NMR and chemical ionization mass spectrometry.

Three nitropropanoyl esters of glucose from Indigofera linnaei

Three 3-nitropropanoyl esters of d-glucose were isolated from Indigofera linnaei, the known 1,2,6-tri- O-3-nitropropanoyl-β- d-glucopyranose (karakin) and 2,3,4,6-tetra- O-3-nitropropanoyl-α- d-glucopyranose, and the apparently previously undescribed 3,4,6-tri- O-3-nitropropanoyl-α- d-glucopyranose. The use of countercurrent chromatography for the isolation of these compounds is described, as well as the spectroscopic evidence which led to their structural elucidation.

Synergistic Use of Countercurrent Chromatography and High Performance Liquid Chromatography for the Purification of Synthetic Peptides

Abstract Examples are given demonstrating how countercurrent chromatography (CCC) and high performance liquid chromatography (HPLC) can be used together to purify synthetic peptides. In one example, CCC provided a preliminary purification of Met-Arg-Asp-Val-Val-Leu-Phe-Glu-Lys by enabling separation of ultraviolet absorbing, ninhydrin-negative material from the desired peptide. Final purification was achieved with HPLC without risk of harming the HPLC column. In a second example Tyr-Ala-Ala-Nle-Ala-Ala was completely purified by CCC with the CCC separation rapidly and conveniently monitored by HPLC. CCC appears to be a very useful technique for the peptide chemist.