Countercurrent Chromatography Fractionation Research Articles

UV-based solvent system screening for high-speed counter-current chromatography fractionation of compounds with similar UV absorption from complex samples followed by preparative HPLC purification: Flavonoids from barley seedlings as sample.

This article proposes a solvent system screening strategy for compounds with similar UV absorption in complex samples by UV spectrophotometer. There is no need to calculate the partition coefficient value of each compound, only the partition coefficient of the whole sample. The partition coefficient value should be close to 1 in order to obtain as many high-speed counter-current chromatography fractions as possible. Then, preparative HPLC was used to purify the high-speed counter-current chromatography fractions. Based on the above strategy, seven c-glycosyl flavonoids and an amino acid were successfully obtained from barley seedlings through high-speed counter-current chromatography fractionation with ethyl acetate/n-butanol/water (8:2:10, v:v:v) system followed by preparative HPLC purification. The research shows that high-speed counter-current chromatography could be well developed as a tool for fractionation before purification, and greatly improves the separation efficiency.

Enrichment and structural assignment of geometric isomers of unsaturated furan fatty acids.

Furan fatty acids (FuFAs) are valuable minor fatty acids, which are known for their excellent radical scavenging properties. Typically, the furan moiety is embedded in an otherwise saturated carboxyalkyl chain. Occasionally, these classic FuFAs are accompanied by low amounts of unsaturated furan fatty acids (uFuFAs), which additionally feature one double bond in conjugation with the furan moiety. A recent study produced evidence for the occurrence of two pairs of E-/Z-uFuFA isomers structurally related to saturated uFuFAs. Here, we present a strategy that allowed such trace compounds to be enrichedto a level suited for structure determination by NMR. Given the low amounts and the varied abundance ratio of the four uFuFA isomers, the isolation of individual compounds was not pursued. Instead, the entire isomer mixture was enriched to an amount and purity suitable for structure investigation with contemporary NMR methods. Specifically, lipid extracted from 150g latex, the richest known source of FuFAs, was subsequently fractionated by countercurrent chromatography (CCC), silver ion, and silica gel column chromatography. Analysis of the resulting mixture of four uFuFAs isomers (2.4mg in an abundance ratio of 56:23:11:9) by different NMR techniques including PSYCHE verified that the structures of the two most abundant isomers were E-9-(3-methyl-5-pentylfuran-2-yl)non-8-enoic acid and E-9-(3-methyl-5-pent-1-enylfuran-2-yl)nonanoic acid. Additionally, we introduced a computer-based method to generate an averaged chromatogram from freely selectable GC/MS runs of CCC fractions without the necessity of pooling aliquots. This method was found to besuitable to simplify subsequent enrichment steps.

Comprehensive two-dimensional countercurrent chromatography × gas chromatography characterization of Artemisia argyi essential oil

A comprehensive two-dimensional (2D) countercurrent chromatography (CCC) × gas chromatography (GC) was investigated for characterization of chemical constituents of Artemisia argyi essential oil, and orthogonality for the 2D chromatographic system was evaluated. A solvent system composed of n-hexane/acetonitrile/methanol (2:2:1, v/v/v) was selected for first dimensional separation of Artemisia argyi essential oil. Then all CCC fractions were analyzed by GC, which provided a wealth of information regarding the composition of the essential oil. Visualization of chemical compositions obtained from the comprehensive 2D CCC × GC separation was achieved by creation of a 2D contour plot map. Total peak capacity was evaluated and approximately 1392 peaks were obtained through a comprehensive 2D CCC × GC separation. A high spatial coverage and a low linear correlation coefficient were achieved. Meanwhile, all compounds were identified by GC-MS. The obtained 2D contour plot could be divided into six zones to show the characteristic chemical compositions. Six zones could be divided into different component groups, including monoterpenes, sesquiterpenes, monoterpene alcohols, phenols, aldehydes, ketones and esters, which could be used to identify compounds that have not been reported, and to predict the structure of unknown compounds in Artemisia argyi essential oil and comprehensively characterize fingerprint peak.

Countercurrent chromatography isolation of 11′‐γ‐tocomonoenol from pumpkin seed oil with detection of novel minor tocochromanols

AbstractA non‐refined, organic pumpkin seed oil (PSO) was chosen for the isolation and structure verification of the rare vitamin E compound γ‐tocomonoenol (γ‐T1). Initial measurements indicated the presence of ~0.4 mg γ‐T1 per 100 g PSO. Saponification of ~2 L of PSO, followed by repeated countercurrent chromatography (CCC) with the solvent system n‐hexane/benzotrifluoride/acetonitrile (v/v/v) and silica gel column chromatography enabled the isolation of 6.8 mg γ‐T1 with a purity of 96.0% according to gas chromatography with mass spectrometry (GC/MS) analysis. Structural analysis by 1H nuclear magnetic resonance (NMR) spectroscopy and GC/MS of the γ‐T1 isolate confirmed the presence of a double bond in C‐11′‐position (11′‐γ‐tocomonoenol). In addition, CCC fractionation enabled the detection of 18 different tocochromanols, many of which were reported for the first time in PSO. This unmatched variety included among others α‐/γ‐tocopherol, α‐/γ‐tocomonoenol, two α‐ and two γ‐tocodienol isomers, α‐/γ‐tocotrienol as well as the rare 11′‐β‐tocomonoenol (β‐T1) and δ‐T1. Three uncommon tocochromanols were also detected whose origins and structure remained unclear.

High-performance countercurrent chromatography fractionation of epimeric pairs intermedine/lycopsamine and amabiline/supinine by an off-line electrospray mass spectrometry injection profiling of the roots of Lappula squarrosa

The toxicity of pyrrolizidine alkaloids (PAs) and how they endanger the health of livestock and humans have been known for decades. However, quantitative measurements of these toxic PAs in food sources and herbal products are still hindered by poor availability of some authenticated reference standards. In this study, a PA fortified extract from a lyophilized Lappula squarrosa root sample (564 g) was separated using high-performance countercurrent chromatography (HPCCC) with the solvent system n-hexane − n-butanol − 1% aqueous ammonia (1:1:2, v/v/v). The HPCCC fractions were initially monitored by an off-line electrospray ionization mass-spectrometry (ESI-MS), and the targeted alkaloid fractions were subsequently elucidated by GC-EI-MS, ESI-MS/MS injection profiling, coupled with 1D/2D-NMR analysis. This approach led to accurate fractionation of some L. squarrosa PAs, while high recovery rates were reached since adsorptive losses of sample is neglected as countercurrent chromatography is an all-liquid chromatographic technique.

Identification of two α-tocodienol isomers in palm oil after countercurrent chromatographic enrichment.

Countercurrent chromatography (CCC) was used for the enrichment of α-tocodienol (α-T2), a rare vitamin E-related minor compound previously tentatively detected in palm oil. Hitherto, only one isomer has been mentioned to occur at traces in palm oil. However, CCC fractionation followed by GC/MS measurements of all fractions resulted in the detection of two α-T2 isomers in five different palm oil vitamin E dietary supplement capsules. Five repetitive CCC separations of ~ 1g sample and additional purification steps by column chromatography provided ~ 2mg of two equally abundant α-T2 isomers with a purity of ~ 85%. The positions of the double bonds in the alkyl side chain could be assigned by means of two characteristic chemical shifts in the 1H NMR spectrum. Accordingly, the structures of the α-T2 isomers were 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-3,11-dienyl)chroman-6-ol (double bonds in 3',11'-position) and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-7,11-dienyl)chroman-6-ol (double bonds in 7',11'-position). Natural occurrence of both isomers was proven by GC/MS screening of crude palm oil after saponification and CCC separation. Moreover, GC/MS analysis allowed the tentative assignment of γ-tocomonoenol (γ-T1) and β-tocomonoenol (β-T1) as trace compounds in palm oil.

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

Thorough study of persistent organic pollutants and halogenated natural products in sperm whale blubber through preparative sample cleanup followed by fractionation with countercurrent chromatography

Marine mammals are top predators of the marine food chain and thus known to bioaccumulate high concentrations of polyhalogenated compounds. Yet, details of the organohalogen pattern are largely unknown. For this reason, we isolated the polyhalogenated compounds from 750 g blubber of a sperm whale (Physeter catodon), which deceased at the German North Sea coast in January 2016. The sample matrix was decomposed by sulfuric acid treatment and the polyhalogenated compounds were then fractionated by countercurrent chromatography (CCC). Seventy-three CCC fractions were taken and analyzed by gas chromatography with electron capture negative ion mass spectrometry (GC/ECNI-MS). The bulk of the polyhalogenated compounds in the sample originated from classic persistent organic pollutants (POPs). Altogether 90 polychlorinated biphenyl (PCB) congeners were detected in the sample including all possible octa- to decachloro congeners except one. The sample also featured 105 toxaphene congeners including 30 chlorobornenes (contribution ∼14% of the total toxaphene content) which were only detected after the CCC fractionation. In addition, several chlordane and mirex related compounds were detected which were never or very scarcely described before in biota. Classic POPs (PCBs, DDT, toxaphene, chlordane, mirex) were predominant, while new emerging contaminants were scarcely detected. The sample featured several halogenated natural products (HNPs) some of which were less stable and destroyed during the sample cleanup involving treatment with sulfuric acid. Sample fractionation by means of CCC was crucial for the detection and assignment of many of the uncommon polyhalogenated compounds.

Analysis and Characterization of Polychlorinated Hydroxybornanes as Metabolites of Toxaphene Using a Polar Bear Model.

Abiotic and biotic transformation of toxaphene (camphechlor) results in the selective enrichment of recalcitrant congeners while other, less persistent compounds of technical toxaphene (CTTs) are degraded. Until now, there has been little knowledge on oxidation transformation of toxaphene. For instance, the existence of hydroxylated CTTs (OH-CTTs) in authentic environmental and food samples has not been proven. For this reason, we synthesized a mixture consisting of tetra- to heptachlorinated OH-CTTs and simplified it by countercurrent chromatography (CCC). Thus, 227 OH-CTTs were detected in the CCC fractions (12 tetra-, 117 penta-, 81 hexa-, and 17 heptachlorinated OH-CTTs), which was >50% more than detected before the fractionation. One CCC fraction consisting of only 18 OH-CTTs was used to develop a sample cleanup method which aimed to remove CTTs, isobaric PCBs, and sample matrix. The final cleanup procedure consisted of (i) gel permeation chromatography (GPC) and adsorption chromatography using (ii) deactivated and (iii) activated silica gel. Hence, up to 320 and 4350 μg/kg lipid weight of octa- and nonachlorinated CTTs were detected in four liver samples and adipose tissue of polar bears, respectively. Furthermore, the presence of one hexachlorinated OH-CTT isomer could be verified in the samples, which was about 1% of the octachlorinated CTTs determined in the liver samples.

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.

Development of an efficient fractionation method for the preparative separation of sesquiterpenoids from Tussilago farfara by counter-current chromatography

A novel application of counter-current chromatography (CCC) to enrich plant extracts using direct and continuous injection (CCC-DCI) was developed to fractionate sesquiterpenoids from the buds of Tussilago farfara L. In this study, an n-hexane-acetonitrile-water (HAcW) solvent system was separately pumped into the CCC column, and an extraction solution (45% acetonitrile) was directly and continuously injected into the CCC column. Since the extraction solution was used as a mobile phase in this method, solvent consumption could be greatly reduced. To enrich the extraction solution (315.9g/5.4L), only 4.2L water, 4.6L acetonitrile, and 1.2L n-hexane were used, including the extraction step. Finally, 6.8g of a sesquiterpenoid-enriched (STE) fraction was obtained from the crude extract (315.9g) of Tussilago farfara (1kg) in a single CCC run with a separation time of 8.5h. The sample injection capacity of CCC-DCI was greater than 300g; this amount of sample could not be handled in conventional CCC or other fractionation methods with the same column volume. Moreover, three major sesquiterpenoids (1: tussilagone, 2: 14-acetoxy-7β-(3′-ethyl cis-crotonoyloxy)-1α-(2′-methylburyryloxy)-notonipetranone, and 3: 7β-(3′-ethyl cis-crotonoyloxy)-1α-(2′-methylburyryloxy)-3, 14-dehydro-Z-notonipetranone) were purified from the STE fraction by CCC, and their chemical structures were elucidated by 1H NMR and 13C NMR. A quantification study was conducted, and the contents of compounds 1–3 in the CCC-DCI fraction were higher than those of conventional multi-step fractionations performed in series: solvent partitioning and open column chromatography. Furthermore, the average CCC-DCI recoveries were 96.1% (1), 96.9% (2), and 94.6% (3), whereas the open column chromatography recoveries were 77.7% (1), 66.5% (2), and 58.4% (3). The developed method demonstrates that CCC is a useful technique for enriching target components from natural products.

Selection of a nonaqueous two-phase solvent system for fractionation of xylo-oligosaccharide prebiotics using a conductor-like screening model for real solvents

ABSTRACTFractionation of xylo-oligosaccharides (XOS) using a liquid–liquid solvent system can be a difficult endeavor due to the high solubility of XOS in water. Use of a nonaqueous solvent system is a solution for XOS fractionation. XOS are gaining attention as a prebiotic food additive and are abundant in agricultural residues. We describe the use of a molecular modeling approach to determine which solvents and at what volume ratio to use for XOS fractionation. The conductor-like screening model for real solvents was used to predict the partition coefficients of xylose and two major functional XOS—xylobiose and xylotriose, based on the structure of the compounds and the composition of solvents in a panel of nonaqueous biphasic solvent systems. Eleven common solvents used in countercurrent chromatography were used to build 12 biphasic solvents model systems that were then evaluated and compared using shake flask experiments to determine which could fractionate the three XOS from a mixture. The model and experimental results indicate that a heptane/n-butanol/acetonitrile system at a volume ratio of 9:4:5 would result in a partition coefficient close to the region of optimal separation for a countercurrent chromatography fractionation.

Enantioseparation of (RS)-ibuprofen by closed recycling high-speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector

High-speed counter-current chromatography combined with closed recycling elution mode was developed to enantioseparate (RS)-ibuprofen by using hydroxypropyl-β-cyclodextrin (HP-β-CD) as the chiral selector. Key parameters for high-speed counter-current chromatography resolution including the concentration of HP-β-CD, the two-phase solvent system composition, equilibrium temperature, and the pH of aqueous phase were extensively investigated. Under the optimized conditions, the enantiomers of ibuprofen were successfully separated by preparative recycling high-speed counter-current chromatography and the resulting enantiopurity of each enantiomer was over 97.5% as determined by HPLC. Moreover, the recovery for (R,S)-ibuprofen from high-speed counter-current chromatography fractions was achieved in the range of 82–89%.

Synthesis, liquid chromatographic fractionation and partial characterization of polybrominated dibenzofuran congeners

Polybrominated dibenzofurans (PBDFs) are a class of highly toxic environmental contaminants which comprises 135 structurally different congeners. While the gas chromatographic separation and analysis of the most polychlorinated dibenzofurans (PCDFs) are well-documented, comparably little data is currently available in the case of PBDFs. In this study dibenzofuran was brominated to give a mixture of ∼40 PBDFs with one to seven bromine atoms. This synthesis mixture was fractionated by both countercurrent chromatography (CCC) with the solvent system n-hexane/toluene/acetonitrile and non-aqueous reversed-phase high performance liquid chromatography (RP-HPLC) with acetonitrile as the mobile phase. All together 80 consecutive CCC fractions and 40 HPLC fractions were taken and analyzed for PBDFs by gas chromatography coupled to mass spectrometry (GC/MS). CCC and RP-HPLC offered orthogonal separation of the PBDF mixture. As a consequence, selected CCC fractions were further fractionated by RP-HPLC. In this way, eight PBDFs could be isolated and the structures of twelve PBDFs were elucidated by proton magnetic resonance spectroscopy (1H NMR).

Fractionation of technical octabromodiphenyl ether by countercurrent chromatography combined with gas chromatography/mass spectrometry and offline and online 1H nuclear magnetic resonance spectroscopy

Countercurrent chromatography (CCC) is a technique, which uses two immiscible liquid phases for a separation process in a long and hollow tube. The technique allows the separation of high amounts of sample (50mg to several grams) with a low consumption of solvents. In this study, we fractionated 50mg technical octabromodiphenyl ether (DE-79) and analyzed the fractions by gas chromatography with mass spectrometry (GC/MS) and proton nuclear magnetic resonance (1H NMR) spectroscopy. CCC separations were performed with n-hexane/acetonitrile as solvent system in tail-to-head (i.e. the upper phase is mobile) mode. Twelve CCC fractions were studied for the PBDE composition. CCC elution of PBDE congeners was dependent both on the degree of bromination and substitution pattern. Higher brominated congeners eluted faster than lower brominated congeners and isomers with vicinal hydrogen atoms eluted last. In addition to several known PBDE congeners in DE-79, we were able to unequivocally identify BDE 195 in DE-79 and we could verify the presence of BDE 184. Finally, we also established the online hyphenation of CCC with 1H NMR. The use of deuterated solvents could be avoided by using n-hexane/acetonitrile as two-phase system. By online CCC-1H NMR in stop-flow mode we were able to detect eight PBDE congeners in the mixture.

Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High performance countercurrent chromatography fractionation of triterpene acids with off-line detection using atmospheric pressure chemical ionization mass spectrometry

‘Countercurrent chromatography’ (CCC) is an ideal technique for the recovery, purification and isolation of bioactive natural products, due to the liquid nature of the stationary phase, process predictability and the possibility of scale-up from analytical to preparative scale. In this work, a method developed for the fractionation of Schinus terebinthifolius Raddi berries dichloromethane extract was thoroughly optimized to achieve maximal throughput with minimal solvent and time consumption per gram of processed crude extract, using analytical, semi-preparative and preparative ‘high performance countercurrent chromatography’ (HPCCC) instruments. The method using the biphasic solvent system composed of n-heptane–ethyl acetate–methanol–water (6:1:6:1, v/v/v/v) was volumetrically scaled up to increase sample throughput up to 120 times, while maintaining separation efficiency and time. As a fast and specific detection alternative, the fractions collected from the CCC-separations were injected to an ‘atmospheric pressure chemical ionization mass-spectrometer’ (APCI-MS/MS) and reconstituted molecular weight MS-chromatograms of the APCI-ionizable compounds from S. terebinthifolius were obtained. This procedure led to the direct isolation of tirucallane type triterpenes such as masticadienonic and 3β-masticadienolic acids. Also oleanonic and moronic acids have been identified for the first time in the species. In summary, this approach can be used for other CCC scale-up processes, enabling MS-target-guided isolation procedures.

Isolation of achyrobichalcone from Achyrocline satureioides by high- speed countercurrent chromatography.

Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.

Preparative enantioseparation of β-blocker drugs by counter-current chromatography using dialkyl l-tartrate as chiral selector based on borate coordination complex

Counter-current chromatography (CCC) was applied for preparative enantioseparation of three β-blocker drugs, including propranolol, pindolol and alprenolol. The two-phase solvent system was composed of chloroform–0.05molL−1 acetate buffer containing 0.10molL−1 boric acid (1:1, v/v), in which 0.10molL−1 di-n-hexyl l-tartrate was added in the organic phase as chiral selector. Influence factors in the enantioseparation of propranolol were investigated. The chromatographic retention mechanism based on borate coordination complex was proposed. 116mg of racemic propranolol was completely enantioseparated using conventional high speed CCC in a single run, yielding 48mg of (+)-propranolol with HPLC purity of 98.9% and 47mg of (−)-propranolol with HPLC purity of 96.3%. Recovery for propranolol enantiomers from CCC fractions was in the range of 75–82%. pH-zone-refining CCC was also successfully applied in enantioseparation of propanolol and it was found that 356mg of racemic propranolol could be completely enantioseparated. 145mg of (+)-enantiomer with HPLC purity of 95.6% and 148mg of (−)-enantiomer with HPLC purity of 98.2% were recovered from pH-zone-refining mode. Separation mechanism about chiral separation by pH-zone-refining CCC was discussed.

Evaluation of different solvent systems for the isolation of Sparattosperma leucanthum flavonoids by counter-current chromatography

Three flavonoids – 2′,4′,6′-trihydroxy-4′-O-β-d-glucopiranosyl dihydrochalcone, 1, pinocembrin-7-O-(-neohesperidoside, 2 and pinocembrin-7-O-(-(6″-O-acetyl) neohesperidoside, 3 – were successfully isolated from the EtOAc extract of leaves of Sparattosperma leucanthum (Vell.) K. Schum (Bignoniaceae) using a two-step counter-current chromatography (CCC). Two different CCC machines were used, with different column axes (P.C. Inc., vertical orientation axis and AECS Quattro HTPrep, horizontal orientation axis). Detailed studies of flavonoids behaviour in several solvent systems made possible the use of the best system for their isolation. HEMWat and its modifications – exchange of alcohol and addition of a fifth solvent – were tested for isolation of the three compounds in a single run, but good K and α values were not achieved. So, HEMWat 4:10:4:10, with upper phase as mobile, was used to isolate compound 3. A mixture of compounds 1 and 2 was recovered and submitted to a new CCC fractionation using a more polar solvent system: EBuWat 8:2:10, upper phase as mobile. Butironitrile–acetonitrile–water (BuCN–ACN–H2O) 5:10:10, upper phase as mobile, was also used for the isolation of the mixture containing compounds 1 and 2, in order to increase the solubility of the compounds in the CCC solvent system. It is the first time that the system BuCN–ACN–H2O is described in literature.

Multi-channel counter-current chromatography for high-throughput fractionation of natural products for drug discovery

A multi-channel counter-current chromatography (CCC) method has been designed and fabricated for the high-throughput fractionation of natural products without complications sometimes encountered with other conventional chromatographic systems, such as irreversible adsorptive constituent losses and deactivation, tailing of solute peaks and contamination. It has multiple independent CCC channels and each channel connects independent separation column(s) by parallel flow tubes, and thus the multi-channel CCC apparatus can achieve simultaneously two or more independent chromatographic processes. Furthermore, a high-throughput CCC fractionation method for natural products has been developed by a combination of a new three-channel CCC apparatus and conventional parallel chromatographic devices including pumps, sample injectors, effluent detectors and collectors, and its performance has been displayed on the fractionation of ethyl acetate extracts of three natural materials Solidago canadensis, Suillus placidus, and Trichosanthes kirilowii, which are found to be potent cytotoxic to tumor cell lines in the course of screening the antitumor candidates. By combination of biological screening programs and preparative high-performance liquid chromatography (HPLC) purification, 22.8 mg 6β-angeloyloxykolavenic acid and 29.4 mg 6β-tigloyloxykolavenic acid for S. canadensis, 25.3 mg suillin for S. placidus, and 6.8 mg 23,24-dihydrocucurbitacin B for T. Kirilowii as their major cytotoxic principles were isolated from each 1000 mg crude ethyl acetate exctract. Their chemical structures were characterized by electrospray ionization mass spectrometry, one- and two-dimensional nuclear magnetic resonance. The overall results indicate the multi-channel CCC is very useful for high-throughput fractionation of natural products for drug discovery in spite of the solvent balancing requirement and the lower resolution of the shorter CCC columns.