Counter-current Chromatography Research Articles

Separation and Purification of Ginsenosides and Flavonoids in From the Leaves and Stems of Panax quinquefolium by High-Speed Countercurrent Chromatography and Online-Storage Inner-Recycling Countercurrent Chromatography.

Study aimed to isolate and purify compounds from the stems and leaves of Panax quinquefolius. By employing a highly innovative separation technique that combined multistage countercurrent chromatography (MRCC), high-speed countercurrent chromatography (HSCCC), and an advanced online-storage inner-recycling countercurrent chromatography (OS-IRCCC) mode for the first time, 12 compounds were successfully isolated, including 10 ginsenosides and 2 flavonoids. First, the crude extract was fractionated into five parts using D101 MRCC, with HPLC analysis revealing that 40% and 60% ethanol eluate contained the highest compound diversity. Overall, 40% ethanol eluate was separated using the solvent system of EtOAc/n-BuOH/H2O (2:1:3, v/v), whereas 60% ethanol eluate underwent traditional countercurrent chromatography coupled with OS-IRCCC separation using the solvent system of methyl tert-butyl ether (MTBE)/n-BuOH/ACN/H2O (4:2:3:8, v/v). Ultimately, various compounds were obtained, including kaempferol 3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-7-O-α-l-rhamnopyranoside (13.2mg), ginsenoside Rc (7.4mg), 20(R)-ginsenoside Rh1 (7.2mg), ginsenoside Re (12.3mg), kaempferol 3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranoside (14.1mg), ginsenoside Rb1 (8.2mg), ginsenoside Rb2 (17.5mg), ginsenoside Rb3 (27.3mg), ginsenoside Rg1 (13.3mg), ginsenoside Rg2 (9.7mg), ginsenoside Rd (11.4mg), and pseudo-ginsenoside F11 (16.7mg). This research highlights the efficacy of the novel separation technique in isolating and purifying valuable compounds from P. quinquefolius stems and leaves.

Recent Progress in Curcumin: Extraction, Purification, and Bioactivity.

Curcumin is a natural plant pigment that has been widely used in food production, drug development, and textile engineering. Gaining a deep understanding of the biological activities of curcumin and obtaining high-purity curcumin are of vital importance for basic research and applications of curcumin. In this review, we summarize recent advances in curcumin, mainly focusing on the methods of extracting and purifying curcumin from turmeric as well as applications based on biological activity. We systematically describe the advantages and disadvantages of traditional and modern extraction technologies. The usual purification methods include conventional methods (such as macroporous resin column chromatography and silica gel column chromatography, etc.) and auxiliary modern technologies (such as high-speed countercurrent chromatography and supercritical fluid chromatography). In terms of biological activity, the phenolic hydroxy group and methoxy group of curcumin are closely related to its antioxidant activity, endowing it with strong antibacterial, anti-inflammatory, and antitumor properties. Moreover, the development direction based on its multiple biological activities is also discussed.

Isolation, Characterization, and Validation of Amide Alkaloids from Piper Guineense Seeds

Introduction: Alkaloids are naturally occurring nitrogen-containing chemicals in plants, fungi, and mammals. These chemicals protect plants from herbivores and pathogens and have a wide range of biological actions, making them useful in pharmacology and medicine. Alkaloids offer potent therapeutic effects, including antifungal, local anaesthetic, anti-inflammatory, antineoplastic, pain-killing, neuropharmacological, and antibacterial properties. Morphine is commonly used to relieve pain, quinine for malaria, and vincristine for cancer. Because of their considerable therapeutic potential, alkaloids have become a focus of study in the development of medications to treat infections, inflammation, neurological diseases, and cancer. Objective: The primary goal of this research is to isolate, purify, and validate amide alkaloids from the seeds of PIPER guineense, a West African species while contrasting traditional alkaloid extraction methods with novel techniques such as molecular distillation, membrane separation, molecular imprinting, and high-speed counter-current chromatography. The study aims to address the challenges posed by conventional methods' high cost and low yield, by providing insights into more efficient, scalable, and advanced technologies for alkaloid extraction and purification, which are critical for the advancement of complementary and Chinese medicine. Materials and Methods: The seeds of Piper guineense were extracted using a Soxhlet device with methanol as the solvent. The extracted extract was purified and separated into amide alkaloids using column chromatography. Thin-layer chromatography (TLC) was used to confirm the presence of alkaloids. The isolated compounds were examined and characterized using an array ofspectrum methods, including Mass spectra, FTIR, and NMR spectroscopy. Natural substances with nitrogen in the exocyclic outlook, such as mescaline, serotonin, & dopamine, were recognized as amines rather than alkaloids in the investigation. Results: The synthesized chemicals, including Piperine, Wisanine, Piperlonguminine, Propiverine, and Piperidine, were satisfactorily characterized using FTIR, NMR, mass spectrometry, and HPLC. The FTIR spectra showed distinct peaks whichwere consistent with conventional standards, demonstrating the effective synthesis. NMR research, performed with a BRUKER AVANCE NEO-500MHz spectrometer, revealed information on the chemical surroundings of the protons, confirming the molecular structure. Mass spectroscopy was done using a MICROMASS Q-TOF micro-mass spectrometer to precisely measure molecular weights and identify fragmentation patterns, verifying the existence of important functional groups and assuring the structural integrity of the compounds. Conclusions: This work effectively extracted, purified, and characterized numerous amide alkaloids from Piper guineense seeds using modern chromatographic and spectroscopic methods. The compounds, which included Piperine, Wisanine, Piperlonguminine, Propiverine, and Piperidine, were validated using FTIR, NMR, mass spectroscopy, and HPLC to ensure structural integrity and composition. The study reveals the efficacy of contemporary extraction and analytical procedures, offering useful insights into how to improve alkaloid purification processes. These discoveries advance our understanding of alkaloid chemistry and have the potential for future uses in alternative medicine and pharmaceutical research.

Screening of Solvent Systems for Countercurrent Chromatography Separation of Polar Constituents from Ginkgo biloba L. Seeds.

The separation of large polar constituents presents a substantial challenge in natural product research when employing column chromatography techniques, as the process is both complex and time-consuming. In this study, an acetonitrile/tetrahydrofuran/di-(2-ethylhexyl) phosphoric acid/aqueous saturated sodium chloride solvent system was developed and utilized for the countercurrent chromatography of polar constituents from Ginkgo biloba L. seeds. Five polar constituents were effectively isolated using an acetonitrile/tetrahydrofuran/di-(2-ethylhexyl) phosphoric acid/aqueous saturated sodium chloride (2:2:0.8:3, v/v) solvent system using a two-step countercurrent chromatography method. In the initial countercurrent chromatography process, three constituents were successfully purified from the methanol extract: compound 1, compound 4, and compound 5. Compounds 2 and 3, co-eluted from the column, were further subjected to three inner-recycling chromatographic procedures. At last, five constituents were purified and identified, including 4'-O-methylpyridoxine (1); two indole alkaloid N-glucosides, ginkgoside B (2) and ginkgoside A (3); 2-(4-hydroxybenzyl) malic acid (4); and coniferyl alcohol (5). The results demonstrated that the acetonitrile/tetrahydrofuran/di-(2-ethylhexyl) phosphoric acid/aqueous saturated sodium chloride solvent system serves as a feasible system for the efficient countercurrent chromatography separation of polar components.

Separation of Three Polar Compounds with Similar Polarities from Swertia mussotii by a Combination of Two Counter-Current Chromatography Modes.

Separation of polar compounds especially with similar polarities is challenging. In the present study, three polar compounds with similar polarities, including gentiopicroside, sweroside and mangiferin, have been successfully separated from Swertia mussotii by a combination of two counter-current chromatography modes. With the selected solvent system of ethyl acetate/n-butanol/water (8/2/10, v/v), a continuous injection mode was firstly employed. As a result, 10.4g of mangiferin with purity higher than 98% and 8g of Peak I mainly composed of gentiopicroside and sweroside could be easily obtained from 24g of the sample by counter-current chromatography through eight injections. Then, a recycling counter-current chromatography mode was introduced for the separation of gentiopicroside and sweroside. Through six cycles, 5.3g of gentiopicroside and 0.8g of sweroside with purity higher than 98% were obtained from 8g of Peak I. The research provides a reference for the large-scale separation of polar compounds with similar polarities.

Harnessing the Power of Black Rice Anthocyanins: A Review in Context of Extraction and Chromatographic Techniques

Black rice, a well‐known grain for its anthocyanin‐rich pigmentation and nutritional benefits, has gained significant attention in scientific research. Anthocyanins, flavonoid pigments responsible for the distinctive coloration of black rice, offer potent antioxidant and health‐promoting properties. Extraction and chromatographic techniques play pivotal roles in isolating and analyzing these valuable compounds. This review comprehensively examines the extraction methodologies, encompassing traditional solvent extraction and advanced techniques such as ultrasound‐assisted extraction and supercritical fluid extraction. In addition, chromatographic techniques, including high‐performance liquid chromatography (HPLC), high‐speed countercurrent chromatography (HSCCC), and preparative HPLC (Prep‐HPLC) are demonstrated in detail for precise identification and quantification of anthocyanins in black rice.

Antifungal Properties of Bioactive Compounds Isolated from Fucus vesiculosus Supercritical Carbon Dioxide Extract.

The exploration of natural antifungal substances from algal origins is significant due to the increasing resistance of pathogens to conventional antifungal agents and the growing consumer demand for natural products. This manuscript represents the inaugural investigation into the antifungal attributes of bioactive compounds extracted from Fucus vesiculosus via supercritical carbon dioxide (scCO2) extraction utilizing contemporary countercurrent chromatography (CCC). In aligning with the prospective utilization of this extract within the agricultural sector, this study also serves as the preliminary report demonstrating the capability of Fucus vesiculosus scCO2 extract to enhance the activity of plant resistance enzymes. The fractions obtained through CCC were subjected to evaluation for their efficacy in inhibiting the macrospores of Fusarium culmorum. The CCC methodology facilitated the successful separation of fatty acids (reaching up to 82.0 wt.% in a given fraction) and fucosterol (attaining up to 79.4 wt.% in another fraction). All CCC fractions at the concentration of 1.0% were found to inhibit 100% of Fusarium culmorum growth. Moreover, Fucus vesiculosus scCO2 extract was able to activate plant resistance enzymes (Catalase, Ascorbic Peroxidase, Guaiacol Peroxidase, Phenylalanine Ammonia-Lyase, and Phenylalanine Ammonia-Lyase Activity).

Phytochemical Profile and Biological Activity of the Ethanol Extracts from the Aerial Parts of Adonis tianschanica (Adolf.) Lipsch. Growing in Kazakhstan.

Adonis tianschanica is a lesser-known plant species belonging to the genus Adonis that grows in Kazakhstan. The aim of this study was to characterize the composition of the ethanolic, water, and hydroethanolic extracts from the aerial parts of A. tianschanica by HPLC-ESI-QTOF-MS/MS to isolate the major compound isoquercitrin by HSCCC (High-Speed Counter-Current Chromatography) and to determine the cytotoxicity and anti-inflammatory potential of the extracts produced with this plant. Fingerprinting of the analyzed extracts showed the presence of a multitude of metabolites comprising polyphenols, organic acids, and coumarins, and only trace quantities of cardiac glycosides in the analyzed samples. Flavonoids were certainly the best-represented group, with kaempferol, quercetin, and their derivatives as the major components of the extracts. Key findings in this paper were that the ethanol: water (50:50 v/v) extract of A. tianschanica and its major compound isoquercitrin were able to reduce the production of NO induced by LPS, in addition to demonstrating anti-inflammatory effects by reducing cytokines such as IL-6, TNF-α, and IL-1β.

Recent Advances in Screening and Separating Active Components From Natural Products Based on High-Speed Countercurrent Chromatography.

Natural products play a vital role in human health because people find that they can reduce the incidence of many diseases, such as cancer and cardiovascular diseases. Therefore, researchers have developed many effective techniques for screening and separating active compounds from natural products. High-speed countercurrent chromatography is a unique unsupported liquid-liquid chromatography technology that has been widely used in the separation of natural products. In this paper, the research progress of screening and separating active components from natural products by combining high-speed countercurrent chromatography technology with other technologies is reviewed from two aspects: pre-column activity assay and post-column activity assay. The purpose of this paper is to provide some theoretical support for the screening and separation of natural active compounds by high-speed countercurrent chromatography. I hope that the contents summarized in this paper can provide more basis for the development of screening and separation of natural active compounds to realize large-scale preparation.

A HPLC-assisted mathematical prediction method of ternary solvent systems to develop an intelligent online selection system for countercurrent chromatography solvent system.

Countercurrent chromatography (CCC) is an efficient technique for purifying bioactive natural compounds, but selecting the solvent system can be a time-consuming and crucial process for successful separation. This paper discussed the HPLC-assisted mathematical prediction method for the n-hexane/alcohol solvents/water (HAWat) and ethyl acetate/n-butanol/water (EBuWat) systems and designed an intelligent online selection system to simplify the separation process. First, the applicable rage of HAWat and EBuWat solvent systems were quantified by the methanol concentration at the column inlet when template molecules peak in a HPLC analysis (B%). Then, we analyzed the relationship between the logarithmic partition coefficient (log K) of template molecules and the alcohol solvent ratio by using different brands of chromatography columns. Moreover, the prediction function was developed by correlating the best solvent composition of CCC solvent system with the B% of template molecules. The results indicated that the HAWat system was suitable for separating compounds with B% > 85 %, while the EBuWat system was effective for compounds with 40 % < B% < 55 %. The quadratic function for HAWat systems and one universal EBuWat system (8.2:1.8:10) were determined and verified to develop the HPLC-assisted mathematical prediction function method. The method could select suitable solvent systems for separfigation easily and quickly. And a step of result correction was required for the occasional discrete value. Finally, an intelligent CCC online selection system that integrates established mathematical functions was designed to enable real-time solvent selection, on-demand solvent configuration, and automated sample separation. It eliminates the iterative process of shake-flask experiments and facilitates intelligent upgrading of CCC instrumentation.

Comparison of Different High-Speed Countercurrent Chromatography Injection Modes for the Separation of Glabridin.

In this work, different injection modes (single injection, overlapping injection, and continuous injection) of countercurrent chromatography were used and compared for the isolation and purification of glabridin from the crude extract of Glycyrrhiza glabra. The two-phase solvent system consisting of n-hexane/ethyl acetate/methanol/water (5:4:5:4, v/v) was employed as stationary and mobile phases. Compared with single injection mode, both overlapping injection and continuous injection modes exhibited higher separation efficiency at the same sample loading capacity. Their total separation times were 89.68% and 53.97% of the single injection mode, and the total solvent consumption were 74.88% and 52.49% of the single injection mode, respectively. Meanwhile, the yield, purity, and recovery rate of these three injection modes were almost the same. It was demonstrated that high-speed countercurrent chromatography in continuous injection mode was the most effective way for separating glabridin in three methods and it can be further applied to the separation of other economically valuable natural products.

Lipid removal by countercurrent chromatography in co-current mode in trace analysis of polyhalogenated compounds in fish

Persistent organic pollutants (POPs) are ubiquitously present in food and environmental samples. Accompanied by similar compounds like halogenated natural products (HNPs), their regular monitoring in fish and their predators is an important task. While different procedures have been developed, the removal of the lipid matrix, mainly carried out by treatment with sulfuric acid or by gel permeation chromatography (GPC), is an indispensable step during sample preparation. Here, we present an alternative, non-destructive method using countercurrent chromatography operated in the co-current mode (ccCCC) for the removal of lipids from the fraction of polyhalogenated compounds in fish. The final method was developed using various POP and HNP standards as well as the most critical lipid compound, i.e. free docosahexaenoic acid (DHA). The recovery rates of polyhalogenated compounds ranged between 79 and 117 %. Applied to six herring (Clupea harengus) fillet samples from the North, the Baltic, and the Norwegian Seas, the results obtained with the new (using ccCCC for lipid removal) method agreed well with those determined with an established method using GPC. The quantitative results indicated that fish from contaminated areas is still a major source in the human uptake of POPs. HNP levels were on average only ∼two times lower than POPs which underscores that they should be monitored on a more regular basis.

Extraction and Counter-Current Separation of Phenylpropanoid Glycosides from Pedicularis oederi Vahl by Deep Eutectic Solvent

Deep eutectic solvents (DESs) are mixtures of organic compounds displaying excellent solvent properties while keeping an ecofriendly character. In this study, DESs have been applied to the extraction of phenylpropanoid glycosides from Pedicularis oederi Vahl, successively separated by means of counter-current chromatography. Firstly, the ultrasonic-assisted extraction conditions were optimized by response surface methodology, and the results showed phenylpropanoid glycosides could be well extracted under the optimized extraction conditions with deep eutectic solvents. Then, the sample was separated by counter-current chromatography using ethyl acetate/aqueous solution of choline chloride and glycerol (6:6, v/v) as the solvent system. In about 360 min, four phenylpropanoid glycosides, including 31.6 mg of echinacoside, 65.3 mg of Jionoside A1, 28.9 mg of Forsythoside B, 74.1 mg of verbascoside, and 21.2 mg of kaempferol-3-O-rutinoside were obtained from about 900 mg of the sample. It revealed deep eutectic solvents could be well employed as a green solvent for the extraction and counter-current separation of natural products.

HSCCC Straightforward Fast Preparative Method for Isolation of Two Major Cytotoxic Withanolides from Athenaea fasciculata (Vell.) I.M.C. Rodrigues & Stehmann.

Athenaea fasciculata belongs to the Solanaceae family and is a promising source of cytotoxic withanolides known as aurelianolides A and B. In the last years, the pharmacological studies of these aurelianolides on different leukemia cell lines have stimulated new studies on their potential as alternative candidates for new lead anticancer drugs. However, the obtention of these two pure compounds by traditional preparative is a costly and long time-consuming process, which is performed in several steps. This study aimed to propose a straightforward approach for isolating aurelianolides A and B using high-speed countercurrent chromatography (HSCCC). In this study, among 10 different solvent systems, the system composed of n-hexane/ethyl acetate/methanol/water 3:6:2:1 (v/v/v/v) was chosen for optimization. This HEMWat system was optimized to 4:8:2:4 (v/v/v/v) and chosen for HSCCC separation in a tail-to-head elution mode. After the HSCCC scale-up procedure, a withanolides mixture (200.0 mg) was separated within 160 min in a single-step purification process. In total, 78.9 mg of aurelianolide A (up to 95.0% purity) and 54.3 mg of aurelianolide B (up to 88.5% purity) was obtained by this fast sequential liquid-liquid partition process. The isolated withanolides were identified by 1H and 13C NMR spectroscopy (this method has proven to be faster and more efficient than classical procedures (CC and Prep-TLC)).

A modeling method based on incomplete mixed cells in counter-current chromatography: Interphase mass transfer ratio

Complex mass transfer processes are highly involved in the performance of counter-current chromatography (CCC), so profound exploration should be conducted to improve the overall performance. To more accurately estimate the interphase mass transfer efficiency in the elution process, this study proposes an incomplete mixed cell model by linking layering-mixing-layering behavior and wave-like mixing. With the model, we proposed a fast estimation algorithm for interphase mass transfer ratio (IMTR), which can independently reflect the magnitude of the interphase mass transfer efficiency. The experimental results show that the IMTR of all samples was relatively small, with an average value of not >10 % under different operating conditions. This indicates that the low column efficiency of CCC may be mainly due to the low efficiency of interphase mass transfer. In addition, by analyzing the influencing factors of IMTR, it is found that IMTR is positively correlated with the number of theoretical plates and the retention of stationary phase (Sf), but negatively correlated with the number of incomplete mixing cells (n). Therefore, in the study of column efficiency optimization, we recommend that the distribution ratio, Sf, and n should remain unchanged in control experiments to ensure that IMTR directly reflects the effect on interphase mass transfer efficiency. This study further completes the evaluation index of the CCC column efficiency, revealing the underlying mechanisms of IMTR on column efficiency and providing a theoretical foundation for future research on high-efficiency CCC.

Preparation of esculin acetates through transesterification reaction catalyzed by Novozyme 435® and their Purification followed by NMR characterization

In this study, biocatalytic transesterification reaction using Novozyme 435® (N435) lipase was employed to enhance the hydrophobicity of esculin, aiming to improve its solubility for commercial applications and enhance its bioactivity and oral viability. The acylation reaction of esculin with vinyl acetate was conducted at 60 °C and 200 rpm for 24 h. After chromatographic and spectroscopic analysis, two products were identified: the first one was monoacylated at the 6′-OH position of the glucosyl moiety of esculin (TR: 10.3 min and m/z 382.93 [M + H]+), and the second one was diacylated at the 6′-OH and 3′-OH positions (TR: 13.0 min and m/z 424.93 [M + H]+). The latter was the major product, with a conversion rate of 53.550 ± 0.368%, while the monoacetylated one showed 8.715 ± 0.064%. Both products were isolated by high-speed counter-current chromatography (HSCCC) using a two-phase system HEMWat 1:9:1:9 and characterized by NMR. In this way, these results improve the practical application of esculin, through the obtention of esculin mono and diacetates by fast and efficient biocatalysis reaction.

Plukenetia volubilis leaves as source of anti-Helicobacter pylori agents.

Helicobacter pylori infection is a major issue worldwide, with widespread prevalence, combined with its link to gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Meanwhile, effectiveness of current treatment protocols is limited by increasing antibiotic resistance and patient compliance issues due to long regimens and side effects. Plukenetia volubilis, or sacha inchi, is a valuable source of bioactive molecules. However, studies on its antimicrobial activity, especially against H. pylori, are lacking. In this study, the anti-H. pylori activity of P. volubilis leaves water extract was explored using in vitro and in silico approaches. High-Performance Liquid Chromatography coupled to Electrospray Ionisation and Quadrupole Time-of-Flight Mass Spectrometry (HPLC-ESI- QTOF-MS-MS) analysis of the water extract from the leaves was used to characterise the chemical composition of the plant and allowed identification of some flavonoids, such as astragalin, and some phenolic compounds. Then, high-speed counter current chromatography (HSCCC) was used to fractionate the ethyl acetate partition obtained from the water extract from the leaves. The presence of flavonoids derived from kaempferol was confirmed and astragalin was isolated for the first time in P. volubilis. The P. volubilis water infusion, ethyl acetate extract and the isolated astragalin exhibited anti-bacterial activity against H. pylori J99 and two clinical isolates (e.g., minimum inhibitory concentrations of 0.53, 0.51 and 0.49μg/mL, respectively, for clarithromycin-resistant clinical isolate SSR366). Then, using molecular docking for potential protein targets for H. pylori, it was verified that astragalin could interact with these proteins by in silico analysis. These findings highlight that P. volubilis and astragalin produce a bacteriostatic activity against H. pylori and may have potential to be used in treatment against H. pylori, after further research.

A New Megastigmane Nor-sesquiterpene Isolated from Pourouma Guianensis by High-Performance Countercurrent Chromatography

Introduction: Pourouma guianensis (Urticaceae) is widespread throughout Brazil and is commonly known as “mapati”. Terpenes are common in Urticaceae species. However, few studies have reported the isolation and characterisation of these compounds in Pourouma genus. Method: The aim of this study was to separate substances from stem barks of P. guianensis using a high-performance countercurrent chromatography gradient elution method. The dichloromethane extract of the P. guianensis stem bark was submitted to silica gel column chromatography, yielding three fractions. Fraction 3 was submitted to a gradient elution system in high-performance countercurrent chromatography using two solvent systems, hexane/ethyl acetate/methanol/water (1:2:x:1, x=1.0 and 1.5). Six compounds were isolated. Results: A new megastigmane nor-sesquiterpene named 2-acetyl-4,7-epoxy-3-oxy-5,5,9- trimethylhexahydrocoumaran (1) was obtained together with five known terpenes: vomifoliol (2), 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (3), 3-β-D-arabinopyranoside-19-hydroxyurs-12-en- 28-oic acid (4), arjunolic acid (5), and tormentic acid (6). Fractions 1 and 2 were purified by binary gradient elution from hexane to ethyl acetate. Conclusion: This process isolated three known compounds: friedelin (7) and 3β-friedelanol (8) from fraction 1, and β-sitosterol (9) from fraction 2. Gradient elution in countercurrent chromatography was a suitable alternative, exploiting variations in the partition coefficient between substances with small structural differences.

Separation and Characterization of Wenjin Tongluo San Essential Oil with a Comprehensive Chromatographic Separation

The essential oil components of traditional Chinese medicine in-hospital preparation were complex, and one-dimensional chromatographic separation was difficult to completely separate them due to the limited peak capacity. This study was carried out to establish a comprehensive two-dimensional chromatographic separation and analysis method based on countercurrent chromatography (CCC) and gas chromatography (GC). In this paper, we focused on the separation of the essential oil of the traditional Chinese medicine in-hospital preparation Wenjing Tongluo San by CCC × GC, and explored the orthogonality between the two chromatographic techniques to provide the new technical support for the screening of the active ingredients. A solvent system composed of n-hexane-ethyl acetate-methanol-water (9.5:0.5:8.5:1.5, v/v) was chosen for the first-dimensional CCC separation. All the fractions collected from CCC were transferred to GC for plotting two-dimensional contours map. The calculated capacity of the two-dimensional separation system exceeded 3000, which was 8 times more than that of the one-dimensional separation system. High orthogonality (r = 0.42) and spatial coverage factor (70.42%) were obtained. Meanwhile, all the fractions were identified by GC-MS. Our research provided a new methodology for separating essential oils in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal in-hospital preparation based on two-dimensional chromatographic fingerprints.

A new consecutive high-performance counter-current chromatography approach based on co-current and overlap mode for isolating active compounds from olive leaves

Conventional consecutive separation methods based on overlapping injection modes produce interactions between samples as the separation time increases. This will lead to a reduction in the purity of the target due to the gradual saturation of the stationary phase distribution. To enhance the purity of the sample preparation and separation efficiency, we devised a consecutive separation method under high-performance counter-current chromatography (HPCCC) utilizing co-current elution and overlapping injection. The method was used for the separation and preparation of olive leaves for their main bioactive polyphenolic constituents. Six HPCCC separations were conducted in reversed-phase mode using a two-phase solvent system consisting of petroleum ether/ethyl acetate/water (1:40:40, v/v). Finally, 302.7 mg of luteolin-4’-O-β-D-glucoside and oleuropein were isolated from 720 mg of crude sample with purities of 93.3% and 96.2%, respectively. The optimization strategy outlined in this study can be used to enhance the extraction of bioactive components from olive leaves.