Counter-current Chromatography Separation Research Articles

Comprehensive separation of iridoid glycosides and triterpenoid saponins from Dipsacus asper with salt-containing solvent by high-speed countercurrent chromatography coupled with recycling mode.

The roots of Dipsacus asper Wall as a commonly used traditional Chinese medicine are used for tonifying liver and kidney and strengthening bones and muscles. However, an effective separation strategy for comprehensive and rapid separation of the main active compounds from the roots of D. asper is nonexistent. This investigation provided an effective separation method based on AB-8 macroporous resin column chromatography using different ratios of ethanol in water and two different modes of high-speed countercurrent chromatography with salt-containing solvent system for rapid enrichment and separation from the roots of D. asper. The macroporous resin column chromatography was performed on AB-8 resin using ethanol in water ratios of 10, 30, 40, 50, and 80% as the optimized enrichment conditions for iridoid glycosides and triterpenoid saponins with different polarities. For high-speed countercurrent chromatography separation, the conventional and recycling modes were combined together to develop a strategy for 12 compounds (1-12) from the enriched parts of 30, 40, and 80% ethanol, including six high-polarity iridoid glycosides (1-6) using inorganic salt-containing solvent system and six triterpenoid saponins (7-12). Recycling high-speed countercurrent chromatography separation was successfully applied to separate two isomers (9 and 10) after 11 cycles.

Separation of acteoside and linarin from Buddlejae Flos by high‐speed countercurrent chromatography and their anti‐inflammatory activities

Buddleja officinalis Maxim., a deciduous, flowering shrub, is used as a traditional Chinese medicine; the bioactivity of B. officinalis is primarily due to flavonoids and phenylethanoid glycosides. In the study, acteoside and linarin were successfully isolated from B. officinalis by high-speed countercurrent chromatography with a two-phase solvent system composed of ethyl acetate: n-butanol: water (5:0.8:5, v/v/v). The purities of acteoside and linarin were determined to be 97.3 and 98.2%, respectively, using one-step high-speed countercurrent chromatography separation. The chemical structures of the two compounds were identified by electrospray ionization-mass spectrometry and nuclear magnetic resonance. After separation, the anti-inflammatory effects of the two compounds were evaluated using lipopolysaccharide-induced human umbilical vein endothelial cells. Acteoside and linarin inhibited the expression of nitric oxide, tumor necrosis factor α and interleukin 1β, which demonstrated that acteoside and linarin possessed anti-inflammatory activity.

High-Speed Counter-Current Chromatography in Separation and Identification of Saponins from Beta vulgaris L. Cultivar Red Sphere

High-Speed Counter-Current Chromatography in Separation and Identification of Saponins from Beta vulgaris L. Cultivar Red Sphere

A biphasic system based on guanidinium ionic liquid: Preparative separation of eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester by countercurrent chromatography

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are high nutritional components. Evidence for unique effects of them is increasing. Further understanding of their independent biological functions urgently needs more efficient separation techniques. Nowadays, most of the commercially available fish oil products are the mixture of eicosapentaenoic acid ethyl ester (EPAEE) and docosahexaenoic acid ethyl ester (DHAEE). It will be convenient to directly separate esterified EPA and DHA without saponification pretreatment. However, it is of great challenge to separate EPAEE and DHAEE because of their extremely fat-soluble nature and the equivalent chain length rule. In this research, the suitability of green guanidinium ionic liquid (IL) in countercurrent chromatography (CCC) solvent system for the separation of them was evaluated for the first time. Compared with imidazolium IL and phosphonium IL, guanidinium IL based non-aqueous biphasic system showed more outstanding separation performance. The separation mechanism was elucidated in depth through quantum mechanical calculations. It was found that guanidinium IL acted a crucial role in the CCC separation, which resulted in difference of partition behavior of EPAEE and DHAEE via different hydrogen-bonding affinity. EPAEE and DHAEE were successfully separated by solvent system (n-heptane/methanol/propylguanidinium chloride ([C3Gun]Cl, 1:1:5%, v/v/m)) with high purity (>95%) in one step, which was not achieved beforehand. Moreover, an easy recycling procedure of IL had also been devised, which significantly reduced waste generated. It opens up a new way for reasonable design water-free two-phase solvent system for efficient separation of very non-polar lipid compounds.

Efficient separation of tocopherol homologues in vegetable oil by ionic-liquid-based countercurrent chromatography using a non-aqueous biphasic system.

Tocopherol homologues are important fat-soluble bioactive compounds with high nutritional value. However, it is of great challenge to separate these homologues because of their high structural similarities. In this work, ionic-liquid-based countercurrent chromatography was used for the separation and purification of tocopherol homologues. Conventional countercurrent chromatography and ionic-liquid-based countercurrent chromatography solvent systems were evaluated in respect of partition coefficient, separation factor, and stationary phase retention factor to separate these targets. Kind of ionic liquids, amount of ionic liquid, and sample amount were systematically optimized. A novel countercurrent chromatography non-aqueous biphasic system composed of n-hexane-methanol-1-butyl-3-methylimidazolium chloride was established. The baseline separation of tocopherol mixtures was obtained in one cycle process. The ionic liquid played a key role in the countercurrent chromatography separation, which resulted in difference of partition behavior of individual tocopherol in the whole system through different hydrogen-bonding affinity. Finally, n-hexane-methanol-1-butyl-3-methylimidazolium chloride (5:5:3, v/v) water-free biphasic system was successfully applied to separate tocopherol homologues from vegetable oil that was not achieved beforehand. This method can be widely employed to separate many similar molecules such as tocotrienols, tocomonoenols, and marine-derived tocopherol in food samples.

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.

Modeling of two semi-continuous methods in liquid–liquid chromatography: Comparing conventional and closed-loop recycling modes

Two high throughput steady-state methods of counter-current chromatography separations: conventional (SS CCC) and closed-loop recycling (SS CLR CCC) are proposed, evaluated and compared. The methods are based on the application of semi-continuous sample loading technique: the CCC setup includes two mobile phase tanks – one with the pure mobile phase and the second – with the sample solution in the mobile phase. The mobile phase pump is periodically switching from one tank to another. The sample solution is continuously loaded into the CCC column over a constant time with the constant volumetric rate equal to the flow rate of the pure mobile phase.Analytical expressions are developed to describe the SS CCC and SS CLR CCC separations with semi-continuous sample loading. Examples of separation of binary and multicomponent mixtures are discussed. The SS CLR CCC has been shown to provide a multiple increase in the performance and effectiveness of CCC devices. Several examples of simulation of SS CLR CCC separation with semi-continuous sample loading are presented in “Mathcad” program.For the experimental verification of the theory, the separation of the binary mixture caffeine/ coumarin was studied. The biphasic solvent system hexane/isopropanol/water (1:1:1), was used. The comparison of experimental and simulated separations demonstrated a reasonable agreement between theory and experiment.

Visualization of Type-J counter current chromatography: Hydrodynamic behavior in a helical column

In counter-current chromatography (CCC), the hydrodynamic motion of a two-phase solvent system in the column/coil is very important. There are some previous visualization studies on CCC using stroboscopic photography. As CCC separation is a continuous liquid-liquid extraction process, observing the distribution and movement of the solvent system on-line will be helpful to understand the hydrodynamic behavior during the whole CCC separation process. In the present study, a high-speed imaging camera was employed to take videos of a running CCC bobbin (visualized continuously on-line). The dynamic motion and phase distribution of conventional quaternary solvent system hexane/ethyl acetate/methanol/water (HEMWat) and ternary solvent system dichloromethane/methanol/water were both investigated. Wave-like mixing was observed in the area where the centrifugal force is minimum in different diameter columns. When the coil rotated, the mixing zone was “fixed” at the minimum centrifugal force position. Several photographs of the rotating coiled tube were taken which revealed that the phase dispersion hardly changes once equilibrium is established. Finally, the sample dispersion process was also recorded. These results will help us to understand the separation process in a CCC column/coil and also present some more interesting questions related to separation efficiency which are shown in discussion part of this paper.

Methanol linear gradient counter-current chromatography for the separation of natural products: Sinopodophyllum hexandrum as samples

Counter-current chromatography (CCC) is a unique, liquid-liquid partition chromatography process. Both the mobile and stationary phases are liquids, so no solid support matrix is used. CCC has gained wide acceptance as a preparative technique in a variety of fields. Because the mobile and stationary phases are both liquids, gradient elution is difficult to perform with CCC. Phase equilibrium must be maintained, so any change in the composition of one phase may induce a compositional change in the other. In this work, a new linear gradient elution method was developed for CCC. Biphasic solvent systems containing heptane, ethyl acetate, methanol, and water (HepEMWat) in various ratios were prepared and used to optimize both isocratic and linear gradient CCC separation with methanol. We first separated a test mixture of four standard compounds with partition coefficients ranging from 0.8 to 7.8. The separation resembled a reversed-phase process, and elution was performed while progressively decreasing the polarity of the mobile phase. Target molecules with small partition coefficients eluted first in the lower phase of the optimized HepEMWat solvent system. Elution of constituents with large partition coefficients was quite slow under isocratic conditions. Separation time was significantly reduced when elution was performed with a linear gradient using methanol and the optimal HepEMWat system. Elution with a 3:7:4:6 (v/v/v/v) HepEMWat system took approximately 200 min. This included an 80-min isocratic step, followed by gradient elution with methanol from 0% to 30%. The optimized methanol linear gradient CCC method was then used to separate a complex mixture of natural products isolated from Sinopodophyllum hexandrum (Royle) Ying roots. Twelve compounds with a wide range of polarities were well-resolved in a single separation. We have developed a convenient and cost-effective strategy for the separation of complex mixtures. No tedious mobile phase preparation step is required. The volume of unused mobile phase is minimal, so little solvent is wasted. The method is an important advance for the separation of mixtures that contain many compounds with a large range of polarities and partition coefficients, which are common features of natural products.

Separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn using solvent and flow-rate gradient high-speed counter-current chromatography target-guided by ultrafiltration HPLC-MS screening.

Potentilla kleiniana Wight et Arn is widely used as a herbal medicine to treat type 2 diabetes. However, detailed information about its active compounds is lacking. To develop an efficient method for the rapid screening and separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn. Potential α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn were rapidly screened out through ultrafiltration high-performance liquid chromatography mass spectrometry (HPLC-MS), and then followed by a target-guided high-speed counter-current chromatography (HSCCC) separation using two-phase solvent systems composed of n-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v and 1:10:5:6, v/v/v/v), and adopting increasing flow-rate from 1.5 to 3.0mL/min after 200min. Their structures were identified by ultraviolet (UV), MS, proton nuclear magnetic resonance (1 H-NMR) and carbon-13 (13 C)-NMR, and their α-glucosidase inhibitory activities were assessed by in vitro assay. Five α-glucosidase inhibitors including gallic acid (25.7mg, 98.2%, 1), brevifolincarboxylic acid (9.86mg, 95.3%, 2), ethyl evifolincarboxylate (13.26mg, 97.6%, 3), 3,3'-di-O-methylellagic acid-4'-O-β-d-glucopyranoside (16.26mg, 95.1%, 4), and 3,3'-di-O-methylellagic acid (10.54mg, 96.8%, 5) were successfully purified from 250mg n-butanol extract in a single run. Compounds 1, 2, 4 and 5 exhibited stronger α-glucosidase inhibitory activities[half maximal inhibition concentration (IC50 ) values at 173.41±6.35, 323.46±8.08, 44.63±2.50, and 20.73±2.56μM, respectively] than acarbose (IC50 value at 332.12±5.52μM, reference compound). Notably, compounds 2-5 were reported in the Potentilla kleiniana Wight et Arn for the first time. The results indicated that the proposed method could be applied for the rapid screening and preparative separation of α-glucosidase inhibitors from a complex matrix.

Development of equivalent chain length (ECL) rules for lipid compounds

Lipid compounds (fatty acids, tocochromanols, phytosterols) are difficult to separate by countercurrent chromatography (CCC) due to the existence of many similar structures and the limited availability of suitable biphasic solvent systems. Here we show that for these compound classes the success of a CCC separation can be directly derived from the chemical structures without the necessity of experimental determinations of K values. In most cases, lipid compounds differ in the total carbon number and the number of double bonds. For each structure the so-called equivalent chain length (ECL) can be calculated by subtracting a distinct value for each double bond from the total carbon number. Empirically, we verified that in the case of unbranched fatty acids (determined as methyl esters) one double bond corresponds with two carbons. Evaluation of CCC data from seventeen phytosterols in five plant oils and nine tocochromanol standards showed that one double bond was equal with one carbon for both lipid classes. Most compounds with different ECL can be separated by CCC but not those with the same ECL. In these cases, the selection of a suitable source for isolation of a particular lipid compound becomes very important. Knowledge of the impact of double bonds may also be a helpful tool for other substance classes.

Three Polymethoxyflavones Purified from Ougan (Citrus reticulata Cv. Suavissima) Inhibited LPS-Induced NO Elevation in the Neuroglia BV-2 Cell Line via the JAK2/STAT3 Pathway.

In order to establish an efficient method for separation of polymethoxyflavones (PMFs) and explore the anti-inflammatory mechanism of PMF monomers, a citrus variety rich in PMFs, Ougan (Citrus reticulata cv. Suavissima), was selected, and three monomers, including nobiletin, tangeretin, and 5-demethylnobiletin, were purified by ultrasonic-assisted extraction, solid phase extraction, and high-speed countercurrent chromatography separation. UPLC-MS was used to identify the three monomers. UPLC determined purities of 99.87% to nobiletin, 99.76% to tangeretin, and 98.75% to 5-demethylnobiletin with the standard curve method. A lipopolysaccharide (LPS)-induced NO releasing model was performed in the mouse microglia BV-2 cell line. Results illustrated that PMF monomers inhibited the NO release and the inflammation-related cytokines, including IL-1β, IL-6, and TNFα elevation. QRT-PCR revealed that PMFs alleviated LPS-induced upregulation of iNOS, IL-6, JAK2, TNFα, IL-1β, and NF-κB and LPS-induced downregulation of IκBα, while they did not affect TLR1, TLR2, TLR4, and TLR6. STAT3 expression was repressed by tangeretin and 5-demethylnobiletin, but not by nobiletin. Western blot assay also showed a suppression of expression and phosphorylation of JAK2 by all three PMF monomers, while STAT3 phosphorylation was restrained by tangeretin and 5-demethylnobiletin. The mechanism was primarily verified by the JAK2 inhibitor Ruxolitinib and the STAT3 inhibitor Stattic.

Ultra-high-pressure-assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high-speed counter-current chromatography.

Ultra-high-pressure extraction combined with high-speed counter-current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra-high-pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid-liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra-high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (3:7:5:5, v/v) was used for high-speed counter-current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one-step separation. This research demonstrated that ultra-high-pressure extraction combined with high-speed counter-current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.

Comprehensive evaluation of effective polyphenols in apple leaves and their combinatory antioxidant and neuroprotective activities

Apple (Malus pumila Mill.) leaves contain many antioxidant polyphenols that have received considerable attention due to their potential prevention of various chronic human diseases. The present study aimed to provide a comprehensive evaluation of polyphenols in apple leaves. The petroleum ether, ethyl acetate, and 75% ethanol extracts of apple leaves were assessed for their total phenolic and flavonoid contents and antioxidant activities. Among the three extracts, the 75% ethanol extract contained the highest phenolic (56.74 mg/g) and flavonoid (37.56 mg/g) contents and had the highest antioxidant activity (EC50 value 50.96 mg/L) by 2, 2-diphenyl-1-picrylhydrazyl assay. According to high performance liquid chromatography profiling and high-speed countercurrent chromatography separation, the 75% ethanol extract contained five major polyphenols phloridzin (P, 66.1 mg/g), isoquercitrin (IQ, 8.4 mg/g), quercetin 3-O-xyloside (9.5 mg/g), quercetin 3-O-arabinoside (10.7 mg/g), and quercetin 3-O-rhamnoside (28.5 mg/g). Additionally, the five separated polyphenols were investigated for their antioxidant activities and protective effects against hydrogen peroxide-induced oxidative stress in rat hippocampal neurons in vitro. The results showed that all the five polyphenols had antioxidant activities like the synthetic antioxidants butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) and IQ had the highest antioxidant activity (EC50 value 2.92 mg/L) among them. The IQ combination with P or with BHT had significant synergism in antioxidant activity at the ratios from 25:1 to 1:25 (w/w) and the strongest synergism at the ratio of 1:1. The IQ showed significant neuroprotective effect at the concentrations of 0.5–1.0 mg/L, while P and BHT had no neuroprotective effect and TBHQ had neurotoxic effect at high concentrations (0.5–5.0 mg/L). These results indicated that apple leaves could be exploited as a cheap and abundant resource of antioxidants and their utilization could be very attractive for pharmaceutical, food, and cosmetic industries.

Efficient extraction and isolation of skimmianine from New Caledonian plant Medicosma leratii and evaluation of its effects on apoptosis, necrosis, and autophagy

This study was aimed at improving the current knowledge about the new Caledonian plant species Medicosma leratii, by developing efficient methods of extraction and isolation of specialized metabolites. Ultrasound-assisted extraction (UAE) significantly gave higher yields of the furoquinoline alkaloid skimmianine (22.34 mg/100 g) than accelerated solvent extraction (ASE, 1.37–12.36 mg/100 g). High-performance countercurrent chromatographic (HPCCC) separation of the methanol leaf extract with n-hexane-ethyl acetate-methanol-water (6:5:6:5 v/v) afforded skimmianine (1, 7.55 mg, HPLC-DAD purity 99.4%), normelicopidine (3, 1.87 mg, 95.6%), normelicopicine (4, 2.63 mg, 97.3%) and a new furoquinoline alkaloid named sarcodifurine C (2, 1.20 mg, 95.8%). The effect of the crude extract and its main constituent, skimmianine, on apoptosis, autophagy, and necrosis induction in tumor cells has also been investigated. The crude extract and skimmianine were found to be effective apoptosis inducers in HeLa cervical cancer cell line, but not in colon cancer SW620 and HT-29 cells. Additionally, skimmianine effectively potentiated the pro-apoptotic properties of the antincancer agent sorafenib in cervical cancer cells.

Industrial countercurrent chromatography separations based on a cascade of centrifugal mixer-settler extractors

In hydrometallurgy, traditional extraction technologies, in particular, for isolation and purification of rare-earth metals include a number of processing steps using up to hundreds of mixer-settler extractors. These technologies could be greatly simplified by using the methods of countercurrent chromatography (CCC) separation. However, the current CCC equipment cannot process large volumes of feed material formed during the industrial production of these metals. In this paper, the cascade of centrifugal mixer-settler extractors assembled as a multi-stage unit is suggested for industrial application of CCC and discussed.

Separation and purification of intermediates for the preparation of naproxen from synthetic mixtures by countercurrent chromatography.

Three key intermediates in the preparation of the nonsteroidal anti-inflammatory drug naproxen were successfully separated and purified with high purity from synthetic mixtures by countercurrent chromatography with a selected biphasic solvent system. The biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (9:1:9:1, v/v/v/v) was selected according to partition performance of the three components using thin-layer chromatography. Fifty milligrams of the synthetic mixture after the three-step reaction was injected into a preparative countercurrent chromatography separation column and yielded 3.5, 14.0, and 8.0mg of three key intermediates with 95.0, 99.0, and 98.0% purity, and the recovery of each component was 65.2, 71.2, and 69.6%, respectively. The results indicated that countercurrent chromatography is an efficient alternative and economical method for the separation and purification of intermediate components from synthetic mixtures.

Multiple injection mode with or without repeated sample injections: Strategies to enhance productivity in countercurrent chromatography

Countercurrent chromatography (CCC) is an all liquid based separation technique typically used for the isolation and purification of natural compounds. The simplicity of the method makes it easy to scale up CCC separations from analytical to preparative and even industrial scale. However, scale-up of CCC separations requires two different instruments with varying coil dimensions. Here we developed two variants of the CCC multiple injection mode as an alternative to increase the throughput and enhance productivity of a CCC separation when using only one instrument. The concept is based on the parallel injection of samples at different points in the CCC column system and the simultaneous separation using one pump only. The wiring of the CCC setup was modified by the insertion of a 6-port selection valve, multiple T-pieces and sample loops. Furthermore, the introduction of storage sample loops enabled the CCC system to be used with repeated injection cycles. Setup and advantages of both multiple injection modes were shown by the isolation of the furan fatty acid 11-(3,4-dimethyl-5-pentylfuran-2-yl)-undecanoic acid (11D5-EE) from an ethyl ester oil rich in 4,7,10,13,16,19-docosahexaenoic acid (DHA-EE). 11D5-EE was enriched in one step from 1.9% to 99% purity. The solvent consumption per isolated amount of analyte could be reduced by ∼40% compared to increased throughput CCC and by ∼5% in the repeated multiple injection mode which also facilitated the isolation of the major compound (DHA-EE) in the sample.

An easy-to-use calculating machine to simulate steady state and non-steady-state preparative separations by multiple dual mode counter-current chromatography with semi-continuous loading of feed mixtures

Multiple dual mode counter-current chromatography (MDM CCC) separation processes with semi-continuous large sample loading consist of a succession of two counter-current steps: with “x” phase (first step) and “y” phase (second step) flow periods. A feed mixture dissolved in the “x” phase is continuously loaded into a CCC machine at the beginning of the first step of each cycle over a constant time with the volumetric rate equal to the flow rate of the pure “x” phase. An easy-to-use calculating machine is developed to simulate the chromatograms and the amounts of solutes eluted with the phases at each cycle for steady-state (the duration of the flow periods of the phases is kept constant for all the cycles) and non-steady-state (with variable duration of alternating phase elution steps) separations. Using the calculating machine, the separation of mixtures containing up to five components can be simulated and designed. Examples of the application of the calculating machine for the simulation of MDM CCC processes are discussed.

Elution-extrusion countercurrent chromatography separation of six pairs of isomeric iridoids from Cornus officinalis Sieb. et Zucc. guided by ion current extraction in mass spectrometry.

A mass spectrometry-guided elution-extrusion countercurrent chromatography protocol was developed to separate chemical components from Cornus officinalis Sieb. et Zucc. In this study, ion current extraction, a mass spectrometry-based data postacquisition method, was utilized to boost the separation power and scope of countercurrent chromatography technique. As a peak repicking and denoising tool, ion current extraction was carried out to process the liquid chromatography with mass spectrometry and the countercurrent chromatography with mass spectrometry data. So the target components were reacquired in the created extracted ion current patterns with enhanced responses and diminished background noise, which facilitated the distribution constant determination (by liquid chromatography with extracted ion current) and the targets fractionation (by countercurrent chromatography with extracted ion current). Under the guidance of the extracted ion currents of the target components and with the support of elution-extrusion mode in the countercurrent chromatography separation, six pairs of minor iridoid isomers were obtained in shortened experimental duration. Besides, a reciprocal shifted symmetry plot was established to represent the elution-extrusion countercurrent chromatography chromatogram. The results demonstrated the capability of the ion current extraction-guided elution-extrusion countercurrent chromatography protocol in discovery, analysis, and fractionation of low-concentration and structurally similar chemicals from a complicated sample.