Large‐scale separation of antipsychotic alkaloids from Rauwolfia tetraphylla L. by pH‐zone‐refining fast centrifugal partition chromatography

pH‐zone‐refining centrifugal partition chromatography was successively applied in the large‐scale separation of close R_f antipsychotic indole alkaloids directly from CHCl₃ fraction of Rauwolfia tetraphylla leaves. Two experiments with increasing mass from 500 mg to 3 g of crude alkaloid extracts ( 1 C) of R. tetraphylla were carried out in normal‐displacement mode using a two‐phase solvent system composed of methyl tert‐butyl ether/ACN/water (4:1:5, v/v/v) where HCl (12 mM) was added to the lower aqueous stationary phase as a retainer and triethylamine (5 mM) to the organic mobile phase as an eluter. The two centrifugal partition chromatography separations afforded a total of 162.6 mg of 10‐methoxytetrahydroalstonine ( 1 ) and 296.5 mg of isoreserpiline ( 2 ) in 97% and 95.5% purity, respectively, along with a 400.9 mg mixture of α‐yohimbine and reserpiline ( 3 and 4 ). Further, this mixture was resolved over medium pressure LC using TLC grade silica gel H (average particle size 10 μm), which afforded 160.4 mg of α‐yohimbine ( 3) and 150.2 mg of reserpiline ( 4) in >95% purities. The purity of the isolated antipsychotic alkaloids was analyzed by high‐performance LC and their structures were characterized on the basis of their 1D, 2D NMR and electrospray ionization‐mass spectroscopic data.     

Application of an efficient strategy based on liquid–liquid extraction,high‐speed counter‐current chromatography,and preparative HPLC for the rapid enrichment,separation, and purification of four anthraquinones from Rheum tanguticum

This study presents an efficient strategy based on liquid–liquid extraction, high‐speed counter‐current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid–liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe‐emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high‐speed counter‐current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe‐emodin, physcione, and chrysophanol.     

A new tropane alkaloid from the leaves of Erythroxylum subsessile isolated by pH‐zone‐refining counter‐current chromatography

Tropane alkaloids are bioactive metabolites with great importance in the pharmaceutical industry and the most important class of natural products found in the Erythroxylum genus. However, these compounds are usually separated by traditional chromatographic techniques, in which the sample is progressively purified in multiple chromatographic steps, resulting in a time‐ and solvent‐consuming procedure. In this work we present the isolation of a novel alkaloid, 6β,7β‐dibenzoyloxytropan‐3α‐ol, together with the two known 3α‐benzoyloxynortropan‐6β‐ol and 3α,6β‐dibenzoyloxytropane alkaloids, directly from the crude alkaloid fraction from the leaves of Erythroxylum subsessile, by using a single run pH‐zone‐refining counter‐current chromatography method. The ethyl acetate/water (1:1, v/v) biphasic solvent system with triethylamine and HCl as retention and eluter agents, respectively, was used to isolate tropane alkaloids for the first time. The structures of the isolated alkaloids were elucidated by spectroscopic methods.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

Large‐scale separation of acetylcholinesterase inhibitors from Zanthoxylum nitidum by pH‐zone‐refining counter‐current chromatography target‐guided by ultrafiltration high‐performance liquid chromatography with ultraviolet and mass spectrometry screening

A new strategy by converging ultrafiltration high‐performance liquid chromatography with ultraviolet and mass spectrometry and pH‐zone‐refining counter‐current chromatography was developed for the rapid screening and separation of potential acetylcholinesterase inhibitors from the crude alkaloidals extract of Zanthoxylum nitidum. An optimized two‐phase solvent system composed of chloroform/methanol/water (4:3:3, v/v) was used in this study. And, in the optimal solvent system, 45 mM hydrochloric acid was added to the aqueous stationary phase as the retainer, while 5 mM triethylamine was added to the organic mobile phase as the eluter. As a result, with the purity of over 95%, five alkaloids including jatrorrhizine ( 1 , 340 mg), columbamine ( 2 , 112 mg), skimmianine ( 3 , 154 mg), palmatine ( 4 , 226 mg), and epiberberine ( 5 , 132 mg) were successfully purified in one step from 3.0 g crude alkaloidals extract. And their structures were identified by ultraviolet, mass spectrometry, ¹H and ¹³C NMR spectroscopy. Notably, compounds 2 , 4 and 5 were firstly reported in Z. nitidum. In addition, acetylcholinesterase inhibitory activities of compounds 1–5 were evaluated, and compounds 3, 4 and 5 exhibited stronger acetylcholinesterase inhibitory activity (IC₅₀ values at 8.52 ± 0.64, 14.82 ± 1.21 and 3.12 ± 0.32 μg/mL, respectively) than berberine (IC₅₀ value at 32.86 ± 2.14 μg/mL, positive control). The results indicated that the proposed method is an efficient technique to rapidly screen acetylcholinesterase inhibitors from complex samples, and could be served as a large‐scale preparative technique for separating ionizable active compounds.     

Separation of chlorogenic acid and concentration of trace caffeic acid from natural products by pH‐zone‐refining countercurrent chromatography

Chlorogenic acid and caffeic acid were selected as test samples for separation by the pH‐zone‐refining countercurrent chromatography (CCC). The separation of these test samples was performed with a two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/water at a volume ratio of 4:1:5 v/v/v where trifluoroacetic acid (TFA; 8 mM) was added to the organic stationary phase as a retainer and NH₄OH (10 mM) to the aqueous mobile phase as an eluter. Chlorogenic acid was successfully separated from Flaveria bidentis (L.) Kuntze (F. bidentis) and Lonicerae Flos by pH‐zone‐refining CCC, a slightly polar two‐phase solvent system composed of methyl‐tert‐butyl‐ether/acetonitrile/n‐butanol/water at a volume ratio of 4:1:1:5 v/v/v/v was selected where TFA (3 mM) was added to the organic stationary phase as a retainer and NH₄OH (3 mM) to the aqueous mobile phase as an eluter. A 16.2 mg amount of chlorogenic acid with the purity of 92% from 1.4 g of F. bidentis, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Lonicerae Flos have been obtained. These results suggest that pH‐zone‐refining CCC is suitable for the isolation of the chlorogenic acid from the crude extracts of F. bidentis and Lonicerae Flos.     

Direct from solid natural products to pure compounds in a single step: Coupling online extraction with high‐speed counter‐current chromatography

Extraction is the most important step in the purification of bioactive compounds from natural products. This study introduces a simple online extraction strategy coupled with high‐speed counter‐current chromatography for efficient extraction and purification of bioactive components from solid natural products. For online extraction strategy, 1.0 g of ground Mangnolia officinalis or Piper nigrum was loaded into a guard column, which was then positioned on the manual injection valve instead of the sample loop. Bioactive components were directly extracted by the mobile phase of high‐speed counter‐current chromatography, and then transferred into high‐speed counter‐current chromatography for purification. In addition, the compatibility of the developed methodology for direct purification of bioactive components from fresh M. officinalis was successfully demonstrated. Obviously, in comparison with traditional offline heat‐reflux extraction, online extraction avoided the instrument, time, solvent, and energy consumption, and purified two phenolic compounds (honokiol and magnolol) from M. officinalis and three alkaloids (piperyline, piperine, and piperanine) from P. nigrum with high extraction efficiency. The superiority of the developed methodology is to establish an easy, rapid, and efficient technique for the purification of a wide variety of bioactive components from solid natural products.     

Isolation and purification of alkaloids from the fruits of Macleaya cordata by ionic‐liquid‐modified high‐speed counter‐current chromatography

Macleaya cordata (Willd) R. Br. is a medicinal plant. The most important bioactive compounds of M. cordata are alkaloids that have many biological activities including antifungal, anti‐inflammatory, and antitumor. In this study, an ionic‐liquid‐modified high‐speed counter‐current chromatography method was established to obtain alkaloids from the fruits of M. cordata. The conditions of ionic‐liquid‐modified high‐speed counter‐current chromatography, including solvent systems, the content of ionic liquid (1‐butyl‐3‐methylimidazolium tetrafluoroborate [C₄mim][BF₄]), and the posttreatment of the ionic liquid, were investigated. Five alkaloids protopine, allocryptopine, sanguinarine, 8‐O‐demethylchelerythrine, and chelerythrine were separated from the extract of the fruits using a high speed counter‐current chromatography with two‐phase solvent system composed of dichloromethane/methanol/0.3 mol/L hydrochloric acid aqueous solution/[C₄mim][BF₄] (4:2:2:0.015, v/v). Their purities were 96.33, 95.56, 97.94, 96.22, and 97.90%, respectively. The results indicated that a small amount of ionic liquids as modifier of the two‐phase solvent system could shorten the separation time and improve the separation efficiency of the alkaloids from the fruits. The ionic‐liquid‐modified high‐speed counter‐current chromatography would provide a feasible way for highly effective separation of alkaloids from natural products.     

Accelerated solvent extraction and pH‐zone‐refining counter‐current chromatographic purification of yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom

This study aimed to seek an efficient method to extract and purify yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH‐zone‐refining counter‐current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L₉ (3)⁴. Then a separation and purification method was established using pH‐zone‐refining counter‐current chromatography with a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8‐deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI‐MS and ¹H and ¹³C NMR spectroscopy.     

Semi‐preparative high‐speed counter‐current chromatography separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn by pH‐gradient elution

A new high‐speed counter‐current chromatography method for semi‐preparative separation and purification of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn was developed by using pH‐gradient elution mode. Diethyl ether was used as the stationary phase of the two‐phase solvent system and Na₂HPO₄/NaH₂PO₄ buffer solution with pH values of 7.5 and 7.2 in gradient mode as the mobile phase. Consequently, 33 mg of liensinine, 42 mg of isoliensinine, and 67 mg of neferine were obtained from 200 mg of crude extracts. The purities of them were all over 98% as determined by HPLC area normalization method, and the structures were identified by ¹H‐NMR and ¹³C‐NMR.     

Application of pH‐zone‐refining countercurrent chromatography in the chiral separation of two β‐adrenergic blocking agents

Two β‐adrenergic blocking agents, 1‐[(1‐methylethyl)amino]‐3‐phenoxy‐2‐propanol ( 1 ) and 1‐[(1‐methylethyl)amino]‐3‐(3‐methylphenoxy)‐2‐propanol ( 2 ; Toliprolol), were enantioseparated by pH‐zone‐refining countercurrent chromatography. A two‐phase solvent system composed of chloroform containing 0.10 mol/L of di‐n‐hexyl l‐ tartrate/0.10 mol/L of boric acid aqueous solution (1:1, v/v) was selected, in which 20 mmol/L triethylamine was added in the organic phase as a retainer and 2 mmol/L HCl was added in the aqueous phase as an eluter. Fifty milligrams of each racemate was completely enantioseparated by pH‐zone‐refining countercurrent chromatography to yield each enantiomer with a purity of more than 98%, and the recovery of each separated enantiomer reached around 76–82%.     

Enrichment and separation of antitumor triterpene acids from the epidermis of Poria cocos by pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography

Triterpene acids were extracted from the epidermis of Poria cocos (Schw.) Wolf. These acids were found to inhibit the growth of lung cancer cells in vitro and in vivo. An efficient method for the preparative separation of antitumor triterpene acids was established that involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography. We used pH‐zone‐refining counter‐current chromatography to concentrate the triterpene acids using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (3:7:5:5, v/v/v/v), trifluoroacetic acid (10 mM) was added to the upper phase as a retainer, and ammonia (10 mM) was added to the lower phase as an eluter. As a result, 200 mg concentrate of triterpene acids was obtained from 1.0 g of crude extract. The concentrate was further separated by conventional high‐speed counter‐current chromatography using a solvent system composed of petroleum ether/ethyl acetate/methanol/water (0.8:1.2:1.2:0.9, v/v), yielding 50 mg of poricoic acid A and 5 mg of poricoic acid B from 120 mg concentrate, respectively. The inhibitory activity of the major compound on lung A549 cells was examined and poricoic acid A was found to significantly inhibit the growth of A 549 cells.     

Separation and purification of five alkaloids from Aconitum duclouxii by counter‐current chromatography

C₁₉‐diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C₁₉‐diterpenoid alkaloids, namely, benzoylaconine ( 1 ), N‐deethylaconitine ( 2 ), aconitine ( 3 ), deoxyaconitine ( 4 ), and ducloudine A ( 5 ), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter‐current chromatography. In the process of separation, the critical conditions of counter‐current chromatography were optimized. The two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water/NH₃·H₂O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1 , 24.1 mg of 2 , 250.6 mg of 3 , 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra‐high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation.     

pH‐Zone‐refining counter‐current chromatography for two new lipo‐alkaloids separated from refined alkaline extraction of Kusnezoff monkshood root

An efficient and refined method for the separation of six aconitine‐type alkaloids from the alkaline prepared “Kusnezoff monkshood root” was established. It is the first study that two new lipo‐alkaloids were successfully isolated from refined sample by pH‐zone‐refining counter‐current chromatography rather than synthetic method. It was of interest that a great deal of lipo‐alkaloids was produced in crude extract from the alkalization of “Kusnezoff monkshood root.” A refined sample method was proposed to enrich two types of alkaloids by liquid–liquid extraction, i.e. lipo‐alkaloids and monoester‐diterpenoid alkaloids. The pH‐zone‐refining counter‐current chromatography was performed with an optimized two‐phase solvent system composed of n‐hexane‐ethyl acetate–methanol–water (3:5:4:5, v/v), where upper organic phase was added to 3 mmol/L triethylamine as a retainer and lower aqueous mobile phase was added to 3 mmol/L hydrochloric acid as an eluter. As a result, six aconitum alkaloids, including two lipo‐alkaloids (8‐lino‐14‐benzoylaconine, 8‐pal‐14‐benzoylaconine), three monoester‐diterpenoid alkaloids (14‐benzoylmesaconine, 14‐benzoylaconine, beyzoyldeoxyaconine), and one aconine alkaloid (neoline) were acquired from the plant at the same time. The anti‐inflammatory activities of the two new lipo‐alkaloids were compared to the six alkaloids in vitro, in cyclo‐oxygen‐ase‐2 inhibition assays. The separation mechanism of six alkaloids by pH‐zone‐refining counter‐current chromatography was illustrated.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Simultaneous separation of triterpenoid saponins and flavonoid glycosides from the roots of Glycyrrhiza uralensis Fisch by pH‐zone‐refining counter‐current chromatography

Glycosides including triterpenoid saponins and flavonoid glycosides are the main constituents of Glycyrrhiza uralensis Fisch (licorice) and exhibit prominent pharmacological activities. However, conventional methods for the separation of glycosides always cause irreversible adsorption and unavoidable loss of sample due to their high hydrophilicities. The present paper describes a convenient method for the simultaneous separation of triterpenoid saponins and flavonoid glycosides from licorice by pH‐zone‐refining counter‐current chromatography. Ethyl acetate/n‐butanol/water (2:3:5, v/v) with 10 mM TFA in the upper organic stationary phase and 10 mM ammonia in the lower aqueous mobile phase was used as the biphasic solvent system. Three triterpenoid saponins and two flavonoid glycosides including licorice‐saponin A3 (63.3 mg), glycyrrhizic acid (342.2 mg), 3‐O‐[β‐d ‐glucuronopyranosyl‐(1 → 2)‐β‐d ‐galactopyranosyl]glycyrrhetic acid (56.0 mg), liquiritin apioside (232.6 mg), and liquiritin (386.5 mg) were successfully obtained from licorice ethanol extract (2 g) in one step. This method subtly takes advantage of the common acidic properties of triterpenoid saponins and flavonoid glycosides, and obviously is much more efficient and convenient than the previous methods. It is also the first time that the separation of acidic triterpenoid saponins by using pH‐zone‐refining counter‐current chromatography has been reported.     

Recent progress in separation prediction of counter‐current chromatography

As a liquid‐liquid partition chromatography, counter‐current chromatography has advantages in large sample loading capacity without irreversible adsorption, which has been widely applied in separation and purification fields. The main factors, including partition coefficient, two‐phase solvent systems, apparatus, and operating parameters greatly affect the separation process of counter‐current chromatography. To promote the applications of counter‐current chromatography, it is essential to develop theoretical research to master the principles of counter‐current chromatographic separations so as to achieve predictions before laborious trials. In this article, recent progress about separation prediction methods are reviewed from a point of the steady and unsteady state of the mass transfer process of counter‐current chromatography and its mass transfer characteristics, and then it is divided into three aspects: prediction of partition coefficient, modeling the thermodynamic process of counter‐current chromatography, and modeling the dynamic process of counter‐current chromatography.     

Separation of high‐purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH‐zone‐refining countercurrent chromatography

The evidence for unique effects of eicosapentaenoic acid and docosahexaenoic acid is growing. Further understanding and exploration of their independent effects in the nutraceutical and pharmaceutical industry is calling for the more efficient separation techniques to overcome the equivalent chain length rule of fatty acids. In this study, free eicosapentaenoic and docosahexaenoic acid were successfully separated by pH‐zone‐refining countercurrent chromatography for the first time. The different solvent systems and the influence of retainer and eluter concentration on the separation efficiency were investigated. A two‐phase solvent system composed of n‐heptane/methanol/water (100:55:45, v/v) was selected with 50 mM of trifluoroacetic acid as retainer in the organic phase and 40 mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500 mg of free fatty acids from a refined fish oil sample. 79.6 mg of eicosapentaenoic acid and 328.3 mg docosahexaenoic acid were obtained with the purities of 95.5 and 96.9% respectively determined by gas chromatography with mass spectrometry after methyl esterification. The scale‐up separation of 1 g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150 mM of retainer, producing satisfactory yields and purities of targets.     

pH‐zone‐refining elution–extrusion countercurrent chromatography: Separation of hydroxyanthraquinones from Cassiae semen

Seven hydroxyanthraquinones were successfully separated from the traditional Chinese medicinal herb Cassiae semen by conventional and pH‐zone‐refining countercurrent chromatography with an environmentally friendly biphasic solvent system, in which elution–extrusion mode was investigated for pH‐zone‐refining countercurrent chromatography for the first time. A two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (5:3:4:4, v/v/v/v) was used for the conventional countercurrent chromatography while the same system with a different volume ratio n‐hexane/ethyl acetate/ethanol/water (3:5:2:6, v/v/v/v) was used for pH‐zone‐refining countercurrent chromatography, in which 20 mmol/L of trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L of ammonia was added to the aqueous phase as an eluter. A 400 mg crude sample could be well separated by pH‐zone‐refining countercurrent chromatography, yielding 53 mg of aurantio‐obtusin, 40 mg of chryso‐obtusin, 18 mg of obtusin, 24 mg of obtusifolin, 10 mg of emodin, and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8, 95.7, 96.9, 93.5, 97.4, 77.1, and 19.8%, as determined by high‐performance liquid chromatography. Furthermore, the difference in elution sequence between conventional and pH‐zone‐refining mode was observed and discussed.     

Isolation of a furan fatty acid from Hevea brasiliensis latex employing the combined use of pH‐zone‐refining and conventional countercurrent chromatography

Furan fatty acids are valuable and bioactive minor fatty acids that usually contribute <0.1% to the fatty acid content of food samples. Their biological role still remains unclear as authentic furan fatty acid standards are not readily available and thorough experimental studies verifying the relevance of furan fatty acids are thus virtually impossible. An efficient protocol for the isolation of the furan fatty acid 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid from hydrolyzed and centrifuged latex of Hevea brasiliensis was developed using countercurrent chromatography. A first run using pH‐zone‐refining countercurrent chromatography provided 48.4 mg of 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid from 210 mg latex extract in a purity of 95%. The purity was increased to 99% by means of one second run in conventional countercurrent chromatography mode. The Structure and purity of 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid were determined by gas chromatography coupled to mass spectrometry and ¹H and ¹³C NMR spectroscopy.