Solvent Systems For Countercurrent Chromatography Research Articles

"Sweet space" strategy for solvent system selection in countercurrent chromatography: A case study on separation of polyunsaturated fatty acids from borage oil.

This study presents a comprehensive strategy for the selection and optimization of solvent systems in countercurrent chromatography (CCC) for the effective separation of compounds. With a focus on traditional organic solvent systems, the research introduces a "sweet space" strategy that merges intuitive understanding with mathematical accuracy, addressing the significant challenges in solvent system selection, a critical bottleneck in the widespread application of CCC. By employing a combination of volume ratios and graphical representations, including both regular and trirectangular tetrahedron models, the proposed approach facilitates a more inclusive and user-friendly strategy for solvent system selection. This study demonstrates the potential of the proposed strategy through the successful separation of gamma-linolenic acid, oleic acid, and linoleic acid from borage oil, highlighting the strategy's effectiveness and practical applicability in CCC separations.

A countercurrent chromatography solvent system based on deep eutectic solvents for separation of cis- and trans-crocetin from Gardenia jasminoides Ellis.

Due to the structural similarity and large difference in concentration, the separation of trans- and cis-crocetin has been challenging, and the cis-crocetin is usually neglected. In this work, a countercurrent chromatography method was developed for the quick separation of trans-crocetin and cis-crocetin from the hydrolytic extract of Gardenia jasminoides Ellis. High purity of trans-crocetin (>95%) and cis-crocetin (>91%) were prepared simultaneously for the first time through a novel biphasic system based on deep eutectic solvents, n-heptane/n-butyl alcohol/13mmol/L Na2 CO3 in water/acetamide-benzyltrimethylammonium chloride (4:1, mol/mol) (4:7:9:1, v/v). The addition of deep eutectic solvent significantly improved the separation efficiency. The two targets can be easily recovered from the separation system through simple acidification and precipitation. It has potential for preparative separations on a large scale.

Nuclear magnetic resonance-based solvent system selection for counter-current chromatography separation of compounds present in the same high-performance liquid chromatography peak: Flavonoids in barley seedlings as an example.

Partition coefficient is a key parameter for counter-current chromatography separation. High-performance liquid chromatography (HPLC) is the most commonly used tool for the screening of partition coefficients. However, HPLC technology is not applicable to the compounds present in the same chromatographic peak. Nuclear magnetic resonance (NMR) technology could easily distinguish compounds according to their characteristic absorption even if they exist in the same HPLC peak. In this study, two flavonoids present in the same HPLC peak were successfully purified by counter-current chromatography with a solvent system screened by NMR to show the great potential of NMR technology in the screening of the partition coefficient of co-efflux compounds. Through NMR screening, an optimized ethyl acetate/n-buthanol/water (7:3:10, v/v/v) system was applied in this study. As a result, two flavonoids, including 4.8mg of 3'-methoxyl-6'''-O-feruloylsaponarin and 9.8mg of 6'''-O-feruloylsaponarin were separated from 15mg of the mixture. There is only one methoxy group difference between the two flavonoids. This study provides a new strategy for the screening of counter-current chromatography solvent systems and broadens the application scope of counter-current chromatography.

GUESS – A simple approach to accelerate optimization countercurrent separation

The generally useful estimate of solvent systems (GUESS) method, which is based on thin layer chromatography, is a simple and practical method for selecting solvent systems for countercurrent chromatography (CCC). However, it is rarely used for complex samples derived from natural products.In this study, GUESS was used for CCC solvent system selection and polarity-adjusted CCC separations of several fractions, which were obtained from a silica gel column containing complex compositions with a broad polarity from Salvia bowleyana Dunn. The GUESS method was performed on five fractions based on solvent systems in the n-hexane–ethyl acetate–methanol-water (HEMWat) family. Based on the GUESS results, the optimal solvent systems were selected for CCC separation.Twelve diterpenoids were obtained from the five silica gel column fractions of S. bowleyana Dunn using elution-extrusion countercurrent chromatography (EECCC). These demonstrate that GUESS guidance and the polarity adjustment of the solvent system accelerate the optimization of CCC separation conditions and simplify the process of accommodating a broad polarity of components in complicated mixture fractions.We therefore confirmed the feasibility and advantage of the GUESS method for complex natural chemical component separations.

A Neglected Issue: Stationary Phase Retention Determination of Classic High-Speed Counter-Current Chromatography Solvent Systems

Obtaining an ideal solvent system for target compounds is still an obstacle to the wide application of high-speed counter-current chromatography (HSCCC). The partition coefficient and retention of the stationary phase are two key parameters for solvent system selection. The retention of the stationary phase of the solvent system is roughly judged by settling time using a test tube, which is subjective and inaccurate. In this study, we demonstrated that high-resolution separation of HSCCC is tightly connected with the retention of the stationary phase. Notably, unlike the in vitro test of settling time, we investigated the retention of the stationary phase of classical biphasic solvent systems by a TBE300C HSCCC apparatus. Our results revealed that settling time is not always inversely proportional to the retention of the stationary phase. The n-hexane–ethylacetate–methanol–water solvent systems showed the highest correlation coefficient of settling time and retention of the stationary phase (r = −0.91, n = 16). N-heptane–n-butanol–acetonitrile–water solvent system showed the lowest correlation coefficient (r = −0.26, n = 7). These results may be helpful for HSCCC solvent system selection and accelerate the application of this technique.

Role of three-phase solvent system in the counter-current chromatography.

In counter-current chromatography, the separation efficiency greatly depends on the partitioning ability of the separated substance between the stationary phase and the mobile phase. Partitioning ability is mainly represented by the parameter partition coefficient which is one of the important parameters to evaluate the separation effect of counter-current chromatography. The scope of the partition coefficient value mainly depends on the solvent system. A suitable solvent system election is, therefore, a critical role in the separation of counter-current chromatography. The existing solvent systems that are widely used are mainly two-phase solvent systems. It is difficult to decide on an appropriate solvent system for the separation of compounds with a wide polarity range, which promotes the development of the three-phase solvent system in counter-current chromatography. This review mainly described the origin, development history of three-phase solvent system, summarized the volume ratios and volume fractions of the upper, middle, and lower phases of nearly 50 three-phase solvent systems, their advantages, and disadvantages in counter-current chromatography. In addition, the challenges and future perspectives on three-phase solvent systems in counter-current chromatography also are discussed in this review.

H2O-Induced Hydrophobic Interactions in MS-Guided Counter-Current Chromatography Separation of Anti-Cancer Mollugin from Rubia cordifolia.

Counter-current chromatography (CCC) is a unique liquid–liquid partition chromatography and largely relies on the partition interactions of solutes and solvents in two-phase solvents. Usually, the two-phase solvents used in CCC include a lipophilic organic phase and a hydrophilic aqueous phase. Although a large number of partition interactions have been found and used in the CCC separations, there are few studies that address the role of water on solvents and solutes in the two-phase partition. In this study, we presented a new insight that H2O (water) might be an efficient and sensible hydrophobic agent in the n-hexane-methanol-based two-phase partition and CCC separation of lipophilic compounds, i.e., anti-cancer component mollugin from Rubia cordifolia. Although the n-hexane-methanol-based four components solvent systems of n-hexane-ethyl acetate-methanol-water (HEMWat) is one of the most popular CCC solvent systems and widely used for natural products isolation, this is an interesting trial to investigate the water roles in the two-phase solutions. In addition, as an example, the bioactive component mollugin was targeted, separated, and purified by MS-guided CCC with hexane-methanol and minor water as a hydrophobic agent. It might be useful for isolation and purification of lipophilic mollugin and other bioactive compounds complex natural products and traditional Chinese medicines.

Development of ultrasound-assisted centrifugal extraction and online solvent concentration coupled with parallel countercurrent chromatography for the preparation of purified phytochemicals: Application to Lycium ruthenicum

To improve the preparation efficiency of natural products, new techniques for extraction and concentration of natural products have been established. Based on this, an on-line technology to improve the yield and preparation efficiency of natural products have been established. In this study, a novel method to extract natural products from crops and plants, termed ultrasound-assisted centrifugal extraction (UACE), was developed as a supplement to a new online solvent concentration (OSC) technique. These techniques were coupled with parallel countercurrent chromatography (PCCC) and evaluated in the automated preparation of phytochemicals from Lycium ruthenicum. The UACE device comprises an extraction chamber and a solution separation chamber and offers the advantages of higher extraction efficiency, fewer impurities, and conduciveness to sample processing. The OSC device comprises a spray nozzle, concentrating cylinder, and hot-blast air nozzle. The mechanical parameters of the UACE and OSC were optimized, and the operation mode of online UACE combined with OSC and PCCC was described. Raw plant materials were extracted with a two-phase extractant comprising petroleum ether‒ethyl acetate‒ethanol‒water (2:2:1:6, v/v/v/v), and the aqueous and organic phases were then concentrated by OSC. After extraction and online concentration, the concentrate was separated. During PCCC separation, the on-line extraction, and concentration were still conducted. When the first cycle of the UACE/OSC/PCCC was completed, the second cycle was initiated, and so on. As a result, six target compounds with purities exceeding 97.03 % (high performance liquid chromatography detection at 250 nm) were successfully separated using the countercurrent chromatography solvent systems comprising n-heptane‒ethyl acetate‒acetonitrile‒water (20:4:17:1, v/v/v/v) and n-butanol–ethyl acetate–water (55:21:70, v/v/v). Compared with conventional extraction methods, the proposed method offers the advantages of efficiency, automation, high-purity separation of the analytes, and systematic preparation of natural products from crops.

A rapid and practical prediction method for the Arizona solvent system family used in high speed countercurrent chromatography.

Selecting the appropriate solvent system is the key to the successful separation of samples by using countercurrent chromatography. Although high-speed countercurrent chromatography has been widely used in the separation and preparation of natural products, the selection of a solvent system has always been a stumbling block to the application of high-speed countercurrent chromatography. In order to explore a rapid and practical prediction method to select countercurrent chromatography solvent system, five linear prediction models of the Arizona solvent system family (HEMW) was established by using fourteen compounds with different structures and five HPLC columns of different brands. And two different solvent system selection methods (The partition coefficient K of the target compound in the solvent system was in the range of 0.25<K<2.5) were proposed for targeted separation of compounds and multi-component separation in a complex sample respectively. The appropriate HSCCC solvent system of five known compounds was determined by a HPLC analysis and a shake flask test and the appropriate HSCCC solvent system of two Chinese herbal extracts was determined by a HPLC analysis to verify the prediction method. In this study, solid-liquid partition chromatography (HPLC) and liquid-liquid partition chromatography (HSCCC) were linked by polarity to simplify the screening process of solvent system. This method reduced the difficulty and workload of solvent system selection, which provided methods and ideas for more solvent system prediction models.

Effective on-line high-speed shear dispersing emulsifier technique coupled with high-performance countercurrent chromatography method for simultaneous extraction and isolation ofcarotenoids from Lycium barbarum L. fruits.

An efficient combination strategy based on high-speed shear dispersing emulsifier technique and high-performance countercurrent chromatography was developed for on-line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high-speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high-performance countercurrent chromatography solvent system composed of n-hexane-dichloromethane-acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high-performance counter-current chromatography process adopts elution-extrusion mode and the upper phase of the solvent system as stationary phase (reverse-phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction-separation operation within 120min. The targeted compounds were analyzed and identified by high-performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on-line combination method could serve as a simple, rapid, and effective way to achieve weak polarand unstable compounds from natural products.

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.

Separation of five naphtho‐γ‐pyrones from Pleurotus ostreatus by high‐speed countercurrent chromatography

A method to optimize a high-speed countercurrent chromatography solvent system from many possible solvent systems was guided by average polarity and K values of crude samples, which was successfully applied in separation of the naphtho-γ-pyrones. This method expandedthescopeof spectrophotometerapplication in the solvent systems selection, and showed the merits of simplicity, rapidity, and low reagent consumption. As a consequence of utilizing the method, an efficient separation procedure was established, and two groups of naphtho-γ-pyrone isomers (fonsecinone B and asperpyrone F; asperpyrone C, fonsecinone A, and aurasperone A) were obtained from Pleurotus ostreatus by high-speed countercurrent chromatography with a solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:5:5, v/v). The results indicate that high-speed countercurrent chromatography could be a powerful technology for separation isomers and analogues, and the method guided by average polarity and K values of crude samples is feasible to optimize solvent system.

Hydrodynamics studies of the behaviour of traditional and two-phase ionic liquid solvent systems in countercurrent chromatography (CCC)

The hydrodynamics and retention of 2D countercurrent chromatographic systems were investigated for the first time for ionic liquid based biphasic solvents using high-speed imaging. The results were compared against 3D chromatographic systems and different solvent systems for a wide range of beta values (0.27–0.83). The studies revealed details of the phase distribution, the mixing and settling areas within the column, as well as the interfacial characteristics that can enhance mass transfer (such as waves). There was no single-phase flooding in the beta range 0.27–0.5 contrary to previous limited visualisation studies, which suggested that for the biphasic solvents they utilized, single-phase flooding at low beta values (less than 0.5) occurs. Previously unseen, stable, six segment phase distributions, involving alternate high and low stationary phase retention were observed for the ionic liquid biphasic solvent system but not for the organic-aqueous and aqueous-aqueous biphasic solvent systems also studied. This unusual behaviour and the resulting considerable variations in interfacial height and thus local velocity of each phase along the coil, are discussed in the text. The ionic liquid biphasic solvent also demonstrated novel behaviour in other aspects. In the mixing zone around the proximal key node, interfacial waves were observed; however, it was found that their amplitude and frequency depended on the chosen elution mode as well as the mobile phase flow rate and beta value. While there was some agreement in the performance of both column geometries, it was found that for the serial 2D column there was always some retention of the stationary phase over the entire range of flow rates tested, unlike the case for the 3D column.

Salting-in counter-current chromatography separation of tanshinones based on room temperature ionic liquids

Ionic liquids have been widely used for the extraction and separation of bioactive natural and synthetic mixtures. In this study, we provided an updated example by using an ionic liquid-based salting-in counter-current chromatography (CCC) strategy for the separation of hydrophobic tanshinones without subsequent column chromatography purification. Several ionic liquids such as 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), 1-methallyl-3-methylimidazolium chloride ([MAMIM]Cl) and 1-butyl-3-ethylimidazolium chloride [BMIM]Cl could significantly decrease the partition coefficients (K) of tanshinones in the selected two-phase solvent composed of hexane-ethyl acetate-methanol-ionic liquid aqueous solution (5:5:6:4, v/v). Typically, K values of three target tanshinones including tanshinone I, 1,2-dihydrotanshinquione and tanshinone IIA were reduced from 3.57, 4.57 and 5.50 to 1.62, 2.33 and 3.08, respectively, by the inclusion of 10% [AMIM]Cl in the solvent system. After salting-in CCC separation, the purified tanshinones were obtained only by simple ethyl acetate extraction. In general, the current results demonstrated that the ionic liquid-based salting-in CCC may be as an alternative strategy for the optimization of CCC solvent systems and separation of lipophilic natural products.

Sugaring-out strategy for counter-current chromatography isolation: podophyllotoxins and flavones from Dysosma versipellis as examples

In this work, we introduced a sugaring-out strategy for optimizing two-phase solvent systems for counter-current chromatography by adding some sugars into the selected two-phase solvent system.

Preparative isolation of a cytotoxic principle of a forest mushroom Suillus luteus by sodium dodecyl sulfate based "salting-in" countercurrent chromatography.

In the course of screening new anticancer natural products, an edible forest mushroom Suillus luteus (L. Ex Franch). Gray was found to have potent cytotoxicity against several human cancer cells. However, the lipophilic sample made some countercurrent chromatography solvent systems emulsify, which caused difficulties in the separation of its cytotoxic components. Here, we found that the addition of an organic salt sodium dodecyl sulfate could efficiently shorten the settling time of the mushroom sample solutions by eliminating the emulsification of two-phase solvent systems. Moreover, we found that sodium dodecyl sulfate could play a new "salting-in" role and made the partition coefficients of the solutes decrease with the increased concentrations. Thus, a sodium dodecyl sulfate based salting-in countercurrent chromatography method has been successfully established for the first time for preparative isolation of a cytotoxic principle of the mushroom. The active component was identified as isosuillin. Whole results indicated that sodium dodecyl sulfate could be used as an efficient salting-in reagent for two-phase solvent system selection and targeted countercurrent chromatography isolation. It is very useful for current natural products isolation and drug discovery.

The Generally Useful Estimate of Solvent Systems (GUESS) method enables the rapid purification of methylpyridoxine regioisomers by countercurrent chromatography

The TLC-based Generally Useful Estimate of Solvent Systems (GUESS) method was employed for countercurrent chromatography solvent system selection, in order to separate the three synthetic isomers: 3-O-methylpyridoxine, 4′-O-methylpyridoxine (ginkgotoxin), and 5′-O-methylpyridoxine. The Rf values of the three isomers indicated that ChMWat+2 (chloroform-methanol-water 10:5:5, v/v/v) was appropriate for the countercurrent separation. The isomer separation was highly selective and demonstrated that the TLC-based GUESS method can accelerate solvent system selection for countercurrent separation. Accordingly, the study re-emphasizes the practicality of TLC as a tool to facilitate the rapid development of new countercurrent and centrifugal partition chromatography methods for this solvent system. Purity and structure characterization of all samples was performed by quantitative 1H NMR.

A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural products

Counter-current chromatography (CCC) is an efficient liquid–liquid chromatography technique for separation and purification of complex mixtures like natural products extracts and synthetic chemicals. However, CCC is still a challenging process requiring some special technical knowledge especially in the selection of appropriated solvent systems. In this work, we introduced a new 9×9 map-based solvent selection strategy for CCC isolation of targets, which permit more than 60 hexane–ethyl acetate–methanol–water (HEMWat) solvent systems as the start candidates for the selection of solvent systems. Among these solvent systems, there are clear linear correlations between partition coefficient (K) and the system numbers. Thus, an appropriate CCC solvent system (i.e., sweet spot for K=1) may be hit by measurement of k values of the target only in two random solvent systems. Besides this, surprisingly, we found that through two sweet spots, we could get a line (“Sweet line”) where there are infinite sweet solvent systems being suitable for CCC separation. In these sweet solvent systems, the target has the same partition coefficient (K) but different solubilities. Thus, the better sweet solvent system with higher sample solubility can be obtained for high capacity CCC preparation. Furthermore, we found that there is a zone (“Sweet zone”) where all solvent systems have their own sweet partition coefficients values for the target in range of 0.4<K<2.5 or extended range of 0.25<K<16. All results were validated by using 14 pure GUESSmix mimic natural products as standards and further confirmed by isolation of several targets including honokiol and magnolol from the extracts of Magnolia officinalis Rehd. Et Wils and tanshinone IIA from Salvia miltiorrhiza Bunge. In practice, it is much easier to get a suitable solvent system only by making a simple screening two to four HEMWat two-phase solvent systems to obtain the sweet line or sweet zone without special knowledge or comprehensive standards as references. This is an important advancement for solvent system selection and also will be very useful for isolation of current natural products including Traditional Chinese Medicines.

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.

Direct purification of tanshinones from Salvia miltiorrhiza Bunge by high‐speed counter‐current chromatography without presaturation of the two‐phase solvent mixture

Tanshen, the rhizome of Salvia miltiorrhiza Bunge, is a famous Traditional Chinese Medicine for multiple therapeutic remedies. This work presents the isolation and purification of tanshinone I and tanshinone IIA from the extract of the rhizome of S. miltiorrhiza by using high-speed counter-current chromatography (CCC) without presaturation of the two-phase solvent mixture. The CCC method combines the results of CCC solvent system selection and components analyses of solvent mixture by GC, and thus it is possible to add accurately each individual solvent to prepare single saturated solvent phase without presaturation. The optimum CCC solvent system is a system of hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v), which has been determined by usual solvent system selection and CCC runs. As a result, over 98% pure tanshinone IIA and over 94% pure tanshinone I have been obtained by using less solvent volume. Their structures have been identified by ESI-MS, NMR spectra.