Recycling Countercurrent Chromatography Research Articles

Enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography using anionic β-cyclodextrin as chiral selector.

Two anionic β-cyclodextrins as chiral selectors were successfully applied in the enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography. Sulfobutyl ether-β-cyclodextrin and carboxymethyl-β-cyclodextrin showed opposite enantioselectivity for both duloxetine and N-methyl duloxetine enantiomers. Two biphasic solvent systems, n-hexane: 0.1mol/L phosphate buffer pH 7.6 with 50mmol/L of sulfobutyl ether-β-cyclodextrin (1:1, v/v) and n-hexane: 0.1mol/L phosphate buffer pH 7.2 with 50mmol/L of carboxymethyl-β-cyclodextrin (1:1, v/v), were selected for N-methyl duloxetine. Enantioseparation of duloxetine was achieved by recycling countercurrent chromatography using a solvent system composed of n-butyl acetate: 0.1mol/L phosphate buffer pH 7.2 with 20mmol/L of sulfobutyl ether-β-cyclodextrin or carboxymethyl-β-cyclodextrin (1:1, v/v). A solvent system composed of n-hexane: n-butyl acetate: 0.1mol/L phosphate buffer pH 7.6 containing 20mmol/L of sulfobutyl ether-β-cyclodextrin (6:4:10, v/v) was selected for enantioseparation of fluoxetine.

Closed-Loop Recycling Dual-Mode Counter-Current Chromatography with Specified Sample Loading Durations: Modeling of Preparative and Industrial-Scale Separations.

We previously reported on a new counter-current chromatography (CCC) operating mode called closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC), which incorporates the advantages of closed-loop recycling (CLR) and dual-mode (DM) counter-current chromatography and includes sequential separation of compounds in the closed-loop recycling mode with the mobile x-phase and in the inverted-phase counter-current mode with the mobile y-phase. The theoretical analysis of several implementations of this separation method was carried out under impulse sample injection conditions. This study is dedicated to the further development of CLR DM CCC theory applied to preparative and industrial separations, where high-throughput operation is required. Large sample volumes can be loaded via continuous loading within a specified time. To simulate CLR DM CCC separations with specified sample loading durations, equations are developed and presented in “Mathcad” software.

Enantioseparation of ondansetron by countercurrent chromatography using sulfobutyl ether-β-cyclodextrin as chiral selector.

Ondansetron, a highly selective 5-hydroxytryptamine 3 receptor antagonist, was successfully enantioseparated by recycling countercurrent chromatography using sulfobutyl ether-β-cyclodextrin as chiral selector. Important factors for the enantioseparation were optimized, including different organic solvent, type of substituted β-cyclodextrin, pH of aqueous phase, concentration of chiral selector, and separation temperature. A biphasic solvent system composed of n-hexane: n-butyl acetate: 0.1mol/L phosphate buffer solution pH 9.2 with 50mmol/L of sulfobutyl ether-β-cyclodextrin (2.5:7.5:10, v/v/v) was selected. Under optimized separation conditions, 5mg of ondansetron was enantioseparated using recycling countercurrent chromatography, yielding 1.2 and 1.5mg of ondansetron enantiomers with 97.5 and 95.8% purity and the recovery reached 48-60%.

Unlimited recycling counter-current chromatography for the preparative separation of natural products: Naphthaquinones as examples.

Recycling counter-current chromatography (CCC) has been developed and widely used in preparative separation. Due to increasingly broader peaks with longer elution times, recycling elution must be stopped before a peak overlap occurs, resulting in the insufficient separation of target compounds. In this study, the concept of in situ concentration was proposed, and the corresponding technique was designed to compress the effluents with the reserved separation effect (peak resolution). By combining this technique with multi-stage recycling elution, a novel unlimited recycling CCC (URCCC) strategy was developed to overcome the recycling time limitation to improve the resolution. The URCCC strategy was successfully applied in the preparative separation of naturally occurring naphthaquinones, where the in situ concentration was used two times with three-stage recycling CCC elution. Finally, isobutyrylshikonin (1), β, β-dimethylacrylshikonin (2) and isovalerylshikonin (3) were separated with high resolutions (R1,2=1.38 and R2,3=1.26). A high yield of pure naphthaquinones was achieved (89.6%), and the purity of each exceeded 98%. In conclusion, the URCCC strategy can improve the recycling elution times until the target compounds achieve sufficient separation, which may enable a broader range of application in structurally related compounds separation, especially in natural product separation.

Preparative Separation of Ginkgolic Acids from the Sarcotesta of Ginkgo biloba L. by β-Cyclodextrin Clathration Coupled with pH-Zone-Refining and Recycling Countercurrent Chromatography

Ginkgolic acids (GAs), bioactive compounds found in Ginkgo biloba L., have received much attention in pharmacological research. Separation of individual GAs from their mixtures is, however, difficu...

Simultaneous concentration and separation of target compounds from multicomponent mixtures by closed-loop recycling countercurrent chromatography

Closed-loop recycling countercurrent chromatography (CLR CCC) with multiple sample injection has been shown to provide simultaneous concentration and separation of target compounds from multicomponent mixtures. Previous analysis of CLR CCC with multiple sample injections has been limited to the ideal recycling model, which neglects the effects caused by the pump and connecting lines. In this study, an analysis of the process is carried out based on the non-ideal recycling model: recycling chromatograms at two points of the closed-loop – the inlet of the column (A) and the outlet of the column (B) – are considered. The sample is repeatedly introduced at the inlet of the column when the circulating peak of target compound passes point A. Analytical expressions are developed, allowing the design and simulation of different variants of simultaneous separation and concentration of target compounds from multicomponent mixtures. Examples of separation of target compounds from three and five-component mixtures are discussed. Experimental results are presented demonstrating a reasonable agreement between the theory and the experiment. Due to its ability to concentrate individual solutes, CRL CCC with multiple sample injections can become an efficient analytical method to determine minor components in complex mixtures.

Bioactivity-guided cut countercurrent chromatography for isolation of lysine-specific demethylase 1 inhibitors from Scutellaria baicalensis Georgi

Countercurrent chromatography (CCC) has gradually become a widely used method for preparative separation of bioactive natural molecules. These molecules generally contain distinct scaffolds and characteristics that cannot be readily isolated from plants. While one-dimensional CCC is typically used for the initial purification with insufficiently resolved peaks after locating bioactive components, two-dimensional (2D) or multi-dimensional CCC strategies are employed to improve the resolution of peaks. However, these methods usually present certain disadvantages, such as complicated procedures and increased time consumption, experimental costs, and equipment requirements. Here, a bioactivity-guided cut CCC strategy was established to isolate lysine-specific demethylase 1 (LSD1) inhibitors from Scutellaria baicalensis Georgi. Gradient-elution CCC coupled with real-time detection of LSD1 inhibition by the collected fractions was developed. Next, an online-storage recycling CCC mode was designed to enable the active fractions to be stored in coils, and these active fractions were further separated to obtain pure compounds by using sequential recycling elution. In this strategy, active fractions are first identified, and then pure LSD1 inhibitors are isolated in the 2D CCC mode through continuous separation on a single instrument. By using our bioactivity-guided cut CCC strategy, we successfully isolated six natural LSD1 inhibitors from S. baicalensis Georgi, five of which were identified for the first time as natural LSD1 inhibitors.

Online-storage recycling counter-current chromatography for preparative isolation of naphthaquinones from Arnebia euchroma (Royle) Johnst

Counter-current Chromatography (CCC) has gradually become a popular method for preparative separation, especially in natural product isolation. As an effective separation method, one-dimensional (1D) CCC often results in insufficiently resolved peaks, due to limitations in the separation efficiency and peak capacity in an equipment. Therefore, two dimensional (2D)/multi-dimensional (multi-D) CCC strategies with recycling elution mode were developed to achieve successful separation of target compounds. However, the reported 2D or multi-D CCC approaches lead to experimental costs, complicated procedures, higher requirements for equipment, and increased time consumption. In this study, an online-storage recycling (OSR) CCC strategy was designed to achieve sequential recycling elution for multi-fractions of effluent in non-stop separation with single instrument using three 6-port valves and two storage loops, which would be realized by introducing 2D or multi-D CCC method before. In this non-stop separation system, the fraction C of effluent was subjected to recycling separation while the other fractions (A and B) were storing online, following which these two fractions were subjected to subsequent recycling separations in order, after the completion of the previous recycling elution. Then, six natural occurring naphthaquinone analogues, namely, shikonin (1), propionylshikonin (2), deoxyshikonin (3), isobutyrylshikonin (4), β, β-dimethylacrylshikonin (5) and isovalerylshikonin (6), were isolated from the crude extract of Arnebia euchroma in single run. The purities of all compounds were > 95.0% as determined by HPLC, and their structures were determined by means of UV, MS, 1H NMR, 13C NMR, and optical rotatory dispersion (ORD).

Multi-channel recycling counter-current chromatography for natural product isolation: Tanshinones as examples

Recycling is a simple and efficient strategy for improving the peak resolution. In this work, a novel multi-channel recycling counter-current chromatography (CCC) was successfully established and three representative three-channel recycling CCC systems including 1R, 2R, and 3R CCC recycling on one, two, and three channel columns were developed for four typical tanshinones fractions. The results demonstrated that all recycling CCC separations were efficient for the isolation of targeted tanshinones. 1R CCC not only provided the single recycling separation, but also offered two parallel recycling separation or repeated preparation for multiple targets. 2R CCC provided the increased peak resolution and less risk of overlapping. 3R CCC offered versatile elution and recycling modes for multiple targets. Compared to 1R and 2R CCC, 3R CCC was easier to separate the more complex natural products with more targets in the complex natural extracts. In summary, the developed multi-channel recycling CCC systems including 1R, 2R, and 3R CCC were successful and efficient for resolving the complex natural tanshinones. To the best of our knowledge, this is the first document to report the multi-channel recycling CCC methods for the separation of tanshinones. They may be used as new multi-dimensional recycling CCC for targeted and non-targeted isolation of natural products. It may be widely used for current natural drug development and metabolome analysis.

Separation of anthraquinone compounds from the seed of Cassia obtusifolia L. using recycling counter-current chromatography.

Recycling counter-current chromatography (CCC) together with step-gradient CCC and medium-pressure liquid chromatography (MPLC) was employed to separate nine anthraquinone compounds from Cassia obtusifolia L. in this study. The results showed that recycling CCC is a powerful tool for compounds that are difficult to separate with common elution mode. CCC was the better option for crude material while MPLC had advantage for the final tuning. The combination of recycling CCC and MPLC could simplify the method exploring process in the separation process. The structures of these compounds were identified according to their mass spectra, by (1)H-NMR and compared with standard compounds.

Recycling High-Speed Countercurrent Chromatography for Separation of Taxol and Cephalomannine

A mixture of taxol and cephalomannine was subjected to recycling countercurrent chromatography (CCC) which allows the use of the same column for repetitive separation of the peak fractions to improve the peak resolution. When the total amount of 50 mg of the two components was recycled twice, peak resolution (Rs) increased from 0.7 to 1.27. The results also showed that Rs value increases according to the formula: Rs-n = n1/2Rs-1 where n is the number of CCC runs, and Rs-1 and Rs-n, peak resolutions after the first and the nth run, respectively.