Scale-up Separation Research Articles

Separation of Flavonoids and Purification of Chlorogenic Acid from Bamboo Leaves Extraction Residues by Combination of Macroporous Resin and High-Speed Counter-Current Chromatography.

Flavonoids are major active small-molecule compounds in bamboo leaves, which can be easily obtained from the bamboo leaves extraction residues (BLER) after the polysaccharides extraction. Six macroporous resins with different properties were screened to prepare and enrich isoorientin (IOR), orientin (OR), vitexin (VI), and isovitexin (IVI) from BLER, and the XAD-7HP resin with the best adsorption and desorption performance was selected for further evaluation. Based on the static adsorption experiments, the experimental results showed that the adsorption isotherm fitted well with the Langmuir isotherm model, and the adsorption process was better explained by the pseudo-second-order kinetic model. After the dynamic trial of resin column chromatography, 20 bed volume (BV) of upload sample and 60% ethanol as eluting solvent was used in a lab scale-up separation, and the results demonstrated that the content of four flavonoids could be increased by 4.5-fold, with recoveries between 72.86 and 88.21%. In addition, chlorogenic acid (CA) with purity of 95.1% was obtained in water-eluted parts during dynamic resin separation and further purified by high-speed countercurrent chromatography (HSCCC). In conclusion, this rapid and efficient method can provide a reference to utilize BLER to produce high-value-added food and pharmaceutical products.

Preparation of reference standard extract of Lonicera hypoglauca Miq. with macroporous resin following a scale-up separation

ABSTRACT Bench-scale and pilot-scale methods were developed for the separation of extracted caffeoylquinic acids (CQAs) also called reference standard extracts (RSE) from defined source Lonicerae species using macroporous resin. Various separation parameters of the process were optimized such as Lonicera hypoglauca Miq. batch No. 4, 75% ethanol as extraction solvent, SD3000 macroporous resin, 3 mg/mL of total CQA loading solutions, 2 BV/h of adsorption and desorption flow rates and ethanol-water (30:70, 80:20, v/v) as eluent in gradient mode. The pilot-scale experiments were evaluated in six cycles. The quantitative analysis of CQA using RSE and mixed external standards were compared.

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 50mM of trifluoroacetic acid as retainer in the organic phase and 40mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500mg of free fatty acids from a refined fish oil sample. 79.6mg of eicosapentaenoic acid and 328.3mg 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 1g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150mM of retainer, producing satisfactory yields and purities of targets.

Enrichment and separation of steroidal saponins from the fibrous roots of Ophiopogon japonicus using macroporous adsorption resins.

In this study, a simple and effective strategy for the enrichment of total steroidal saponins (TSS) from the fibrous roots of Ophiopogon japonicus (L. f.) Ker-Gawl. (FROJ) using macroporous adsorption resin was systematically developed. XAD-7HP resin was selected from six macroporous resins for further study because of the highest static adsorption and desorption capacities. The static adsorption of TSS on XAD-7HP resin fitted well to the Langmuir isotherm model and pseudo second-order kinetic model; the thermodynamics test showed that the adsorption process was spontaneous and exothermic. The dynamic tests on XAD-7HP resin columns demonstrated that the breakthrough volume was 16 bed volume (BV), and 6 BV of 80% ethanol was suitable for dynamic desorption. In a lab scale-up separation under optimal dynamic conditions, the content of TSS in the resin-enrichment fraction increased from 1.83% in the crude extracts to 13.86% by 7.59-fold with a recovery yield of 82.68%. Three steroidal saponins were obtained from the resin-enrichment fraction, and showed protective effects against oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cell (HUVEC) injury. Overall, these results suggested that XAD-7HP resin chromatography was an effective strategy for the large scale enrichment of TSS from FROJ, which showed the potential for functional food and pharmaceutical application.

A simple and efficient method for enrichment of cocoa polyphenols from cocoa bean husks with macroporous resins following a scale-up separation

The aim of this study is to develop an efficient method for the decaffeination and enrichment of the polyphenols from cocoa husks extracts. LX-17 was selected for the exploration of optimal processing parameters from eight kinds of macroporous resins. The adsorption kinetics and thermodynamics of (−)-epicatechin (EC) were studied prior to the scale-up separation. The optimum parameters for separation were as follow: 6.0 mg/mL cocoa extracts, pH 2.0, 25 °C column temperature, flow rates of adsorption and desorption 1.6 BV/h (Bed Volume, the volume of the resin) and ethanol-water (20:80, 50:50, 95:5, v/v) solutions in the gradient elution. In the scale-up separation, 2 kg of cocoa husks were extracted in 20 L 50% ethanol solutions, separated on 3 L LX-17 resins and yielded 34.99 g cocoa polyphenols. This method significantly increased the total polyphenol contents from 2.23% to 62.87% with a recovery yield of 78.57%.

Rational approach to solvent system selection for liquid-liquid extraction-assisted sample pretreatment in counter-current chromatography.

A rational liquid-liquid extraction approach was established to pre-treat samples for high-speed counter-current chromatography (HSCCC). n-Hexane-ethyl acetate-methanol-water (4:5:4:5, v/v) and (1:5:1:5, v/v) were selected as solvent systems for liquid-liquid extraction by systematically screening K of target compounds to remove low- and high-polarity impurities in the sample, respectively. After liquid-liquid extraction was performed, 1.4g of crude sample II was obtained from 18.5g of crude sample I which was extracted from the flowers of Robinia pseudoacacia L., and then separated with HSCCC by using a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v). As a result, 31mg of robinin and 37mg of kaempferol 7-O-α-l-rhamnopyranoside were isolated from 200mg of crude sample II in a single run of HSCCC. A scale-up separation was also performed, and 160mg of robinin with 95% purity and 188mg of kaempferol 7-O-α-l-rhamnopyranoside with 97% purity were produced from 1.2g of crude sample II.

Enrichment of total steroidal saponins from the extracts of Trillium tschonoskii Maxim by macroporous resin and the simultaneous determination of eight steroidal saponins in the final product by HPLC.

An effective and simple method was established for the separation and enrichment of steroidal saponins from Trillium tschonoskii Maxim. The adsorption and desorption properties of seven macroporous resins were investigated. Among the tested resins, AB-8 resin showed the best adsorption and desorption capacities. The adsorption of steroidal saponins on AB-8 at 25°C was quite consistent with both the Freundlich isotherm model and the pseudo-second-order kinetics model. By optimizing the dynamic adsorption and desorption parameters, the content of steroidal saponins increased from 5.20% in the crude extracts to 51.93% in the final product, with a recovery yield of 86.67%. Furthermore, by scale-up separation, the concentration and recovery of total steroidal saponins were 43.8 and 85.5%, respectively, which suggested that AB-8 resin had great industrial and pharmaceutical potential because of its high efficiency and cost-effectiveness. In addition, a high-performance liquid chromatography method for the simultaneous determination of eight steroidal saponins was established for the first time, which was employed to qualitatively and quantitatively analyze the final product. Based on the methodological validation results, the high-performance liquid chromatography method can be widely applied to the quality control of steroidal saponins from Trillium tschonoskii Maxim due to its excellent accuracy, stability, and repeatability.

Neolignans as xanthine oxidase inhibitors from Hyptis rhomboides

The active fraction from the EtOH extract of Hyptis rhomboides against xanthine oxidase was identified by use of an HPLC microfractionation–centrifugal vacuum evaporation–bioassay hyphenated technique. Scale-up separation of the active subfractions using semi-preparative RP-HPLC provided 13 phenylpropanoid compounds, including O-styrenylneolignans, hyprhombins A–C, epihyprhombin B, and hyprhombin B methyl ester, and O-caffeoylnorneolignans, hyprhombins D and E. All of these compounds shared a common 1,4-benzodioxane skeleton, as established by spectroscopic analyses. Hyprhombin C and epihyprhombin B exhibited better anti-xanthine oxidase activity than allopurinol, with IC50 values of 0.6 and 2.0μM, respectively.

Enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino by column chromatography technique

In present study, the performance and separation characteristics of nine macroporous resins for the enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino have been evaluated. The adsorption and desorption properties of crude gardenia yellow solution on macroporous resins including HPD722, HPD100, HPD100A, HPD400, HPD400A, D101, AB-8, XAD-16, and NKA-9 have been compared. Then, HPD722 was chosen to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. Column packed with HPD722 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process of gardenia yellow. The optimal conditions were as follows: The crude gardenia yellow solution with concentration of 15mg/mL was loaded in column packed with HPD722 resin at the flow rate of 1.0mL/min, and the adsorbate-laden column was washed with 800mL water, 600mL 15% ethanol water solution respectively at the speed of 2.5mL/min, then desorbed with 200mL 80% ethanol water solution at the speed of 3.5mL/min. The colority of the product obtained were up to 300. The method developed in this study provides a new approach for scale-up separation and purification of gardenia yellow from G. jasminoides var. radicans Makino.

Separation of scutellarin from crude extracts of Erigeron breviscapus (vant.) Hand. Mazz. by macroporous resins

Scutellarin, a flavone glycoside, popularly used in the treatment of heart disease, has been efficiently separated using macroporous resins from crude extracts of Chinese medicinal plant Erigeron breviscapus (vant.) Hand. Mazz. HPD-800 resin offered the best adsorption and desorption capacity for scutellarin among the eight macroporous resins tested, and its adsorption data at 25 °C fit best to the Langmuir isotherm. The dynamic adsorption and desorption experiments have been carried out on a HPD-800 resin packed column to optimize the separation process of scutellarin from the crude extracts of E. breviscapus. After one run treatment with HPD-800 resin, the scutellarin content in the product was increased 15.69-fold from 2.61% to 40.96% with a recovery yield of 95.01%. The preparative separation process via adsorption–desorption method developed in this study provides a new approach for scale-up separation and purification of scutellarin for its wide pharmaceutical use.

Optimization of the capillary zone electrophoresis loading limit and resolution of proteins, using triethylamine, ammonium formate and acidic pH.

Capillary zone electrophoresis (CZE) of five model proteins (lysozyme, myoglobin, ribonuclease A, alpha-lactalbumin, and trypsinogen), using ammonium formate as the electrophoretic buffer and triethylamine (TEA) as a buffer additive at pH 2.5, was used for protein separation. The electrophoretic behavior of these proteins was examined with respect to various concentrations (10-40 mM) of TEA and of ammonium formate. Based on the experimental parameters of electrophoretic resolution, current, and peak separation time, an electrolyte (30 mM each of TEA and ammonium formate) was empirically derived as the optimum for scale-up separation. The loading limit for proteins, covering a wide range of injection volumes (60-990 nl) and amount of protein (1-21 pmol of each protein), was investigated on 75 and 100 microns I.D. untreated fused-silica capillaries. Protein adsorption (average < 15%) was experimentally determined using this volatile buffer system.