Efficient separation of high‐purity compounds from Oxytropis falcata using two‐dimensional preparative chromatography

The separation of high‐purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two‐dimensional reversed‐phase preparative method was successfully developed for the separation of high‐purity compounds from Oxytropis falcata . Based on the analysis results, an ODS C18 prep column was used for first‐dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high‐purity compounds in the second‐dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans‐cinnamic acid ( 1 ) and trifolirhizin ( 2 ) from Oxytropis falcata . This method has the potential to be an efficient separation method of high‐purity compounds from Oxytropis falcata and it shows great promise for the separation of high‐purity compounds from complex samples.     

Preparative isolation of flavonoid glycosides from Sphaerophysa salsula using hydrophilic interaction solid‐phase extraction coupled with two‐dimensional preparative liquid chromatography

An offline preparative two‐dimensional reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography coupled with hydrophilic interaction solid‐phase extraction method was developed for the preparative isolation of flavonoid glycosides from a crude sample of Sphaerophysa salsula . First, the non‐flavonoids were removed using an XAmide solid‐phase extraction cartridge. Based on the separation results of three different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAqua C18 prep column, and 15 fractions were obtained from the 5.2 g target sample. Then, three representative fractions were selected for additional purification on an XAmide preparative column to further isolate the flavonoid glycosides. In all, eight flavonoid glycosides were isolated in purities over 97%. The results demonstrated that the two‐dimensional liquid chromatography method used in this study was effective for the preparative separation of flavonoid glycosides from Sphaerophysa salsula . Additionally, this method showed great potential for the separation of flavonoid glycosides from other plant materials.     

Purification of compounds from Lignum Dalbergia Odorifera using two-dimensional preparative chromatography with Click oligo (ethylene glycol) and C18 column

Purification of compounds from traditional Chinese medicines (TCMs) is an important task for understanding the chemical composition of TCMs. However, it is difficult to obtain compounds with high enough purity for identification by NMR due to the complexity of TCMs in chemical composition. In this study, a two‐dimensional purification method based on a Click oligo (ethylene glycol) column and a C18 column was developed to realize an orthogonal separation in preparative level for purifying compounds efficiently. The first dimensional preparation was performed on a Click oligo (ethylene glycol) column to simplify the sample into the fractions with good separation repeatability. On the first dimension, 7.2 g sample was separated into 11 fractions with a recovery of 86% within 6 h. A C18 column was taken as the second dimension to realize the high‐performance separation and rapid preparation from the fractions collected from the first dimension. Eight compounds in fraction 6 and 2 compounds in fraction 8 were isolated and identified after optimizing the separation and collection parameters. This method is a high‐efficient and orthogonal preparation method to improve the separation of a complex sample and increase the purity of the compounds, which benefits from the application of novel materials in the preparation and purification.     

A novel two‐dimensional preparative chromatography method designed for the separation of traditional animal Tibetan medicine Osteon Myospalacem Baileyi

Animal medicine is an important part in traditional Tibetan medicine. However, information about the chemical composition of animal medicine is very limited, and there is a lack of comprehensive chromatographic purification methods. In the present work, animal medicine Osteon Myospalacem Baileyi was taken as an example and a novel two‐dimensional preparative chromatographic method was established for the preparation of single compounds with high purity from the extract of Osteon Myospalacem Baileyi. The first‐dimension preparation was carried on a DAISO Silica prep column, and ten fractions were obtained from the 112.3 g crude sample within 12 injections. A diol prep column used in nonaqueous mobile phase was selected for the second‐dimension preparation. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single compounds of high purity from the extract of Osteon Myospalacem Baileyi. Additionally, this method showed great potential in the purification of weakly polar chemicals and it could act as a good example in the purification of other traditional animal medicines.     

Two‐dimensional hydrophilic interaction chromatography × reversed‐phase liquid chromatography for the preparative isolation of potential anti‐hepatitis phenylpropanoids from Salvia prattii

Traditional Tibetan medicine is important for discovery of drug precursors. However, knowledge of the chemical composition of traditional Tibetan medicines is very limited due to the lack of appropriate chromatographic purification methods. In the present work, Salvia prattii was taken as an example, and an off‐line hydrophilic interaction liquid chromatography/reversed‐phase liquid chromatography preparative method was developed for the purification of phenylpropanoids with high purity from a crude sample of Salvia prattii. Based on the separation results of four different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAmide preparative column with the crude sample concentration of 62.0 mg/mL, and five main fractions were obtained from the 12.4 g crude sample with a recovery of 54.8%. An XCharge C₁₈ preparative column was applied in the second‐dimensional preparation to further isolate the phenylpropanoids from the redissolved first‐dimensional fractions with concentration of approximately 50.0 mg/mL. The purities of the phenylpropanoids isolated from the crude sample of Salvia prattii were higher than 98%, indicating that the method was efficient for the purification of phenylpropanoids with high purity from Salvia prattii. Additionally, this method showed great potential in the preparation of phenylpropanoids and can serve as a good example for the purification of phenylpropanoids from other plant materials.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Three‐phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high‐speed counter‐current chromatography

A three‐phase solvent system was efficiently applied for high‐speed counter‐current chromatography to separate secondary metabolites with a wide range of hydrophobicity in Dicranostigma leptopodum. The three‐phase solvent system of n‐hexane/methyl tert‐butyl ether/acetonitrile/0.5% triethylamine (2:2:3:2, v/v/v/v) was selected for high‐speed counter‐current chromatography separation. The separation was initiated by filling the column with a mixture of intermediate phase and lower phase as a stationary phase followed by elution with upper phase to separate the hydrophobic compounds. Then the mobile phase was switched to the intermediate phase to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with the lower phase. In this research, 12 peaks were eluted out in one‐step operation within 110 min, among them, eight compounds with acceptable purity were obtained and identified. The purities of β‐sitosterol, protopine, allocryptopine, isocorydione, isocorydine, coptisine, berberrubine, and berberine were 94.7, 96.5, 97.9, 86.6, 98.9, 97.6, 95.7, and 92.8%, respectively.     

Preparative separation of capsaicin and dihydrocapsaicin from Capsicum frutescens by high‐speed counter‐current chromatography

Capsaicin and dihydrocapsaicin are two main bioactive components of Capsicum frutescens and are widely used as food additives and drugs in China and India. Due to their similarity in structures, isolation of capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography, normal‐phase thin‐layer chromatography (TLC) becomes difficult. This study involves separating capsaicin and dihydrocapsaicin with sufficient purity and recovery using high‐speed counter‐current chromatography (HSCCC) with a solvent system composed of n‐hexane–ethyl acetate–methanol–water–acetic acid (20:20:20:20:2, v/v/v/v/v). Separation parameters such as sample volume, and sample concentration were first optimized on analytical HSCCC, and then scaled up to preparative HSCCC. 0.65 g capsaicin and 0.28 g dihydrocapsaicin were obtained from 1.2 g crude extract and their purities were 98.5 and 97.8%, respectively. The recoveries of the two compounds were 86.3 and 85.4%, respectively. The purity of the isolated compounds was analyzed by high‐performance liquid chromatography (HPLC) and their structures were identified by ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analysis.     

Purification of lignans from Fructus Arctii using off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high‐purity compounds. In total, 12 high‐purity compounds were isolated from Fructus Arctii . Their structures were identified by using high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method for the purification of lignans from Fructus Arctii . The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products.     

Online hyphenation of extraction,Sephadex LH‐20 column chromatography,and high‐speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH‐20 column chromatography combined with high‐speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH‐20 column directly to cut the nontarget fractions followed by the second‐dimensional high‐speed countercurrent chromatography, hyphenated by a six‐port valve equipped at the post‐end of Sephadex LH‐20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant.     

Research on the change of chemical composition in productive process of Re Du Ning injection by HPLC/Q‐TOF MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (HPLC/Q‐TOF MS) was developed for the analysis of chemical composition change in the production process of Re Du Ning injection, a Chinese medicine preparation with a combination of Lonicera japonica Thunb., Gardenia jasminoides Ellis and Artemisia annua L. A total of 90 compounds from raw materials–intermediates–Re Du Ning injection were detected; among them, 55 compounds were identified or tentatively characterized, and the characteristic ions of different types of compounds were described. Based on these studies, the different types of compounds in the various process routes were analyzed. A total of 28 compounds, including seven iridoid glycosides and six monoterpenes from G. jasminoides Ellis, five iridoid glycosides, nine phenolic acids and one unknown compound from L. japonica Thunb., were transferred to Re Du Ning injection, and two unknown compounds were generated in the production process of Re Du Ning injection. The results indicated that the Chinese Medicine Pharmaceutical process control is very important. This method could provide some reference for other Chinese medicine preparations. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of high‐speed counter‐current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high‐speed counter‐current chromatography using a two‐phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high‐performance liquid chromatography with an Agilent ZORBAX® SB‐C18 column (4.6 mm × 150 mm, 5 μm). After separation by high‐speed counter‐current chromatography and high‐performance liquid chromatography, we obtained 3.5 mg of platetyolin A, 4.1 mg of platetyolin B, and 18.1 mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high‐performance liquid chromatography and the chemical structures were evaluated by high‐resolution electrospray ionization time‐of‐flight mass spectrometry and one‐ and two‐dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.     

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

In this study, two mixed‐mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two‐dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed‐mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed‐mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off‐line 2D‐LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine—Uncaria rhynchophylla. The two‐dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed‐mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.     

Purification of alkaloids from Corydalis yanhusuo W. T. Wang using preparative 2‐D HPLC

Two‐dimensional preparative multi‐channel parallel high performance liquid chromatography was successfully applied for the first time to isolate and purify alkaloids from Corydalis yanhusuo. The experiments were performed in off‐line mode using the same preparative chromatographic column with pH 3.5 in the first and pH 10.0 in the second separation dimension. In the preparative process, UV‐triggered fraction collection was used in the first dimension while UV and MS‐triggered collection were used in the second dimension for reasons of sensitivity and complementarity. Two pure compounds and nine fractions were obtained in the first dimension. Then two representative fractions were further purified in the second dimension and six pure compounds were obtained. The results demonstrated that this procedure is an effective approach for the preparative isolation and purification of alkaloids from Corydalis yanhusuo. Based on the different pH values of the mobile phase in this method, it is also suitable for the preparative isolation and purification of other compounds from TCMs which are sensitive to the pH of the solutions. Moreover, this method will be a promising tool for the purification of low content compounds from natural products.     

Test compounds for detecting the silanol effect on the elution of ionized amines in reversed‐phase LC

The effectiveness of several basic compounds for testing silica‐based stationary phases was reviewed by applying them to recent columns for reversed‐phase HPLC. Most octadecylsilylated (C18) stationary phases, prepared as a base‐deactivated material from high‐purity silica gel with endcapping, provided excellent peak shape and column efficiency for the bases including benzylamine and amitriptyline that once caused problems and were subsequently employed for testing silanol activities. However, a cyclic tertiary amine, dextrometorphan, was eluted as an acceptable peak from only a few columns at neutral pH. Such a more sensitive probe is expected to contribute to further improvement of the stationary phase for reversed‐phase HPLC.     

Separation of five oligostilbenes from Vitis amurensis by flow‐rate gradient high‐performance counter‐current chromatography

A rapid and efficient high‐performance counter‐current chromatography (HPCCC) method was developed to separate five oligostilbenes from the roots of Vitis amurensis. An n‐hexane/ethyl acetate/methanol/water system (4:8:4:10, v/v/v/v) was selected as an optimal two‐phase solvent system of which the upper phase was used as the stationary phase and the lower phase was used as the mobile one. Partition coefficient values for the target compounds under these optimized conditions were 0.28 ( 1 , ampleosin A), 7.12 ( 2 , (+)‐g‐viniferin), 2.26 ( 3 , vitisin A), 5.38 ( 4 , wilsonol C), and 11.23 ( 5 , vitisin B). Flow‐rate gradient HPCCC (4 mL/min in 0–70 min, 8 mL/min in 70–250 min) was applied to isolate the target compounds in as high purity as possible within the shortest possible run time. Under these conditions, ampelopsin A (12.1 mg), (+)‐g‐viniferin (10.4 mg), vitisin A (2.8 mg), wilsonol C (3.2 mg), and vitisin B (37 mg) were isolated with >95% purity from 150 mg of enriched oligostilbene extract. Although the K_D of the last eluted compound, vitisin B (K_D = 11.23), was relatively large, it was eluted in 115–145 min using the two‐phase solvent system. This study shows that HPCCC is an efficient tool for the isolation and purification of natural products.     

Simultaneous quantification of five proteins and seven additives in dairy products with a heart‐cutting two‐dimensional liquid chromatography method

A heart‐cutting two‐dimensional high‐performance liquid chromatography method was developed to simultaneously quantify five major proteins and seven food additives (maltol, ethyl maltol, vanillin, ethyl vanillin, benzoic acid, sorbic acid, and saccharin sodium) in milk and milk powders. In this two‐dimensional system, a Venusil XBP‐C4 column was selected in the first dimension for protein separation, and a Hypersil ODS‐2 C18 column was employed in the second dimension for additive separation; a two‐position, six‐port switching valve was used to transfer the targets (additives) from the first dimension to the second dimension. Method validation consisted of selectivity, response function, linearity, precision, sensitivity, and recovery. In addition, a conventional one‐dimensional high‐performance liquid chromatography method was also tested for comparison. The two‐dimensional method resulted in significantly improved recovery of the food additives compared to the conventional method (90.6–105.4% and 65.5–86.5%, respectively). Furthermore, this novel method has a simple one‐step sample preparation procedure, which shortens the analysis time, resulting in more efficient analysis and less solvent usage.     

A consecutive preparation method based upon accelerated solvent extraction and high‐speed counter‐current chromatography for isolation of aesculin from Cortex fraxinus

A consecutive preparation method based upon accelerated solvent extraction (ASE) coupled with high‐speed counter‐current chromatography (HSCCC) was presented and aesculin was obtained from Cortex fraxinus. The extraction condition of ASE was optimized with response surface methodology; some significant parameters such as the solvent system and its stability, the amount of loading sample in HSCCC were also investigated. The original sample was first extracted with methanol at 105°C and 104 bar for 7 min using ASE, then the extracts were consecutively introduced into the HSCCC system and separated and purified with the same ethyl acetate/n‐butanol/water (7:3:10, v/v/v) solvent system for five times without further exchange and equilibrium. About 3.1 ± 0.2 mg/g in each time and total of 15.4 mg/g aesculin with purity over 95% was isolated from Cortex fraxinus. The results demonstrated that the consecutive preparation method was time and solvent saving and high throughput, it was suitable for isolation of aesculin from Cortex fraxinus, and also has good potential on the separation and purification of effective compounds from natural product.     

Preparative isolation and purification of harpagoside and angroside C from the root of Scrophularia ningpoensis Hemsley by high‐speed counter‐current chromatography

In this study, the bioactive component harpagoside and angroside C in the root of Scrophularia ningpoensis Hemsley was simultaneously separated by high‐speed counter‐current chromatography (HSCCC). A two‐phase solvent system containing chloroform/n‐butanol/methanol/water (4:1:3:2, v/v/v/v) was selected following consideration of the partition coefficient of the target compound. The crude extract (200 mg) was loaded onto a 280‐mL HSCCC column and yielded 22 mg harpagoside and 31 mg angroside C with the purity of higher than 98 and 98.5%, respectively. It is feasible to isolate active compounds harpagoside and angroside C from S. ningpoensis using HSCCC.     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.