Separation of alkylnaphthalenes by preparative liquid chromatography using column switching systems

Summary The paper describes the separation of the mixture of alkynaphthalenes from distillation cuts of a pyrolysis oil, by preparative liquid chromatography on silica. The design of the system permits the connection of the columns to form multicolumn systems.The samples were first separated on a single column. The mixtures were further separated using two-column chromatography systems.The obtained fractions were analyzed by capillary gas chromatography. In most cases a substantial increase in the concentrations of the individual components was achieved. In several cases, pure compounds have been obtained. Separation efficiency increases in the following order: single column, two directly coupled collumns, two-step switching chromatography, heartcutting.     

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.     

Optimization of throughput in preparative column luquid chromatography by column overloading and partial fractionation of the effluent

Summary The criteria for the optimization of preparative chromatography are sample throughput and product purity. It is shown for column liquid chromatography that the throughput has been maximized for a given purity by column overloading and appropriate fractionation. The size and position of the fraction must be determined by chemical analysis, since overlapping peaks under overloading conditions are usually distorted compared to the peaks of the same amounts of pure compounds. With overloading of the column larger particles can be used as column packings without reduction of the separation effect.     

Rapid prediction and optimization of concentration conditions for preparative fractions by solid-phase extraction

SPE is an effective tool for concentrating preparative fractions isolated from a complex sample. To guarantee high efficiency and recovery of concentration, the concentration conditions could be optimized by predicting the breakthrough volume (V(B)). In this study, a method of predicting V(B )of unknown compounds in preparative fractions at any isocratic mobile phase composition with the analytical retention parameters a and c is described. The a and c values and the relationship between half peak width (W(1/2)) and retention time of a model analyte were measured using the analytical elution mode on an SPE column, and the V(B )and retention volume (V(R)) predicted with the a and c values were validated with breakthrough experiments. However, it is impossible to measure the a and c values of multiple compounds in a complex system directly on an SPE column with a low number of theoretical plates. The correlation of the a and c values between the SPE and analytical columns was developed so that the analytical data could be transferred to the SPE column. With the calculated a and c values, we could optimize the concentration conditions on the basis of the predicted V(B )and the volume of the preparative fraction.     

Purification of flavonoids and triterpene saponins from the licorice extract using preparative HPLC under RP and HILIC mode

A four‐channel preparative HPLC was employed to isolate and purify compounds from licorice extract. Two separation modes, RP and hydrophilic interaction LC (HILIC), were used in preparative HPLC. HILIC mode was adopted to resolve the purification of the compounds with similar hydrophobicity, which were co‐eluted under RP mode. Using the two separation modes during the purification process, fifteen compounds were isolated from licorice extract. The results indicated that preparative HPLC performed under HILIC mode is an efficient method for the isolation and purification of compounds from natural products.     

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 high-speed counter-current chromatography for purification of shikonin from the Chinese medicinal plant Lithospermum erythrorhizon

The bioactive compound shikonin was successfully isolated and purified from the crude extract of the traditional Chinese medicinal plant Lithospermum erythrorhizon Sieb. et Zucc. by preparative high-speed counter-current chromatography (HSCCC). The preparative HSCCC was performed using a two-phase solvent system composed of n-hexane-ethylacetate-ethanol-water (16:14:14:5 (v/v)). A total amount of 19.6 mg of shikonin at 98.9% purity was obtained from 52 mg of the crude extract (containing 38.9% shikonin) with 96.9% recovery. The preparative isolation and purification of shikonin by HSCCC was completed in 200 min in a one-step separation.     

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.     

Separations of enantiomers by preparative capillary isotachophoresis.

The use of capillary isotachophoresis (ITP), operating in a discontinuous fractionation mode, for preparative separations of enantiomers of chiral compounds was studied. The ITP separations were carried out in the column-coupling configuration of the separation unit provided with the preseparation column of a 1.0 mm ID and the trapping column of a 0.8 mm ID. Such a configuration of the CE separation unit offers several working regimes suitable to preparative separations of enantiomers. 2,4-Dinitrophenyl-DL-norleucine (DNP-Norleu) was employed as a model analyte in our experiments with beta-cyclodextrin serving in the electrolyte solutions as a chiral selector. The preparative separations lasting about 20 min were evaluated by ITP and (more often) by capillary zone electrophoresis (CZE). It was found that one preparative run provided up to 14 microg of pure DNP-Norleu enantiomers. This corresponded to a 75 times higher production rate of ITP relative to a maximum value of this parameter as estimated for preparative CZE runs in cylindrical capillaries (0.5 pmol/s). About 75% of the DNP-Norleu enantiomers loaded into the preparative equipment could be recovered in pure enantiomer fractions. Contiguous natures of the zones in the ITP stack and adsorption losses of the enantiomers in the isolation step were found to set practical limits for a further enhancement of the recovery rates in the isolation of pure enantiomers.     

Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography

Positively charged reversed‐phase liquid chromatography was employed for the efficient preparative separation of isoquinoline alkaloids from Corydalis impatiens. Ten commercially available columns were compared for isoquinoline alkaloids analysis. While tailing, overloading, lower resolution, and buffer salts limited the application in purification of isoquinoline compounds of many of these columns, one positively charged reversed‐phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading isoquinoline compounds on a larger, preparative scale. The general separation process is as follows. First, isoquinoline alkaloids are enriched with Corydalis impatiens extract via a middle chromatogram isolated gel column. After column selection, separation is performed on an XCharge C18 analytical column, from which two evident chromatographic peaks are readily obtained. Finally, two isoquinoline alkaloids (protopine and corydamine) are selectively purified on the XCharge C18 preparative column. These results demonstrate that a middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography is effective for the preparative separation of isoquinoline alkaloids from Corydalis impatiens.     

反相液相色谱法制备纯化柠檬苦素类似物配糖体

 利用反相制备液相色谱结合吸附树脂柱色谱和离子交换色谱方法 ,从甜橙种子的提取物中纯化制备了一种柠檬苦素类似物配糖体 ,经 NMR测定为奥巴叩酮配糖体。     

Multidimensional preparative liquid chromatography to isolate flavonoids from bergamot juice and evaluation of their anti‐inflammatory potential

Important objectives of a high‐performance liquid chromatography preparative process are: purity of products isolated, yield, and throughput. The multidimensional preparative liquid chromatography method used in this work was developed mainly to increase the throughput; moreover purity and yield are increased thanks to the automated collection of the molecules based on the intensity of a signal generated from the mass spectrometer detector, in this way only a specific product can be targeted. This preparative system allowed, in few analyses both in the first and second dimensions, the isolation of eight pure compounds present at very different concentration in the original sample with high purity (>95%) and yield, which showed how the system is efficient and versatile. Pure molecules were used to validate the analytical method and to test the anti‐inflammatory and antiproliferative potential of flavonoids. The contemporary presence, in bergamot juice, of all the flavonoids together increases the anti‐inflammatory effect with respect to the single compound alone.     

反相高效液相色谱法制备松果菊苷标准品

 利用反相制备高效液相色谱结合溶剂萃取、大孔吸附树脂柱色谱和葡聚糖凝胶LH 2 0柱色谱方法 ,从管花肉苁蓉的乙醇提取物中纯化制备了苯乙醇苷类化合物松果菊苷的标准品 ,纯度达到 98%以上。方法操作简便 ,重现性好 ,可用于松果菊苷及其他苯乙醇苷类化合物的大量制备。     

Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis

This feasibility study deals with the use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, to the separations and isolations of glycoforms of recombinant human erythropoietin (rhEPO). The preparative CITP separations were monitored by capillary zone electrophoresis (CZE) with a hydrodynamically closed separation unit. Such a CZE system, suppressing fluctuations of the migration data linked with fluctuations of EOF and hydrodynamic flow, made possible to evaluate and compare the preparative CITP separations performed within a longer time frame. Preparative CITP, carried out in the separation unit with coupled columns of enhanced sample loadability, separating 100 microg of rhEPO in a run lasting ca. 30 min, gave the production rate higher than 55 ng/s for the rhEPO glycoforms. The preparative separations included valve isolations of the glycoforms from the ITP stack into four or six fractions. Such numbers of the fractions corresponded to typical numbers of the major glycoform peaks as resolved in CZE of rhEPO. With respect to close effective mobilities of the glycoforms and a multicomponent nature of rhEPO, the fractions contained mixtures of glycoforms with the dominant glycoforms enriched 10-100-fold, relative to the original rhEPO sample.     

A simple and efficient protocol for large‐scale preparation of three flavonoids from the flower of Daphne genkwa by combination of macroporous resin and counter‐current chromatography

In this paper, macroporous resin column chromatography and counter‐current chromatography (CCC) were applied for large‐scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. Nine kinds of resins were investigated by adsorption and desorption tests and D101 macroporous resin was selected for the first cleaning‐up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The crude extract after the first step was directly subjected to the preparative CCC purification using the solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:5:4:5, v/v). The compounds apigemin (823 mg), 3‐hydroxyl‐genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, determined by HPLC were produced from 3‐g crude extract only in one CCC run. Their chemical structures were identified by MS, UV and the standards.     

Large-scale isolation and purification of geniposide from the fruit of Gardenia jasminoides Ellis by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of geniposide from Gardenia jasminoides Ellis. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). According to the above solvent system, preparative HSCCC was successfully performed with the optimal solvent system composed of ethyl acetate-n-butanol-water (2:1.5:3, v/v/v) yielding 389 mg of geniposide at over 98% purity from 1g of the partially purified extract with 38.9% recovery in a one-step separation.     

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.     

Automated free-solution isotachophoresis: Instrumentation and fractionation of human serum proteins

An automated free-solution isotachophoresis system (FS-ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200-fold higher sample load compared to separation in capillaries of 180 μm inner diameter as used in analytical capillary isotachophoresis (C-ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C-ITP, which is essential for preparative-scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C-ITP and agarose gel electrophoresis.     

Liquid chromatographic fractionation of small peptides from wine

Peptides are difficult to isolate from wine because they are present in a complex mixture together with non-peptidic compounds. A method for the isolation, separation and purity assessing of small peptides is proposed. Small peptides (Mr<3000) were isolated from wine by hollow fibre ultrafiltration followed by column chromatography using the gel matrix Sephadex LH20. Fractions obtained by gel filtration on Sephadex LH20 were subjected to HPLC on a porous graphitic carbon column in order to isolate small peptides. Peak purity was then analysed by capillary electrophoresis.